U.S. Department of Commerce Volume 102 Number 1 January 2004 Fishery Bulletin U.S. Department of Commerce Donaid L. Evans Secretary National Oceanic and Atmospheric Administration Vice Admiral Conrad C. Lautenbacher Jr., USN (ret.) Under Secretary for Oceans and Atmosphere National Marine Fisheries Service William T. Hogarth Assistant Administrator for Fisheries .^TOFCo. X K1^ / The Fishery' Bulletin (ISSN 0090-0656) is published quarterly by the Scientific Publications Office, National Marine Fish- N( >AA, 7600 Sand Point Way NE, BIN C15700, Seattle. WA 981 15-0070. Periodicals postage is paid at Seattle, WA, and at additional mailing offices. POST- MASTER: Send address changes for sub- scription- to Fishery Bulletin. Superin- tendent of Documents, Attn.: Chief. .Mail List Branch, Mail Stop SSOM, Washing- ton. DC 20402-9373. Although the contents of this publica- tion have not been copyrighted and may be reprinted entirely, reference to source is appreciated. The Set if Commerce has deter- mined that the publication of tin ording to law for the transaction of public business of this Department. Use of funds for printing of nodical has been approved by the oroftheOffii cement and Budget. For sale by the Superintendent of nuts. US. Government Printing I mice, Washington, DC 20402. Subscrip- tion pi i it: $55.00 domestic and $68.75 foreign. Cost per single issue: $28.00 dome ,5.00 foreign. See back for order form. Scientific Editor Dr. Norman Bartoo Associate Editor Sarah Shoffler National Marine Fisheries Service, NOAA 8604 La Jolla Shores Drive La Jolla, California 92037 Managing Editor Sharyn Matriotti National Marine Fisheries Service Scientific Publications Office 7600 Sand Point Way NE, BIN C15700 Seattle, Washington 981 15-0070 Editorial Committee Dr. Harlyn O. Halvorson Dr. Ronald W. Hardy Dr. Richard D. Methot Dr. Theodore W. Pietsch Dr. Joseph E. Powers Dr. Harald Rosenthal Dr. Fredric M. Serchuk Dr. George Watters University of Massachusetts, Boston University of Idaho, Hagerman National Marine Fisheries Service University of Washington, Seattle National Marine Fisheries Service Universitat Kiel, Germany National Marine Fisheries Service National Marine Fisheries Service Fishery Bulletin web site: www.fishbull.noaa.gov The Fishery Bulletin carries original research reports and technical notes on investigations in fishery scien ring, and economics. It began as the Bulletin of the United States Pish Commission in 1881; it became the Bulletin of the Bureau of Fisheries in 1904 and the Fishery Bulletin of the Fish and Wildlife Service in 1941. Separates were issued as documents tl volume 46; the last document was No. 1103. Beginning with volume 47 in 1931 and continuing through volume 62 in 196.1 ired as a numbered bulletin. A new system began in 1963 with volume 6:3 in which papers are bound together in a single issue of the bulletin. Beginning with volume 70. number 1. January 1972, the Fishery Bulletin became a lieal, issued quarterly. In this form, it is available by subscription from the Superintendent of Documents. U.S. Government Printing Office, Washington, DC 20402. It is also available free in limited numbers to libl irch institutions. State and Federal agencies, and in exi for other scientific publications. U.S. Department of Commerce Seattle, Washington Volume 102 Number 1 January 2004 Fishery Bulletin Contents ary MAR 5 2004 The conclusions and opinions expressed in Fisher)' Bulletin are solely those of the authors and do not represent the official position of the National Marine Fisher- ies Service (NOAA) or any other agency or institution. The National Marine Fisheries Service (NMFS) does not approve, recommend, or endorse any proprietary product or pro- prietary material mentioned in this pub- lication. No reference shall be made to NMFS. or to this publication furnished by NMFS, in any advertising or sales pro- motion which would indicate or imply that NMFS approves, recommends, or endorses any proprietary product or pro- prietary material mentioned herein, or which has as its purpose an intent to cause directly or indirectly the advertised product to be used or purchased because of this NMFS publication. Articles 1-13 Alonzo, Suzanne H., and Marc Mangel The effects of size-selective fisheries on the stock dynamics of and sperm limitation in sex-changing fish 14-24 Baba, Katsuhisa, Toshifumi Kawajiri, Yasuhiro Kuwahara, and Shigeru Nakao An environmentally based growth model that uses finite difference calculus with maximum likelihood method: its application to the brackish water bivalve Corbicula /aponica in Lake Abashiri, Japan 25-46 Brodeur, Rick D., Joseph P. Fisher, David J. Teel, Robert L. Emmett, Edmundo Casillas, and Todd W. Miller Juvenile salmomd distribution, growth, condition, origin, and environmental and species associations in the Northern California Current 47-62 Garcia-Rodrfguez, Francisco J., and David Aurioles-Gamboa Spatial and temporal variation in the diet of the California sea lion (Zalophus californianus) in the Gulf of California, Mexico 63-77 Jung, Sukgeun, and Edward D. Houde Recruitment and spawning-stock biomass distribution of bay anchovy (Anchoa mitchilli) in Chesapeake Bay 78-93 Kellison, Todd G., and David B. Eggleston Coupling ecology and economy: modeling optimal release scenarios for summer flounder (Paralichthys dentatus) stock enhancement 94-107 Kritzer, Jacob P. Sex-specific growth and mortality, spawning season, and female maturation of the stripey bass (Lut/anus carponotatus) on the Great Barrrier Reef Fishery Bulletin 102(1) 108-117 Orr, Anthony J., Adria S. Banks, Steve Mellman, Harriet R. Huber, Robert L. DeLong, and Robin F. Brown Examination of the foraging habits of Pacific harbor seal (Phoca vitulina richardsi) to describe their use of the Umpqua River, Oregon, and their predation on salmonids Companion paper with Purcell et al., see "Notes" below. 118-126 Park, Wongyu, R. Ian Perry, and Sung Yun Hong Larval development of the sidestriped shrimp (Pandalopsis dispar Rathbun) (Crustacea, Decapoda, Pandahdae) reared in the laboratory 127-141 Pearson, Donald E., and Franklin R. Shaw Sources of age determination errors for sablefish (Anop/opoma fimbria) 142-155 Powell, Allyn B., Robin T. Cheshire, Elisabeth H. Laban, James Colvocoresses, Patrick O Donnell, and Marie Davidian Growth, mortality, and hatchdate distributions of larval and juvenile spotted seatrout (Cynoscion nebulosus) in Florida Bay, Everglades National Park 156-167 Santana, Francisco M., and Rosangela Lessa Age determination and growth of the night shark (Carcharhinus signatus) off the northeastern Brazilian coast 168-178 Smith, Keith R„ David A. Somerton, Mei-Sun Yang, and Daniel G. Nichol Distribution and biology of prowfish (Zaprora silenus) in the northeast Pacific 179-195 Ward, Peter, Ransom A. Myers, and Wade Blanchard Fish lost at sea: the effect of soak time on pelagic longlme catches 196-206 Watanabe, Chikako, and Akihiko Yatsu Effects of density-dependence and sea surface temperature on interannual variation in length-at-age of chub mackerel (Scomber japonicus) in the Kuroshio-Oyashio area during 1970-1997 Notes 207-212 Llanos-Rivera, Alejandra, and Leonardo R. Castro Latitudinal and seasonal egg-size variation of the anchoveta (Engrauhs nngens) off the Chilean coast 213-220 Purcell, Maureen, Greg Mackey, Eric LaHood, Harriet Huber, and Linda Park Molecular methods for the genetic identification of salmonid prey from Pacific harbor seal (.Phoca vitulina richardsi) scat Companion paper with Orr et al., see "Articles" above. 221-229 Weng, Kevin C, and Barbara A. Block Diel vertical migration of the bigeye thresher shark (Alopias superciliosus), a species possessing orbital retia mirabilia 231 Subscription form Abstract— Fisheries models have tradi- tionally focused on patterns of growth, fecundity, and survival offish. However, reproductive rates are the outcome of a variety of interconnected factors such as life-history strategies, mating patterns, population sex ratio, social interactions, and individual fecundity and fertility. Behaviorally appropriate models are necessary to understand stock dynamics and predict the success of management strategies. Protogynous sex-changing fish present a challenge for management because size-selective fisheries can drastically reduce repro- ductive rates. We present a general framework using an individual-based simulation model to determine the effect, of life-history pattern, sperm production, mating system, and man- agement strategy on stock dynamics. We apply this general approach to the specific question of how size-selective fisheries that remove mainly males will impact the stock dynamics of a protogynous population with fixed sex change compared to an otherwise identical dioecious population. In this dioecious population, we kept all aspects of the stock constant except for the pattern of sex determination (i.e. whether the species changes sex or is dioecious). Protogynous stocks with fixed sex change are predicted to be very sensitive to the size-selective fishing pattern. If all male size classes are fished, protogynous populations are predicted to crash even at relatively low fishing mortality. When some male size classes escape fishing, we predict that the mean population size of sex-chang- ing stocks will decrease proportionally less than the mean population size of dioecious species experiencing the same fishing mortality. For protogynous spe- cies, spawning-per-recruit measures that ignore fertilization rates are not good indicators of the impact of fishing on the population. Decreased mating aggregation size is predicted to lead to an increased effect of sperm limitation at constant fishing mortality and effort. Marine protected areas have the poten- tial to mitigate some effects of fishing on sperm limitation in sex-changing populations. Manuscript approved for publication 23 July 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull 102:1-13(2004). The effects of size-selective fisheries on the stock dynamics of and sperm limitation in sex-changing fish Suzanne H. Aionzo Institute of Marine Sciences and the Center lor Stock Assessment Research (CSTAR) University of California Santa Cruz 1156 High Street Santa Cruz, California 95064 E-mail address shalonzoiS'ucscedu Marc Mangel Department of Applied Mathematics and Statistics Jack Baskin School of Engineering and the Center for Stock Assessment Research (CSTAR) University of California Santa Cruz 1156 High Street Santa Cruz, California 95064 Fisheries models are generally used to predict the impact of fishing on stock dynamics and yield (Quinn and Deriso, 1999; Haddon, 2001). Classic models have focused mainly on growth, fecundity, and survival of species, with- out considering the impact of mating patterns on reproduction, survival, and recruitment. It is now recognized that life-history strategies and mating behavior will affect stock dynamics. Even so, general quantitative predic- tions regarding the effect of specific life-history patterns on fished popula- tions are limited and further theory is needed (Levin and Grimes. 2002). It is likely that management strategies taking into account a species' reproduc- tive behavior will greatly improve our ability to manage stocks (e.g. Beets and Friedlander, 1999). We would also like to know when the mating behavior and reproductive strategies of a stock will be worth investigating and when tradi- tional management techniques will be sufficient. For example, in a manage- ment context, how do sex-changing stocks differ from separate-sex species? Here, we take an initial step toward generating a theory of the combined effect of life history and mating pat- terns on stock dynamics by focusing on the potential for and effect of sperm limitation in a protogynous (female to male) sex-changing stock. We focus on protogyny for this article because numerous protogynous species are com- mercially important, namely red porgy {Pagrus pagrus), gag grouper iMyc- teroperca microlepis), and California sheephead iSemicossyphus pulcher). Sex-changing fish present a unique challenge for management because size- selective fisheries have the potential to drastically reduce reproductive rates and population size at levels of fishing that would not pose a problem for dioe- cious (separate-sex) species (Huntsman and Schaaf, 1994; Armsworth, 2001; Fu et al., 2001). On the other hand, pro- togynous stocks may be less sensitive to the removal of large individuals if females are not fished and fertilization rates remain high. Many commercially important species are known to change sex (Bannerot et al., 1987; Shapiro, 1987; Coleman et al., 1996; Brule et al., 1999; Adams et al., 2000; Armsworth, 2001; Fu et al., 2001). Previous models have shown that sex-changing fish may be vulnerable to fishing (Bannerot et al., 1987; Huntsman and Schaaf, 1994; Armsworth, 2001; Fu et al.. 2001). Complications arise because the ef- fect of fishing on a sex-changing spe- cies is mediated by many aspects of their reproductive biology, such as sex ratio, size-dependent fecundity, spawn- ing aggregation size, and reproductive skew. Furthermore, patterns of sex change have cascading effects on the sex ratio, social interactions, population Fishery Bulletin 102(1) fecundity, and male sperm production — all of which can affect stock dynamics. Thus, we cannot treat sex change as an isolated aspect of a species. Instead, we must consider sex change within the context of the mating system and the life history of the species to make general predictions. Behaviorally appropriate models are required to gener- ate constructive qualitative and quantitative theory. Past theory has indicated that sex-changing populations exhibit stock dynamics that often differ from those of dioecious populations (Bannerot et al., 1987; Huntsman and Schaaf, 1994; Armsworth, 2001; Fu et al, 2001 ). Furthermore, pro- togynous stocks are predicted to be sensitive to fishing pat- tern and may exhibit nonlinear dynamics that could lead to population crashes (Armsworth, 2001). However, it is not known which aspects of the mating behavior and life his- tory pattern of sex-changing stocks drive these differences. Here we focus on comparing a protogynous stock with an otherwise identical dioecious population to determine the effect of mating aggregation size, fertilization rates, and life history pattern on stock dynamics. Size-selective (or age-selective) fisheries can impact a species through a decrease in spawning stock biomass, in general and through the removal of highly fecund larger and older individuals, in particular (Sadovy, 2001). How- ever, in protogynous species, fisheries that preferentially remove large males can also change the population sex ratio; however, the exact effect of fishing pressure on stock dynamics in a protogynous species is complex. At one extreme, the complete removal of males from the popula- tion would cause a stock to crash, potentially making sex- changing species more vulnerable than dioecious species in the face of high fishing pressures. At the other extreme, sex-changing species may be less affected by size-selective fisheries if female fecundity limits recruitment and males are not removed in such numbers as to reduce mating or fertilization rates. Currently, there is no theory that predicts the potential for sperm limitation in protogynous stocks as a function of gamete production, fertilization rates, and mating pattern. It has been suggested that marine reserves may be a vi- able management option for species where highly fecund older individuals are critical to reproduction (Levin and Grimes, 2002). However, no theory exists that can predict the impact of marine reserves on stock dynamics in sex- changing species. We consider the impact of a no-take marine reserve on the stock dynamics. We compare the effect of setting aside 0-30% of the spawning population in a reserve. We assume that larval production is exported from within the reserve to the rest of the population and determine whether the reserve can mediate some of the ef- fects of fishing outside the reserve because this represents the optimal scenario for marine reserves. We also compare mean catch rates in the presence and absence of a reserve as a function of fishing mortality. Spawning-per-recruit (SPR) measures are often used to estimate the impact of fishing on a stock (Parkes, 2000; Jennings et al., 2001). Ideally, a spawning-per-recruit mea- sure would keep track of per-recruit production of larvae or eggs (Jennings et al., 2001). However, spawning stock biomass per recruit (SSBR) is commonly used to estimate the reproductive output per recruit at different intensities of fishing. One assumes that the biomass of mature fish is linearly related to reproductive output, which may be the case when egg production limits biomass and fecundity in- creases linearly with biomass. In protogynous stocks, over- fishing of males alone may decrease fertilization rates and hence reproductive output without affecting either female biomass or egg production. Thus, in protogynous stocks or sex-selective fisheries, classic measures of spawning per re- cruit may misrepresent the impact of fishing on the stock's reproduction and hence population stability (Punt et al., 1993). We examine a variety of per-recruit measures and determine their ability to predict changes due to exploita- tion in mean population size. In this study, we describe a general approach using sex- and size-dependent individual-based simulation models that predict reproduction, size distribution, and sex ratio in fished populations as a function of mating system and sex-change pattern. We examine the case where sex change occurs at a specific size threshold. We recognize that plastic and socially mediated sex-change patterns have been ob- served, and our results will apply only to species with fixed sex change. We explore the impact of mating aggregation size, sperm production, and asymptotic fertilization rates on the predicted stock dynamics in the presence of exploita- tion. We make predictions regarding the effects of fishing on population size, reproduction, sex ratio, size distribu- tion, and fertilization rates. We also compare our results to previous work and discuss future directions. Methods We used an individual-based simulation to predict the size distribution, individual and population fecundity, popula- tion sex ratio, fertilization rate, and population size as a function of fishing mortality (Fig. 1). Individuals vary in age, size, sex, and mating site. Population size varies as a function of baseline survival, fishing mortality, reproduc- tion, and larval recruitment. Reproduction depends on the pattern of sex change, mating system, sex ratio, mating site, and fecundity (or fertility) of individual males and females. For each annual time period, we determined individual survival, the size and age of these individuals in the next time period, and the total production of surviving offspring by those individuals. Initial analyses showed that a station- ary size, sex, and age distribution is found within approxi- mately 50 time periods and is independent of the initial population conditions. Thus, we simulated 100 time periods prior to examining the impact of fishing on stock dynamics to ensure that the population had already reached the sta- tionary size and sex distribution for that scenario and set of parameters. We then examined the model for 100 repro- ductive seasons in the presence of fishing with a constant mean fishing mortality. Because a number of elements of the model were stochastic, we examined 20 simulations for each scenario and set of parameter values. Initial analyses indicated that 20 simulations were more than sufficient to lead to low variability in the key measures of interest. We assumed that reproduction occurs at the level of the mating Alonzo and Mangel: The effects of size-selective fisheries on the stock dynamics of and sperm limitation in sex-changing fish 3 group at different reproductive sites. Individual sur- vival, maturation, sex change, and mating site were determined stochastically as described below. Fishing and adult survival We assumed that adult survival is density indepen- dent but depends on fishing selectivity, fishing mor- tality, and baseline adult mortality in the absence of fishing. For simplicity, we assumed that age and size do not affect nonfishing adult mortality p.A. We assumed that the fishery is size selective; we let L represent fish size, F represent annual fishing mortality, Lf represent the size at which there is 50% chance an individual of that size will be taken, and r represent the steepness of the selectivity pattern. Then fishing selectivity per size class siL) is given by siD- l + exp-HL-L,)) and adult annual survival becomes cr(L) = exp(-/iA-Fs(L)) (1) (2) We assumed that fishing does not differentially affect the sexes independent of size. We recognize, however, that for some species this may not be the case. We also assumed that fishing occurs each year prior to reproduction and can represent either pulse or continuous fishing with an annual mortality F. We let N it) represent the number of individuals in age class a at time t so that population size N(t)= Sa Na(t). Population dynamics We assumed that the number of larvae that enter the popu- lation is determined by the production of fertilized eggs Pit) and the probability that those larvae will survive to recruit. Pit) is determined by the adult fecundity and fertilization rates described below. For computational tractability, we also assumed that a population ceiling Nmax exists (Mangel and Tier, 1993, 1994 ). However, we chose NmBX large enough that the stable population size was below the ceiling. Larval survival has both density-independent and density-depen- dent components (e.g. Cowen et al., 2000; Sale, 2002). We used a Beverton-Holt recruitment function to determine larval survival to the next age class (Quinn and Deriso, 1999; Jennings et al., 2001). Larvae represented the zero- age class N0(t) and thus the number of larvae surviving to recruit in any year t is given by Nnit) = (oPit))/(l+pPit)) if (ctP(t))/(l+pP(t)) +JjNjt)exp(-A). (4) Mating system We assumed that reproduction occurs at the level of the mating group, and we examined the effect of varying mating group size and the number of mating sites. We assumed Fishery Bulletin 102(1) that juveniles and adults exhibit site fidelity but that larvae settle randomly among mating sites. We also assumed that the population carrying capacity is split equally among the mating sites and that the total capacity of all mating sites exceeds the maximum population size in the absence of fish- ing as determined by adult mortality and the recruitment function. Therefore, mating sites do not limit recruitment but may affect reproductive rates. We examined three cases: 1 ) the entire population mates at one site (one mating site with up to 1000 individuals); 2) a few large mating groups exist ( 10 sites with a maximum of 100 individuals per site); and 3) many small mating aggregations exist (20 mating sites with a maximum of 50 individuals per site). For sim- plicity, we assumed that within a mating site, individuals mate in proportion to their fertility and fecundity. Therefore, large males and females have higher expected reproductive success. However, we assumed that all males that are large enough to change sex have a chance of reproducing propor- tional to their fertility. This is equivalent to assuming that females exhibit a mate choice threshold I Janetos, 1980) that has evolved with the size-at-sex change and that females have an equal probability of mating with males above this size threshold. However, a large male mating advantage clearly still exists. We also assumed that fishing mortality remains constant as mating aggregation size varies. Thus, we assumed that fishing effort per site does not increase as the number of mating sites decreases. An alternative would be to assume that total fishing mortality increases as the number of mating aggregations decreases. Maturity The probability that an individual matures pm(L) is deter- mined by size. Once an individual matures, she remains female until sex change (see below). We let Lm represent the length at which 50% of the individuals will have matured. EiL)=aLh, (7) P,JL)- 1 where a and b are constants. Once an individual has changed sex (as determined by the sex change rule described above) sperm production (in millions) S(L) is given by S{L)=cLd , (8) l + exp(-q(L- Lm (5) where c and d are constants. Size-dependent fecundity has been measured in many fish species (e.g. Gunderson, 1997). A general allometric relationship between sperm production and size has not been established. Therefore, we assumed that male gamete production increases with size at the same rate as that for females ib=d). We also assumed that males produce many more sperm at any body length than females produce eggs. Clearly, other possible patterns exist. We examined the case where males produce from 102 to 106 sperm for every egg produced by a female. In the pelagic spawning wrasse (Thalassoma bifasciatum ), large males release ap- proximately 1000 times more sperm than females release eggs (Schultz and Warner, 1991; Warner et al., 1995). We used recently published data on sperm production and fertilization rates in the bluehead wrasse (Thalas- soma bifasciatum) to generate a biologically appropriate fertilization function for our model (Warner et al., 1995; Petersen et al., 2001). It is critical to consider a biologically appropriate form for the function to express fertilization rates when considering the potential for sperm limitation. The probability an egg will be fertilized is an increasing function of the number of sperm available for that mat- ing (Fig. 2). The number of eggs released per mating also affects the fertilization rate (Fig. 2). For simplicity, we cal- culated the average expected fertilization rate per mating site based on the total production of sperm and eggs at the site. We let S represent the number of sperm released (in millions) and £ the number of eggs released at each mating site. We assumed that the proportion of eggs fertilized per mating site pF is given by where q determines the steepness of the probability function. Sex change The probability of sex change, pciL), is a logistic function of absolute size L P,.(L) = l + exp(-p(L-L, )) (6) where Lr represents the size at which 50% of the indi- viduals will change sex from female to male and p is a constant. Reproduction We assumed that female fecundity E(L) depends on indi- vidual size according to the allometric relationship Pf l + iisE + X)S (9) where k and % are constants fitted to the data. The number of eggs fertilized per group is ph-E and the total production of fertilized eggs. Pit), is the sum of the number of eggs fertilized in all mating groups. Measures of spawning stock biomass per recruit To measure the impact of fishing on stock dynamics, we computed the total spawning stock biomass per recruit starting from the beginning of fishing for the next 50 years. We used the generally recognized pattern that fish wet weight tends to be approximately proportional to the cube offish length (Gunderson, 1997) to convert fish length, L, into relative biomass, B(L)~L\ Then we calculated total female and male spawning stock biomass Alonzo and Mangel: The effects of size-selective fisheries on the stock dynamics of and sperm limitation in sex-changing fish per recruit (SSBR). We also kept track of the total fecundity (egg production per recruit I, fertility (sperm production per recruit), and eggs fertilized per recruit. Marine reserves ^=150 (about 60 females) OS- 'S 0.6 ■s 0.4 02 We examined the effect of no-take marine reserves on the predicted stock dynamics by comparing the stock dynamics in the presence and absence of reserves. Without a reserve, individuals at all mating sites are subject to fishing. In the presence of a no-take marine reserve, we "protect" a percentage of the mating sites (and thus the population) from fishing. We examined cases in which 09c, 10%, 20%, and 30% of mating sites were protected from fishing. We assumed that the population is completely open among mating sites. Thus, eggs produced from all mating sites enter one larval pool and recruitment occurs randomly between mating sites. Clearly other possibili- ties exist and could be considered in future analyses, but this case represents a reasonable baseline situation to con- sider because many marine fish have pelagic larval phases. We also recognize that these analyses ignore the effect of interactions between species within the reserve on stock dynamics. We examined two situations. In the first case, reduced fishing effort occurs when mean fishing mortality is decreased in the presence of reserves because fishing mortality (F) at the unprotected sites remains the same as before the reserve. In the second case, the redistribution of fishing effort occurs when mean fishing mortality across all sites remains the same because fishing mortality increases at the unprotected sites. Comparison of sex-changing stocks and dioecious stocks Ideally, we would like to distinguish the effects of sex change in isolation from the confounding effects of mating pattern, sex ratio, survival, growth, and population fecundity on stock dynamics. To differentiate whether sex change in iso- lation or other aspects of the mating system determine the predicted stock dynamics, we also examined a version of the model described above for a population where sex is fixed at birth. In this dioecious population, we keep all aspects of the stock constant except for the pattern of sex determi- nation (whether the species changes sex or is dioecious). One would generally expect a dioecious population with no differences between the sexes in mortality to exhibit a 50:50 sex ratio ( Fisher, 1930; Trivers, 1972; Charnov, 1982 ). However, we wanted to control for all differences between the dioecious and protogynous stocks other than the sex- determination pattern. Therefore, we considered the same sex ratio at maturity (0.67=the proportion of adults that are female) as found in the sex-changing population in the absence of fishing. Assuming no sex-specific differences in survival to maturity, this is the same as assuming a 0.67 sex ratio at birth. In this model, individuals remain one sex (determined randomly at birth) throughout their lifetime. Km=1750 (about 700 females) K>750 (about 300 females) 5.000 10.000 1 5.000 20,000 Sperm number (S) (in millions, about 1 to 100 males) Figure 2 Fertilization rate as a function of the number of eggs and sperm per mating site. The saturation parameter Km=\E+x is taken from Equation 9. Fishing is size but not sex selective. We assumed that males mature at the same size as females. Parameter values We used previous research on California sheephead (Lab- ridae, Semicossyphus pulcher), a commercially important sex-changing fish, to provide evolutionarily and ecologi- cally reasonable parameters for the model. Although the growth, survival, and reproduction of this species have been studied, less is known about the factors that induce sex change and mating behavior. In this species, sex change occurs at approximately 30 cm although the exact pattern varies among populations (Warner, 1975; Cowen, 1990). It is not known whether sex change is fixed or socially medi- ated. Because nothing is known about fertilization rates in the California sheephead, we generated k and y Lc), the general patterns remain the same, but for the same fishing mortality (.F), the effect of fishing on the population is less (Fig. 4). Female biomass does not decrease much with fish- ing mortality when Lf=Lc even though some females are removed by the fishery because the probability of a female changing sex is the probability of it being fished. Therefore, female loss due to the fishery affects male biomass rather than female biomass in the population. Sperm limitation and production The removal of large males from the population is pre- dicted to cause sperm limitation and decreased fertiliza- tion rates (Fig. 3, A and C), leading to a decrease in mean population size (Fig. 4A). The degree to which the fertiliza- tion rate and thus the population size decreases depends to a great extent on the pattern of sperm production and fertilization. We assumed that only a few males are needed to fertilize the eggs of many females (Fig. 2). We also assumed that per-capita reproduction and recruitment are high even at a low population size (Barrowman and Myers, 2000). Thus, protogynous populations with lower sperm production or fertilization rates would experience greater effects from fishing than predicted in the present study. Similarly, populations with lower production or sur- vival would experience larger decreases in population size even with the same level of sperm limitation and fishing. In general, however, the removal of males alone from a pro- togynous population with a fixed sex change is predicted to cause decreased fertilization rates and lower mean popula- tion size even when the fertilization rate function is asymp- totic and individual male sperm production is high. Mating aggregation size As mating aggregation size decreased and fishing mortality and effort remained constant, the effect of fishing on the pop- Eggs produced 1 1.5 2 Fishing mortality (F) Figure 3 Spawning-per-recruit measures. Results are presented for the sex-changing stock with one mating site when L^= Lc and r=l. Means across 20 simulations are given. For details see the general text. ulation increased. As described above, we assumed that fish- ing effort would not be concentrated on the few large mating aggregations and thus increase total fishing mortality. The sex ratio, mean size, mean fecundity, and mean fertility all remained the same across different mating aggregation sizes with constant fishing mortality. However, the mean fertilization rate and number of fertilized eggs per recruit decreased with mating group size ( Fig. 5 ) even though male biomass and SSBR remained the same. Both predicted mean population size and biomass taken decreased as fish- ing mortality increased (Fig. 5). This pattern was generated by sperm limitation in small mating groups. Smaller groups have higher probabilities that sperm production within the group will not be sufficient to fertilize the eggs produced within the mating group. Small mating aggregations may not only be sperm limited but also be male limited and fail to reproduce completely; populations with small group sizes (50 individuals or less) were predicted to become extinct in Fishery Bulletin 102(1) 5-25% of the simulations as fishing mortality (F) increased from 0 to 1. The impact of mating group size on stock dynam- ics is thus predicted to be nonlinear. A threshold mating aggregation size appeared to exist below which sperm limi- tation and reproductive failure become common. Spawning-per-recruit measures For size-selective fishing, the spawning stock biomass per recruit of females is not predicted to decrease significantly with increased fishing mortality as long as some male size classes escape fishing (Lr>Lv). However, male biomass per recruit and sperm production per recruit are both predicted to decrease. Although egg production is not predicted to 900 800 A L,>LC CD n 700 to ^\ L,=LC <= 600 J o 'ra 500 " 3 g- 400 " Q. c 300 " ra | 200 ' \l,, ra £? 300.000 D T3 a a> c > < ° 200.000 CD 100,000 U/< 0 0 0.5 1 15 2 2.5 3 Fishing mortality (F) Figure 4 The effect of size-s ilect ive fishing on stock dynamics. We present results for the sex-changing stock with one mating site when r=l. Means across 20 simulations are given. For details see the general text. decrease with increasing size-selective fishing pressure, the number of fertilized eggs is predicted to decrease. When all male size classes are fished iL.>Lc), the stock is predicted to crash and therefore clearly female biomass and egg production are predicted to decrease with fishing mortality. In general, the predicted decrease in mean popu- lation size and reproduction is driven for the most part by decreased sperm production and consequently a reduction in the number of eggs fertilized per recruit. The relation- ships between fishing pressure and the classic spawning- per-recruit measures do not indicate the true effect that fishing is predicted to have on the protogynous population (Fig. 6). When Lf>Lc, female spawning stock biomass per recruit and eggs produced per recruit showed almost no effect of fishing on the population, even as mean population size decreased. Because of the size-selec- tive fishing pattern, total and male biomass per recruit decreased with fishing mortality and decreasing mean population size. However, male and total biomass per recruit did not reflect the increased effect of fishing on populations with smaller mating aggregations. The production of fertilized eggs per recruit decreased with increased fishing pressure and decreased more sharply for smaller mating aggregations. Only the number of fertilized eggs per recruit could assess the predicted effect of fishing on the protogynous population. Thus, classic SPR measures were predicted to fail in the presence of sperm limitation to assess the impact of fishing on a protogynous stock. Marine reserves and fishery management In the situation considered in this study, the pattern of fishing is more important to stock dynamics than the presence of marine reserves. We assumed a size- selectivity that allowed on average 50% of individuals of sex-changing size to escape the fishing gear. Thus, although the sex ratio does increase (become more female) by 20-40%, all males are not lost from the population (when Lfs.Lt. and r=l ). If fishing selectivity occurs at a smaller size, then the effects on the popula- tion are predicted to be much greater and the protogy- nous stock would suddenly become more affected than the dioecious population. For example, at L^=25 cm the protogynous stock is predicted to crash whenever F^l. This occurs not because of a reduction in the produc- tion of eggs but rather because of a failure to fertilize the eggs produced by surviving females. When males of all size classes are fished, populations can become male limited and fertilization rates drop drastically. A decrease in the production of fertilized eggs can lead to a decrease in female biomass, but it is the removal of males rather than females that causes this decline. When fishing effort is not redistributed after the formation of a reserve, the impact of fishing on the mean population size and SPR measures is predicted to decrease (e.g. Fig. 7A). However, if fishing effort is redistributed among unprotected areas, the benefit of the reserves to the protogynous stock decreases (Fig. 8A). Protecting some sites allows large males to Alonzo and Mangel: The effects of size-selective fisheries on the stock dynamics of and sperm limitation in sex-changing fish 9 escape fishing and thus increases the pro- duction of fertilized eggs at the population level. However, yield decreased proportion- ally to the percentage of sites protected by the reserve unless fishing effort is redis- tributed among the remaining sites. We as- sumed that fish do not move between sites after the larval stage, and thus larger and older individuals do not leave the reserve and become exposed to fishing. Although this assumption is clearly appropriate for some species, it is important to realize that the dy- namics and predictions would differ for more closed populations or migratory species. For the fishing pattern and biological scenario examined in this study, marine reserves are not predicted to increase biomass available to the fishery (Figs. 7B and 8B). Dynamics of dioecious versus protogynous stocks In the dioecious stock with a single ran- domly mating aggregation, both male and female biomass per recruit and fecundity or fertility per recruit are predicted to decrease as fishing mortality increases ( Fig. 6). Because both egg production and sperm production decrease with increased fishing pressure in the dioecious stock, the number of eggs fertilized per recruit did not differ much from the other SPR measures. Thus, SSBR and eggs per recruit also indicated the impact of fishing on the stock in dioecious stocks with large mating aggregations. The percent drop in population size and fertil- ized egg production is predicted to be much greater in dioecious species and occurred more quickly than in the sex-changing stock because of a reduction in overall population fecundity even in the absence of decreased fertilization rates. However, dioe- cious stocks are predicted to exhibit larger mean population size for the same fishing mortality and to support a larger fishery because of the additional egg production of large fecund females. At very small mating aggregations, sperm limitation is predicted even in the dioecious stock and fertilized eggs per recruit become a better indicator of stock dynamics in the presence of fishing. Dioecious stocks are also predicted to benefit from marine no-take reserves through the protection of large fecund females ( Fig. 7 ). Discussion In this study we developed a general frame- work that examines the consequences to 0.95 0.9 0.85 Egg production (per recruit) Fertilized eggs (per recruit) Mean population size Figure 5 Mating aggregation size affects the response to fishing. Large (one large mating aggregation ) and small ( 10 smaller mating aggregations I situations are compared. Percent change in the presence of fishing (from F=0 to F=l> in egg production per recruit, mean fertilized egg production per recruit, and mean population size are given. Total population fecundity and mean body size are lower for the smaller mating aggregations. PROTOGYNOUS POPULATION 1 1 F=3 Eggs produced F=0 r& S 0.9- 0.8- Eggs fertilized _n .a St' »■" 0.7- a® x o Eggs produced and fertilized DIOECIOUS POPULATION 0.6- 0.5. ft ti * 0.4 F=3 F=0 600 650 700 750 800 850 900 950 Mean population size Figure 6 Spawning-per-recruit (SPR) measures in a protogynous (squares) and dioe- cious (triangles) stock: Mean egg production per recruit (filled) and mean fertilized eggs per recruit (open) are shown for a randomly mating popula- tion with one large mating group. Error bars indicate the standard error of the mean. For the dioecious population, the two SPR measures overlap. 10 Fishery Bulletin 102(1) fisheries management of a behaviorally and evolution- ary reasonable life-history and sex-change pattern. We based our assumptions and parameter values on patterns observed in natural populations that have presumably evolved given the life history tradeoffs and expected repro- ductive success associated with these behaviors. However, we made various assumptions that affect the predicted patterns such as a fixed sex-change pattern, male mating success proportional to sperm production, and a very resil- ient recruitment function. Despite these assumptions, a number of general patterns emerge. Life-history pattern is important but not sufficient to predict stock dynamics In general, we predicted that a protogynous stock with fixed sex change will respond to the same fishing pressure o o ^ fl Q.— ' °> S3, -= cr> X 0) 0 (yi + 72s, <0) and t, = 0 s, - 7i > 0) (s, -/, <0) (7) (8) where ti = dRIRS on the /th day from the first sampling; Yv Yz = coefficients of the equations; and Sj = dRIRL on the /th day from the first sampling. Model estimation Likelihood function The location and scale parameters at the first sampling (o0 and fe0), the coefficients of Equa- tion 6 (smax, a, and pk), and the coefficients of Equations 7 and 8 (y-j and y2) are estimated as values that maximize total log-likelihood. The total log-likelihood is evaluated by the adequate probability density function selected in the first step. The log-likelihood functions take the follow- ing forms: Normal distribution log, L„ormal (aQ,b„, smax , a j , pk , yv y2) = X2>g* -Arexp[-(Z<7i-a,)/242] 2nb (9) Largest extreme value distribution s, =smax/ 1 + exp 2>a+£ab* (61 where si = dRIRL on the /th day from the first sampling; smax = potential maximum dRIRL of the animal; a., Pk = coefficients of each independent variable; A = categorical variable ( a dummy variable indi- cating animal ages ) that takes the value 1 orO; Ekl = the kt\\ environmental factor on the /th day from the first sampling; nA = number of age categories; and nE = number of environmental factors. The categorical variable takes the value of 1 when the animal is the category, otherwise it takes 0. The multivari- ate logistic function with smax = 1 is used for logistic regres- sions (Sokal and Rohlf, 1995). A method of giving a value to the categorical variable is described by Zar ( 1999). Modeling the change in scale The daily relative increase rate of scale parameter (dRIRS) and dRIRL must be cor- loge-L,argcs/o0,fe0,smax,a,,^„71,)'2) N nq =XZ1°g«{(1/Vexp[-^-«,>/4] xexp{-exp[-(Z9i-<59)/feJU, (10) where a0, 60 = values of the location and scale param- eters, respectively, at the first sampling; smax> aj> Pk = coefficients of Equation 6; Yvy2 - coefficients of Equations 7 and 8; N = number of samplings; nq = number of data at the qth sampling; aq = location parameter at the qth sampling estimated by Equation 5 (r,=s, ); bq = scale parameter at the qth sampling esti- mated by Equation 5 (r~^); and / = length of the /th individual at the fiO ; M 4^%, -^— Turbidity -•— Salinity -■ 40 - 1/ \ 20- l"'"l I ■ 1 1 1 ~wj Mode (estimated by model 4.1) 90% confidence interval (estimated by model 4.1) ° Mode (sample) Date Figure 3 Environmental fluctuations and prediction of the growth oiCorbiculajapon- ica juveniles spawned in 1997 in Lake Abashiri by the best model (Model 4.1 in Tablel). (Al Insignificant environmental factors (factors excluded in the model selection), turbidity (equivalent to kaolin density, ppmi and salinity (psu, practical salinity unitl. (Bl Significant environmental factors (factors included in the model selection I, temperature (°C) and water fluorescence (equivalent to uranin density, /'g/L>. (Cl Daily relative increase rate of loca- tion parameter (dRIRLl and daily relative increase rate of scale parameter (dRIRS) estimated by the model. (Di Growth of Corbicula japonica; verti- cal bars represent 90% confidence intervals for the shell lengths of the samples. length distribution becomes asymmetric during growth, skcwness of the distribution would increase according to growth. However, there is no correlation between the skewness and the means of the shell lengths. Therefore, we thought that the shell length distribution of the cohort was already asymmetric just after settlement. Such a distribu- tion might be influenced by fluctuations in larval settle- ment during the spawning season; and larval settlement would be influenced by fluctuations in larval supply from the water column. During the spawning season of 1997. the average planktonic larval density gradually increased from 26 ind/m3 on 25 July to a maximum of 603 ind/m3 on Baba et al.: An environmentally based growth model for |uvenile Corbicula japonica 21 Table 2 95% confidence limits of location and scale parameters at the first sampling and coefficients of the best model constructed based on the largest extreme value distribution (models 4.1 in Table 1) estimated by profile likelihood method. dRIRL = daily relative increase rate of location parameter. dRIRS = daily relative increase rate of scale parameter. Temp. = water temperature, WF = water fluorescence, Sal. = salinity, Turb. = turbidity. Parameters at 1st sampling Max. dRIRL Age categorization Environmental factors Expressing relationship between dRIRS and dRIRL A, a, a. Temp. ft WF ft Sal. ft Turb. ft Lower 95 % Upper 95 % 0.294 0.304 0.039 0.045 0.010 -26.6' 0.013 -11.5' -14.6 -6.4 0.41 1.00 0.27 0.64 0.0027 0.0039 0.734 0.793 1 One common coefficient for the two categorical variables. 13 August. Then it sharply decreased to 3 ind/m3 on 19 August (Baba et al., 1999). Such a pattern of larval-density fluctuation might have caused the asymmetric distribution of shell lengths of the settled juveniles. Another possible factor that influenced the shapes of the shell length distri- butions and the relationship between dRIRL and dRIRS is size-dependent mortality, e.g. predations and fisheries. Size-dependent mortality has been reported in several marine bivalves (e.g. Nakaoka, 1996). Potential predators of C.japonica are fishes, such as Japanese dace (Tribolo- don hakonensis) (also known as big-scaled Pacific redfin, FAO), Pacific redfin (Tribolodon brandtii), common carp (Cyprinus carpio), and the So-iny mullet (Liza haemato- cheila ) (Kawasaki4). In our study, the size-dependent mor- tality was negligible because the range of the shell lengths observed in this study was very narrow. The shape of the distribution to describe a single cohort should be determined from the data. In contrast, single cohorts are usually separated from multicohort data by as- suming a normal distribution of lengths in a single cohort (e.g. Fournier and Sibert, 1990). Therefore, it is possible that multicohort analysis done without selection of an adequate distribution to describe a single cohort causes substantial bias in estimations of various stock features of animal populations, such as age composition, growth, mortality, and recruitment. In our preliminary analyses, we also tested smallest extreme value distribution, inverse Gaussian distribution, and lognormal distribution. The in- verse Gaussian distribution was the best for two samples; the lognormal distribution, was the best for two samples; the largest extreme value distribution was the best for ten samples. Therefore, it is reasonable to select the largest ex- treme value distribution. We selected a single distribution for our analyses, otherwise a discontinuous point would have appeared in the growth curve. Relatively large confidence intervals were obtained in the coefficients of the linear component of Equation 6, i.e. a , and /3;, (Table 2). The relatively large confidence inter- vals may indicate that the number of estimated coefficients is somewhat larger than the number of samplings. There- fore, to estimate these coefficients more precisely, we may need to investigate more cohorts spawned in other years in future investigations. Growth of C. japonica We identified extremely slow growth in C. japonica juve- niles, which grew to a modal shell length of 0.7 mm during the first year in Lake Abashiri, which lies at 43.7°N. Spats of C. japonica collected from 1992 to 1997 in Lake Shinji, which lies at 35.5°N, grew to a mean shell length of 6.7 mm in natural conditions by the first winter (Yamane et al.2). Using environmental factors measured in Lake Shinji from 1990 to 1998 at monthly intervals (Seike5), we simu- lated the growth of C. japonica with model 4.1. Corbicula japonica grew to a mean shell length of 1.4 mm (standard error, 0.37 ) by the first winter in the simulations. Therefore, the large difference in juvenile growth between the two habitats cannot be explained by environmental differences because the results of the simulation were apparently an underestimate. We think that the extremely slow growth of the juveniles (prolonged phase of meiobenthic develop- ment ) in Lake Abashiri is probably a geographical varia- tion, which is genetically determined, within C. japonica. However, there remains a possibility that the juvenile growth differences depend on other environmental factors not measured in this study. Therefore, the geographical 4 Kawasaki, K. 1997. Lagoon structure and fish produc- tion in Ogawara-ko Lagoon. /;; Final reports on fisheries in Ogawara-ko Lagoon (Tohoku Construction Corporation ed.), p. 4-33. Unpubl. rep. Construction Office for Takasegawa General Development of Tohoku Regional Construction Bureau, 3 Ishido, Hachinohe, Aomori 039-1165, Japan. 6 Seike, Y. 1990-98. Gobiusu: monthly report of water quality in Lake Shinji and Lake Nakaumi. Unpubl. rep. Faculty of Science and Engineering. Shimane University, 1060 Nishi- kawatsu, Matsue, Shimane 690-0S23, Japan. 22 Fishery Bulletin 102(1) 10 Sep 1997; mode: 0.30mm, scale: 0.04, n=341 13 May 1998; mode: 0.41mm, scale: 0.06, n=38 11 Jun 1998; mode: 0.51 mm, scale: 0.12, n=292 10 Jul 1998; mode: 0.57mm. scale: 0.10, n=610 13 Aug 1998; mode: 0.64mm, scale: 0.12. n=456 1 1 Sep 1998; mode: 0.70mm. scale: 0.17, n=202 14 Oct 1998; mode: 0.76mm. scale: 0.17. n=162 0.0 22 Apr 1999; mode: 0.74mm. scale: 0.15, n=265 + + 13 May 1999; mode: 0.81mm, scale: 0.20. n=241 0.2 t t H 0.1 00 28 Jul 1999; mode: 2.14mm, scale: 1.06, n=63 ^#T>fffi^^ 3456 0123456 Shell length (mm) Figure 4 Shell-length compositions of a single cohort of Corbicula japonica spawned in 1997. The raw data (shell lengths) are shown by +. The largest extreme value distribution estimated by the best model ( model 4. 1 in Table 1 1 is shown by a solid line. The largest extreme value distribution independently fitted by the maximum likelihood method is shown by a dashed line. The sampling date and values of location parameter I mode) and scale parameter independently fitted by the maximum likelihood method are shown in each panel. variation should be validated by reciprocal transplanta- tions or laboratory experiments (or both) in future inves- tigations. Prolonged phases of meiobenthic development have been reported in some marine bivalves (Nakaoka, 1992; Harvey and Gage, 1995). However, a prolonged phase of meiobenthic development as a geographical variation is rarely reported. In many species of bivalve, populations from higher lati- tudes have a slower initial growth rate; but longevity and ul- timate size in these populations are frequently greater than at lower latitudes (Newell, 1964; Seed, 1980). The extremely slow growth of C. japonica juveniles in Lake Abashiri may be an extreme example of this phenomenon. In Lake Abashiri, C. japonica failed to spawn in ten out of 21 years for which Baba et al An environmentally based growth model for juvenile Corbicu/a japonica 23 data were available because of low water temperatures dur- ing the summer spawning season (Baba et al., 1999). This means that a long life span is essential to sustain popula- tions of C. japonica in northern habitats. We think that a long life span is the ultimate factor for the extremely slow growth rate of C. japonica juveniles in Lake Abashiri. The growth response of C. japonica juveniles is much less susceptible to environmental factors before the second win- ter than after and is the proximate factor for an extremely slow growth rate. Nuculoma tenuis, a detritus feeder, de- velops its palp proboscides, its feeding apparatus, during the prolonged phase of meiobenthic development (Harvey and Gage, 1995). The change of growth susceptibility to en- vironmental factors in young ages may suggest that some functional morphological changes occur in C. japonica, also a filter feeder. In our preliminary analyses, we could not find a better model when we used different values of smax in Equation 6 between ages instead of categorical variables indicating ages. Therefore, we conclude that the difference in growth rates between ages is not due to a difference in potential maximum growth rate, at least in the range of the shell length observed in our study. When our model is ap- plied to a wider range of the shell lengths or other species, it is best to examine the age dependence of smax. Acknowledgments We express our thanks to T Kato, Vice-Head of the River Improvement Section in the Abashiri Local Office of the Hokkaido Development Bureau, for providing environmen- tal data on Lake Abashiri. We also thank the reviewers of Fishery Bulletin for providing helpful suggestions on our manuscript. 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Prentice Hall, Upper Saddle River, NJ. 25 Abstract— Information is summarized on juvenile salmonid distribution, size, condition, growth, stock origin, and species and environmental associations from June and August 2000 GLOBEC cruises with particular emphasis on differences related to the regions north and south of Cape Blanco off Southern Oregon. Juvenile salmon were more abundant during the August cruise as compared to the June cruise and were mainly distributed northward from Cape Blanco. There were distinct differ- ences in distribution patterns between salmon species: chinook salmon were found close inshore in cooler water all along the coast and coho salmon were rarely found south of Cape Blanco. Dis- tance offshore and temperature were the dominant explanatory variables related to coho and chinook salmon distribution. The nekton assemblages differed significantly between cruises. The June cruise was dominated by juve- nile rockfishes, rex sole, and sablefish, which were almost completely absent in August. The forage fish community during June comprised Pacific herring and whitebait smelt north of Cape Blanco and surf smelt south of Cape Blanco. The fish community in August was dominated by Pacific sardines and highly migratory pelagic species. Esti- mated growth rates of juvenile coho salmon were higher in the GLOBEC study area than in areas farther north. An unusually high percentage of coho salmon in the study area were preco- cious males. Significant differences in growth and condition of juvenile coho salmon indicated different oceano- graphic environments north and south of Cape Blanco. The condition index was higher in juvenile coho salmon to the north but no significant differences were found for yearling chinook salmon. Genetic mixed stock analysis indicated that during June, most of the chinook salmon in our sample originated from rivers along the central coast of Oregon. In August, chinook salmon sampled south of Cape Blanco were largely from southern Oregon and northern Cali- fornia; whereas most chinook salmon north of Cape Blanco were from the Central Valley in California. Manuscript approved for publication 30 June 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull 102:25-46 (2004). Juvenile salmonid distribution, growth, condition, origin, and environmental and species associations in the Northern California Current* Rick D. Brodeur Northwest Fisheries Science Center National Marine Fisheries Service, NOAA 2030 S. Marine Science Drive Newport, Oregon 97365 E-mail address: Rick-Brodeuriffinoaa-gov Joseph P. Fisher College of Ocean and Atmospheric Sciences Oregon State University Corvallis, Oregon 97331 David J. Teel Northwest Fisheries Science Center National Marine Fisheries Service, NOAA Seattle, Washington 98112 Robert L. Emmett Northwest Fisheries Science Center National Marine Fisheries Service, NOAA 2030 S Marine Science Drive Newport, Oregon 97365 Edmundo Casillas Northwest Fisheries Science Center National Marine Fisheries Service, NOAA Seattle, Washington 98112 Todd W. Miller Cooperative Institute for Marine Resources Studies Oregon State University Newport, Oregon 97365 The need to understand the direct and indirect linkages between oceano- graphic conditions and salmon sur- vival in the marine environment has increased with the listing of many West Coast salmon stocks as threat- ened or endangered. Recent studies have shown that long-term changes in climate affect oceanic structure and produce abrupt differences in salmon marine survival and returns (Francis and Hare, 1994: Mantua et al., 19971. A major regime shift in the subarctic and California Current ecosystems during the late 1970s may have been a factor in reducing ocean survival of salmon in the Pacific Northwest and in increas- ing marine survival in Alaska ( Hare et al., 1999). Fluctuations in mortality of salmon in the freshwater and marine environments have been shown to be almost equally significant sources of annual salmonid recruitment variability ( Bradford, 1995 ). Unlike in the freshwa- ter environment, the physical and bio- logical mechanisms and factors in the marine environment that cause mor- tality of salmon are largely unknown. Predation, inter- and intraspecific competition, food availability, smolt quality and health, and physical ocean conditions likely influence survival of salmon in the marine environment. Thus, increasing our understanding of nearshore ocean environments, their linkages to oceanographic conditions, and the role they play in salmonid survival, could provide management options for increasing adult returns. Characterization of the space-time vari- ability of the environmental conditions that smolts encounter when they enter the nearshore ocean, and the eventual survival of these smolts will allow us to identify which biotic and abiotic ocean conditions are correlated with various ocean survival levels. Many anadromous salmonid popula- tions along the west coast of the United States have declined over the last few decades (Nehlsen et al., 1991), and most stocks show a regional north-south pat- tern in degree of extinction risk (Kope and Wainwright, 1998). This pattern suggests that both marine habitat con- ditions and mesoscale climate patterns affect salmonid population status (e.g. Lawson, 1993). A dramatic example is the population trend of coho salmon (Oncorhynchus kisutch) along the Or- egon coast. Populations along the coast north of Cape Blanco (43°N) have exhib- ; Contribution number 364 of the U.S. GLOBEC program. NEP Office, Oregon State University, Corvallis. OR. 26 Fishery Bulletin 102(1) ited a strong decline in size and survival in the mid-1990s; whereas populations south of Cape Blanco have not shown this trend (Lewis1). This finding suggests that these two populations have experienced different ocean conditions. The quality of the marine habitat (in terms of habitat complexity, prey density, and temperature) undoubt- edly influences fish growth and condition. Growth and indices of condition can be used as measures of habitat quality for juvenile salmon and to identify essential links between oceanographic conditions and survival of salmon populations during the critical juvenile life history phase. Measures such as growth (growth rate, size variation, and allometric relationships) (Lorenzen, 1996; McGurk, 1996) and accumulation of energetic reserves used in growth and sustenance during the low-productivity winter periods have been used previously to characterize habitat quality and to describe how it ultimately affects the individual and the population (Perry etal., 1996; Paul and Willette, 1997). Environmental factors are known to affect growth, repro- duction, survival, and ultimately population recruitment (Hinch et al., 1995; Marschall and Crowder, 1995; Fried- land and Haas, 1996). As such, fish condition, growth rate, and size in the pre-adult stages are parameters that can be used to identify the influence of natural and anthropogenic ocean conditions on marine survival. Much of our current knowledge of the dominant nekton of the pelagic ecosystem off the coasts of Oregon and Wash- ington is derived from a series of 17 cruises conducted by Oregon State University (OSU) from 1979 to 1985. These collections, consisting of >900 quantitative purse seine sets in the northern California Current, were made to examine geographic distributions and temporal trends of the domi- nant nekton and how these relate to physical and biotic conditions at the time of capture. The primary purpose of these cruises was to collect data for assessment of the abundance, distribution, growth, migration, and ecology of juvenile salmon in coastal waters. Data on the distribution, migration and growth of juvenile salmon from these cruises have been summarized in Fisher and Pearcy (1988; 1995). Pearcy and Fisher ( 1988, 1990), and Pearcy ( 1992). Analy- sis of the nonsalmonid data includes studies on their abun- dance and distribution (Brodeur and Pearcy, 1986; Emmett and Brodeur, 2000), feeding habits (Brodeur et al., 1987) and interannual variability in relation to oceanographic conditions (Brodeur and Pearcy, 1992). In addition, the distribution of juvenile salmon (mainly coho and chinook salmon [O. tshawytscha}) has been studied more recently as a component of a multiyear Columbia River Plume study (Emmett and Brodeur, 2000; Teel et al., 2003; Brodeur et al., 2003). However, all these cruises extended only as far south as Cape Blanco, with the exception of one cruise (July 1984), which extended as far south as Eureka, California, but included only a few collections south of Cape Blanco (Pearcy and Fisher, 1990). Thus, the region south of Cape Blanco is almost completely unknown in terms of juvenile 1 Lewis, M. A. 2002. Stock assessment of anadromous salmo- nids 2001. Monitoring program report OPSW-ODFW-2002-04, 57 p. Oregon Dept. Fish Wildlife, Portland. OR 97207. salmon distribution, pelagic nekton, and biological ocean- ography in general, despite being an area of very strong upwelling and high productivity. Also, the fine-scale dis- tribution of juvenile salmon in relation to environmental variables has not been studied in any detail. The California Current is not homogeneous but rather can be divided into distinct subunits or regions, each with its own physical and biological characteristics (U.S. GLO- BEC, 1994). A break between the northernmost two regions occurs at Cape Blanco, where the equatorward upwelling jet veers sharply off the shelf and into the California Cur- rent (Barth et al., 2000). The upwelling zone north of the cape is narrow, extending out about 30 km, whereas south of Cape Blanco, it can extend up to 100 km offshore. This area also appears to represent a faunal break for some zoo- plankton communities (McGowan et al., 1999; Peterson and Keister, 2002) and is a break point for alternative salmon migration strategies (Weitkamp et al., 1995; Weitkamp and Neely, 2002). During the summer of 2000, we conducted broad-scale sampling and fine-scale process studies from central Or- egon to northern California to examine the distribution of juvenile salmon and associated species in relation to environmental conditions. This was one component of a multidisciplinary U.S. Global Ocean Ecosystem Dynamics (GLOBEC) Northeast Pacific study examining the north- ern California Current ranging in scope from the physics up to the top trophic levels (Batchelder et al., 2002). We were interested in examining the distribution of juvenile salmon north and south of Cape Blanco, the origin of these fish, and any regional differences in growth and condition of salmon across the range of sampling. Evidence exists that the physical conditions and the associated biota are different within this geographical scale. Thus, analyses of the relationship between oceanographic conditions and the response of resident biota can provide insights into the linkages associated with physical and biological processes that shape the biological community, and in particular, those associated with salmon recruitment. Methods Field surveys Surveys were conducted over two time periods — early summer (29 May-18 June, 2000) and late summer (28 July-15 August, 2000). Each survey consisted of a meso- scale grid along designated GLOBEC transects that had been monitored for several years and by fine-scale pro- cess sampling at stations of interest based on features observed in the physical environment (fronts or eddies) or by acoustic sampling conducted by two accompanying oceanographic vessels (RV Wecoma and RV New Horizon). Further details on the physical and biological conditions occurring at the time of our sampling have been reported by Batchelder et al. (2002). For the mesoscale survey, stations were established at 1, 5, 10, 15, 20, 25 and 30 nautical miles from shore on each of five transects. Inclement weather, particularly Brodeur et al.: Distribution, growth, condition, origin, and associations of |uvenile salmonids 27 during the first cruise, prevented us from sampling all the stations along each transect. At each station, a Nordic 264 rope trawl built by Nor'Eastern Trawl Systems, Inc. (Bainbridge Island, WA) was towed in surface waters by a chartered fishing vessel (FV Sea Eagle) at a speed of 6 km/h. This rope trawl has a maximum mouth opening of approximately 30 m x 18 m. Mesh sizes ranged from 162.6 cm in the throat of the trawl near the jib lines to 8.9 cm in the codend. To maintain catches of small fish and squid, a 6.1-m long, 0.8-cm mesh knotless liner was sewn into the codend. All tows were 30 minutes in duration. All fish and squid caught were counted and measured at sea. After fork length (FL) was measured to the nearest mm, all juvenile salmon were immediately frozen for later determinations of growth, condition, food habits, genetic analysis, and as- sessment of pathological condition. The physical and biological environment was monitored and sampled at each station immediately prior to setting the trawl. A CTD (conductivity, temperature, and depth) cast was made with a Sea-Bird SBE 19 Seacat profiler to 100 m at deep stations or within 10 m of the bottom at shallow stations. Chlorophyll and nutrient samples were collected from 3 m depth with a Niskin water sampler. A neuston tow with a 1-m2 mouth containing 333-,(im mesh net was towed for 5 minutes out of the wake of the vessel at each station. General Oceanics flow meters were placed inside the net to measure the amount of water sampled. Additional details on the analysis of these neuston trawls are available in Reese et al.2 Condition and growth analysis Each salmonid was remeasured (FL to the nearest mm) and weighed (to the nearest 0.1 g) in the laboratory. A por- tion of hepatic and muscle tissue was excised, placed in individual capsules, frozen in liquid nitrogen, and stored at -80°C until analyzed. The bioenergetic health of juve- nile salmon was evaluated by assessing changes in water content (as a surrogate measure of fat accumulation) of liver and muscle to estimate dry tissue weight. The water content was determined by drying tissue samples to a con- stant weight at 105°C. The accumulation of energy reserves during the growth season ( energy reserves of salmon in August in relation to salmon collected in June) that would enhance survival of juveniles during the winter when food availability is lower was also measured. The condition of juvenile salmon was assessed by examining weight residu- als (by using either the wet weight or dry weight) derived from the allometric relationship between length and weight of individual juvenile salmon after logarithmic transforma- tion (Jakob et al., 1996) of salmon captured in June and August. Wet-weight residuals are representative of the traditional condition index of animals and are a reflection 2 Reese, D.C., T.W.Miller, and R.D. Brodeur. 2003. Community structure of neustonic zooplankton in the northern California Current in relation to oceanographic conditions. 22 p. Unpubl. manuscript. Northwest Fisheries Science Center, NMFS. 2030 S. Marine Science Drive, Newport, OR 97365. of somatic tissue growth. Dry-weight residuals are respon- sive to accumulation of fat stores and are a reflection of the bioenergetic health of the individual animal (Sutton et al., 2000; Post and Parkinson, 2001). To contrast growth characteristics during 2000 in differ- ent latitudinal ranges of the California Current, we com- pared ocean growth rates of juvenile coho salmon south and north of Cape Blanco in the GLOBEC study area, and in the area from Newport, Oregon, north to northern Washington. The physical and biological characteristics of these three regions of the coastal ocean differ greatly (U.S. GLOBEC, 1994), and these differences may impact the dis- tribution and abundance of prey of juvenile salmonids and therefore may also affect salmonid growth. Data north of Newport, Oregon, were collected during a separate study of the Columbia River plume and the adjacent coastal ocean (hereafter called the "plume study") using the same trawl and a similar sampling strategy as in the GLOBEC study (see Emmett and Brodeur [2000] and Teel et al. [2003] for details). Scales were examined from 45 juvenile coho salmon caught during the June and August 2000 GLOBEC cruises and 252 juvenile coho salmon caught during the 2000 plume cruises. The scales were mounted on gummed cards from which acetate impressions were made. Using a video camera attached to a compound microscope and Optimas® imaging software (vers. 5.1, Optimas Inc., Se- attle, WA) we measured the distance (scale radius) along the anterior-posterior axis of each scale from the focus (F) to the ocean entry mark (OE) and to the scale margin (Fig. 1). The fork-length of each fish at the time of ocean entry (FL0E) was estimated from the scale radius (SR0E) at ocean entry using the Fraser and Lee back-calculation method (Ricker, 1992): FL„ (FL- 36.07) SR xSRof. +36.07, where FL = length at capture; SR = scale radius at capture; and 36.07 = the intercept from a regression of SR on FL for juvenile coho salmon caught in the ocean (Fig. 2A). In an analogous fashion, fish weight at time of ocean entry (Wr0£) was back-calculated f length at ocean entry (FL0E): (Wt0E) was back-calculated from the estimated fish fork \ni Wt0E) = (ln(Wr 1 + 12.633) ln(FL) xln(FLr,F 1-12.633, where Wt = weight at capture; and -12.633 = the intercept from a linear regression of ln(Wr) on ln(FL) for juvenile coho salmon caught in the ocean (Fig. 2B). The growth rate in FL, (FL-FL0E)lAd, 28 Fishery Bulletin 102(1) Figure 1 Scale from a 352-mm FL male juvenile coho salmon (Oncorhynchus kisutch) caught during the August 2000 GLOBEC cruise showing the axis of measurement (black line), the focus (F), the mark of ocean entry (OE), and the scale margin (SM). and the instantaneous growth rate in weight: G = (MWt)-MWt0E))/M, where Ad = estimated days between ocean entry and cap- ture, were estimated for each salmon. The meaning of the instantaneous growth rate G can be stated as follows: if salmon growth is exponential between ocean entry and capture, then Wt Wt„ and at any instant the fish's weight increases at the rate of G of its body weight per day. G can be multiplied by 100 to give the instantaneous growth rate in terms of percentage of body weight per day. Although the dates of ocean entry of individual lish were unknown, seaward migration of coho salmon smolts in California, Oregon, and Washington rivers occurs mainly between mid-April and mid-June, and there is no consis- tent latitudinal trend in timing of the migration ( Weitkamp et al., 1995). Peak downstream migration of coho salmon smolts was between mid-May and very early June in the Columbia River estuary, 1978-83 (Dawley et al., 1985), and in the lower Trinity River, California, 1997-2000 (US- A FL (mm) vs scale radius (mm) GM Regression: FL = 152.22 SR +36.07 r2 = 0.94, n=370 1 2 Scales radius (mm) B Wt(g)vs FL(mm) In(WI) = 3.2273'ln(FL) - 12 6329 or Wt(g) = 3.263x1 (T5 FL(mm)32273 n=1018V = 0.99 s — 5 In (FL) Figure 2 (A) Regression of fork length (FL) on scale radius and. 'Bi regression of ln(WY) on ln(FL) for juvenile coho salmon {On- corhynchus kisutch) caught during the May 1998-September 2000 Columbia River plume study. FWS3). In 2000, peak downstream migration of mainly nonhatchery coho salmon smolts at 13 monitoring sites in coastal Oregon rivers north of Cape Blanco occurred from April 2 to May 20; median peak migration occurred 26 April ( Solazzi et al.4) From the information available on timing of seaward migration of coho salmon smolts. we used an ocean entry date of 15 May when calculating Ad and estimating ocean growth rates of unmarked coho salmon from scales. In addition to estimating growth rates of juvenile coho salmon from scales, we also estimated instantaneous growth rates in weight between hatchery release and cap- ture in the ocean of 28 coded-wire-tagged (CWT) juvenile coho salmon: USFWS (U.S. Fish and Wildlife Service). 2001. Juvenile sal- monid monitoring on the mainstem Klamath River at Big Bar and mainstem Trinity River at Willow Creek, 1997-2000, 106 p. Annual report of the Klamath River Fisheries Assessment Pro- gram. Areata Fish and Wildlife Office, Areata, CA 9552 1 . Solazzi, M.F., S.L.Johnson, B.Miller, and T.Dalton. 2002. Sal- monid life-cvele monitoring project 2001. Monitoring program report OPSW-ODFW-2002-2, 25 p. Oregon Dept. Fish and Wildlife, Portland. OR 97207. Brodeur et al.: Distribution, growth, condition, origin, and associations of juvenile salmonids 29 G = (MWt)-MWtR))/M, where Wt = weight of the CWT fish at capture; WtR = the average weight of fish in the CWT group at time of release; and Ad = days between hatchery release and capture in the ocean. Estimated growth rates of these CWT fish, of known release date and known average release weight were used to vali- date the growth rates estimated from scale analysis Our analysis of the growth of chinook salmon based on scale characteristics is not far enough advanced to report in this article. We plan to present these data in a later article. Contribution of hatchery coho salmon to catches The total numbers, percentages of marked fish ( any exter- nal fin clips or internal tags) and grand average weights of hatchery coho salmon smolts released in 2000 are sum- marized for different release regions in Appendix Table 1. These data were used to compare the estimated average weights of fish at time of ocean entry (from scale analy- sis ) with the average weights of hatchery fish at time of release, and also to estimate the proportions of hatchery coho salmon in our catches. We calculated the expected percentage (E%) of marked fish in each catch if 100% of the fish were hatchery fish: E% X*.*4, where i?, = the proportional contribution of region i to the catch (this paper for the GLOBEC catches, and from Teel et al., 2003 for the plume study catches); and A, = the percentage of hatchery fish that were marked in region i ( from Appendix Table 1 ). The percentage of hatchery fish in each catch sample (H%) was then estimated as 0% H% = — xlOO, E% where OcA = observed percentage of marked fish. Genetic analysis The freshwater origins of juvenile chinook and coho salmon and steelhead (O. my kiss) were studied by using standard methods of genetic mixed stock analysis (Milner et al., 1985; Pella and Milner, 1987). According to the methods described by Aebersold et al. (1987), samples of eye, liver, heart, and skeletal muscle were extracted from frozen whole juvenile salmon and analyzed with horizontal starch-gel protein electrophoresis. Data from previous studies char- acterizing genetic (allozyme) differences among spawning populations in California and the Pacific Northwest were then used as baseline data to estimate the stock composi- tions of our ocean caught mixed-stock samples. Baselines consisted of 32 gene loci and 116 populations for chinook salmon (Teel et al.5), 58 loci and 49 populations for coho salmon (Teel et al., 2003), and 55 loci and 57 populations for steelhead (Busby et al., 1996). Estimates of stock com- positions were made by using the maximum likelihood procedures described by Pella and Milner (1987) and the Statistical Package for Analyzing Mixtures (Debevec et al., 2000). Estimates of individual baseline populations were then summed to estimate contributions of regional stock groups. Precision of the stock composition estimates was estimated by bootstrapping the estimates 100 times with resampling of the baseline and mixture genetic data as described in Pella and Milner (1987). Habitat and assemblage analysis The raw numbers offish and squid caught from each trawl were converted to densities based on the volume filtered per trawl to standardize for differences in effort between tows. Density contours of juvenile salmon and other nekton were produced using specialized graphics programs. We then tested whether the habitat associations of the domi- nant salmonids were significantly different from the total habitat sampled by following the methods outlined in Perry and Smith ( 1994). This procedure involved comparing the cumulative distributions of salmon catch with observed environmental conditions (temperature, salinity, chloro- phyll-a at one meter, water depth, and neuston displace- ment volume). We performed 5000 randomizations of the data and used the Cramer-von Mises test statistic recom- mended by Syrjala ( 1996) as being robust to the effects of inordinately large catches. To explore the relationship between juvenile salmon and other fish species and environmental variables, we used several types of multivariate analyses (McCune and Grace, 2002 ). Original data from each of the two cruises formed complimentary species and environmental matrices. The June and August cruises were analyzed individually to look at spatial patterns of species composition in relation to environmental gradients (Gauch, 1982). To avoid spurious effects of rare species, we excluded species from the data matrix that had a frequency of occurrence of less than 10% of the possible occurrences for each cruise (McCune and Grace, 2002). To minimize the effect of very large catches, the data were log transformed. Stations with no species present were eliminated from the data set to allow for anal- ysis of sample units in species space. Data transformations and their effects on the summary statistics were examined prior to analysis. Analyses of data were performed by using PC-ORD version 4.28 (McCune and Mefford, 1999). Agglomerative hierarchical cluster analysis (AHCA) using the Bray-Curtis dissimilarity measure and Wards Teel, D. J„ P. A. Crane. C. M. Guthrie, III, A. R. Marshall. D. M. Van Doornik, W. D. Templin, N. V. Varnavskaya, and L. W. Seeb. 1999. Comprehensive allozyme database discriminates chinook salmon from around the Pacific Rim. (NPAFC docu- ment 440), 25 p. Alaska Department of Fish and Game, Divi- sion of Commercial Fisheries, 333 Raspberry Road, Ancorage, AK 99518. 30 Fishery Bulletin 102(1) linkage function was applied to arrange the nekton spe- cies assemblages and stations into cluster groups. The cutoff level to form optimal groups within the species and station dendrograms was based on several criteria: 1) biological meaning; 2) significance tests of groups using a multi-response permutation procedure (MRPP); and 3i comparison of cutoff level MRPP results with those groups obtained from one cutoff level below and above the level of interest. A nonparametric procedure, MRPP compares the a priori groupings from AHCA and tests the hypothesis of no difference between the groups. For cluster analysis of stations, indicator species analysis (ISA) was used to determine nekton species strongly associated with indi- vidual groups. ISA assigns indicator values to each spe- cies according to relative abundance and frequency, then tests the significance (Monte-Carlo permutation test) of the highest species-specific indicator value assigned to a particular group. Nonmetric multidimensional scaling (NMS; Kruskal, 1964) was used to ordinate sample units in species space and to compare station cluster groups to environmental gradients. NMS was chosen for this analysis because it is robust to data that are non-normal and that have high numbers of zeros. Initial runs of NMS from both cruise da- tasets resulted in three-dimensional solutions. Subsequent reapplication of NMS using a three-dimensional solution (Sorensen distance, 400 maximum iterations, and 40 runs with real data) was applied for the final ordinations. To examine the environmental or station factors associated with each NMS axis that may have affected the distribu- tion of the dominant taxa, we correlated the NMS station and species scores to a suite of environmental variables including water depth, distance offshore, latitude, surface temperature, surface salinity, chlorophyll-a concentration, and neuston zooplankton settled volumes. Pearson and Kendall correlations with each ordination axis were used to measure strength and direction of individual species and environmental parameters. Results Distribution of juvenile salmon and other species We collected a total of 18,852 nekton individuals: two ceph- alopod, one agnathan, two elasmobranch, and 57 fish taxa from 163 surface trawls (see Table 1 for scientific names of all species). With the exception of market squid in June and blue shark in August, most of the nonteleost nekton occurred in only a few collections. Substantially fewer fish were caught in the June cruise than in the August cruise, but the diversity was much higher in the June cruise. The catch in June was dominated by forage fishes such as Pacific herring, surf and whitebait smelt, and juvenile rock- fishes, sablefish, and flatfishes. Salmonids, mainly juvenile chinook and coho salmon and steelhead, comprised a rela- tively minor proportion of the catches (only 114 juvenile salmonids; 1.9 % of the total). The August cruise was dominated by several large catches of Pacific sardine (Table 1 ). Jack mackerel was the most common nonsalmonid caught. Many of the juvenile fish taxa caught during the June cruise were absent during the August cruise; those that did occur ( sablefish. rex sole) were much lower in abundance. Mesopelagic fishes of the family Bathylagidae and Myctophidae were collected only during the August cruise, mainly because of the inclusion of more offshore stations and occasional collections during nondaylight hours. As in the earlier cruise, salmonids com- prised a relatively minor percentage of the catch (3.19f ) but were more common and abundant during this survey. Juvenile chinook salmon were broadly distributed lati- tudinally during both cruises, but their distribution was mainly restricted to nearshore stations within the 100-m isobath (Fig. 3). Coho salmon juveniles were more common north of Cape Blanco during both cruises and were found generally farther offshore than chinook salmon juveniles (Fig. 3). In contrast, steelhead juveniles were found mainly south of Cape Blanco, especially in June, but their zonal distribution overlapped that of coho salmon juveniles. Size and condition of juvenile salmon Fork length of yearling chinook salmon averaged 227 ±42 mm FL in June and 230 ±30 mm FL in August and aver- aged 135 ±12 mm FL for subyearling chinook salmon in August, whereas juvenile coho salmon averaged 162 ±32 mm FL in June and 286 ±46 mm FL in August ( Table 2 ). No significant differences in fork length of juvenile chinook or coho salmon north or south of Cape Blanco were evident. Juvenile coho salmon weighed significantly more on a wet-weight basis for a given fork length in the region north of Cape Blanco compared to juveniles captured south of Cape Blanco (Fig. 4). This pattern was also similar and significant when evaluated on a dry-weight basis (bioen- ergetic growth). Although the stock composition in the two regions could account for some of these differences, the growth responses likely reflect habitat-specific features in the region north of Cape Blanco that benefit coho salmon. No difference in condition of yearling chinook salmon cap- tured north or south of Cape Blanco, on either a wet- or dry- weight basis, was evident (Fig. 4). Information regarding size and condition of subyearling chinook salmon are not presented because few subyearling chinook salmon were caught in June and all but one subyearling chinook salmon in August were caught in the region south of Cape Blanco, OR. Insufficient subyearling chinook salmon were avail- able for an analysis comparable to that done for yearling chinook and coho salmon. Proportions of wild and hatchery coho salmon Most of the juvenile coho salmon caught during the plume study north of Newport, Oregon, originated in hatcher- ies (Table 3). In June and September 2000 we estimated that wild fish comprised only W9i and 25r< . respectively, of the catch. Wild fish, however, comprised a proportion- ally much higher percentage of the catch of coho salmon in the GLOBEC study area in June north of Cape Blanco (67$ I, and in August south of Cape Blanco (619! I, than in the plume study area farther to the north. Most jacks and Brodeur et al.: Distribution, growth, condition, origin, and associations of |uvenile salmonids 31 Table 1 Phylogenetic listing of nekton catch in numerical composition, frequency of occurrence (F.O.) and size range cau ght for each cruise. (j) indicates juvenile stage; (a) adult. ML = mantle length, TL = total length. FL = fork length, SL = standard length ( in mm). Class and Family Common name June (84 stations) August (79 stations) Scientific name dumber F.O. Size range Number F.O. Size range Cephalopoda Onychoteuthidae Pacific clubhook squid Onychoteuthis borealijaponicus 19 6 21-80 ML 302 6 21-227 ML Loliginidae Market squid Loligo opalescens 301 14 33-122 ML 1 1 35 ML Agnatha Petromyzontidae Pacific lamprey Lampetra tridentata 1 1 625 TL Chondrichthyes Alopiidae Thresher shark Alopias vulpinus 1 1 36-576 TL Carcharhinidae Blue shark Prionace glauca 18 10 1300-1660 TL Osteichthyes Xenocongridae Eel leptocephalus Thalassenchelys coheni 3 1 214-243 TL 2 2 260-305 TL Clupeidae Pacific herring Clupea pallasi 1022 9 127-195 FL Pacific sardine Sardinops sagax 7 2 237-260 FL 10,327 15 178-290 FL Engraulididae Northern anchovy Engraulis mordax 49 12 148-165 FL Salmonidae Chinook salmon (j,a) Oncorhynchus tshawytscha 56 18 121-780 FL 252 26 109-910 FL Coho salmon (j,a) Oncorhynchus kisutch 35 15 122-580 FL 111 25 210-736 FL Cutthroat trout (j,a) Oncorhynchus clarki 1 1 186 FL 3 3 258-341 FL Steelhead trout (j,a) Oncorhynchus mykiss 22 8 176-284 FL 36 13 261-430 FL Osmeridae Smelt (j) Osmeridae 14 4 37-52 SL 74 5 31-50 SL Surf smelt Hypomesus pretiosus 846 8 128-184 FL 351 7 140-187 FL Whitebait smelt Allosmerus elongatus 946 6 60-114 FL 79 3 76-132 FL Bathylagidae Popeye blacksmelt Bathylagus ochotensis 1 1 76 SL Paralepidae Slender barracudina Lestidium ringens 3 1 72-76 SL Myctophidae Northern lampfish Stenobrachius leucopsarus 96 4 14-70 SL Bigfin lanterfish Symbolophorus californiensis 61 4 89-102 SL Blue laternfish Tarletonbeama crenularis 10 3 33-87 SL Gadidae Gadid(j) Gadidae 10 3 42-58 SL 13 3 53-57 SL Pacific cod 1 j ) Gadus macrocephalus 23 1 38-60 SL Pacific tomcod ( j ) Microgadus proximus 6 4 35-55 SL 8 2 49-80 SL Scomberesocidae Pacific saury Cololabis saira 26 1 182-229 FL 66 6 131-194 FL Atherinidae Jacksmelt Atherinopsis californiensis 1 1 302 FL Trachipteridae King-of-the-salmon (j ) Trachipterus altivelis 2 2 71-270 SL 12 2 40-83 SL Gasterosteidae Threespine stickleback Gasterosteus aculeatus 1 1 60 SL Scorpaenidae Pacific ocean perch (j ) Sebastes alutus 1 1 33 SL Darkblotched rockfish (j Sebastes crameri 154 14 29-54 SL 1 1 53 SL Yellowtail rockfish (j) Sebastes flavidus 1350 24 20-63 SL 1 1 18 SL Shortbelly rockfish (j ) Sebastes jordani 1 1 37 SL Black rockfish (j,a) Sebastes melanops 1 1 30 SL 1 1 335 FL Bocaccio (j ) Sebastes paucispinis 20 5 21-36 SL Canary rockfish (j ) Sebastes pinniger 27 5 22-39 SL Bank rockfish (j ) Sebastes rufus 8 1 16-28 SL Stripetail rockfish (j) Sebastes saxicola 13 3 32-37 SL Hexagrammidae Lingcod (j) Ophiodon elongatus 20 9 76-81 FL Anoplopomatidae Sablefish (j ) Anoplopoma fimbria 182 14 55-136 FL 4 2 173-241 FL continued 32 Fishery Bulletin 102(1) Table 1 (continued) Class and Family Common name Scientific name June (84 stations) August 179 stations) Number F.O. Size range Number F.O. Size range Cottidae Irish lord Ij) Hemilepidotus spp. 2 1 38-40 FL Cabezon (j ) Scorpeanichthys marmoratus 12 7 33-38 SL Pacific staghorn sculpin Leptocottus armatus 1 1 180 TL Agonidae Sturgeon poacher (j) Podothecus acipenserinus 1 1 80 TL Cyclopteridae Pacific spiny lumpsucker Eumierotremus orbis 1 1 253 TL Carangidae Jack mackerel Trachurus symmetricus 111 3 364-583 FL 839 20 227-589 FL Bramidae Pacific pomfret Brama japonica 5 2 387-434 FL Anarhichadidae Wolf-eel (j) Anarrhichthys ocellatus 15 13 215-555 TL 8 7 442-582 TL Ammodytidae Pacific sandlance Ammodytes hexapterus 4 4 45-82 SL Zaprodidae Prowfish (j) Zaprora silenus 1 1 68 SL Scombridae Chub mackerel Scomber japonicus 74 6 266-421 FL Centrolophidae Medusafish Icichthys lockingtoni 3 3 37-50 SL 8 6 87-129 FL Bothidae Sanddabs (j) Citharichthys spp. 23 13 35-43 SL 3 2 269-288 TL Pacific sanddab (j ) Citharichthys sordidus 32 4 32^4 SL Speckled sanddab (j ) Citharichthys stigmaeus 60 10 30-43 SL Pleuronectidae Dover sole (j) Microstomas pacificus 2 2 40-50 SL 3 1 27-34 SL Sand sole (j) Psettichthys melanostictus 3 3 22-39 SL Slender sole (j) Eopsetta exilis 1 1 66 SL Starry flounder Platichthys stellatus 2 1 349-399 TL Curlfin sole (j) Pleuronichthys decurrens 5 3 25-31 SL English sole Parophrys vetulus 1 1 303 TL Rex sole (j ) Errex zachirus 581 12 34-79 SL 48 11 44-70 SL Molidae Ocean sunfish Mola mola 1 1 620 TL Total 5974 12,878 about one half of the nonjacks caught north of Cape Blanco in August were hatchery fish. Two factors, however, may have lead to inaccuracies in estimation of hatchery-wild ratios of coho salmon in the GLOBEC study area. First, because of low sample sizes, the data were pooled from both June and August catches for the genetic stock analysis; therefore we do not know the proportional contributions of the different release areas to the catches in either month alone. Second, all the fish released from Klamath River and Trinity River hatcheries had been clipped on the maxillary. We were unaware that the maxillary clip was being used, did not look for it, and consequently may have classified fish with this mark as unmarked. Therefore, the proportion of hatchery fish in the catch of coho salmon during GLOBEC may have been higher than is shown in Table 3. Age and growth of juvenile coho salmon Forty-three percent (24 of 56) of the juvenile coho salmon caught during the August GLOBEC cruise were preco- cious males ("jacks") according to the testes-weight to body-weight criteria of Pearcy and Fisher ( 1988). This is a much higher percentage of jacks than found among juve- nile fish caught in September 2000 in the plume study off Oregon and Washington, where only 4.5% offish (6 of 132) were precocious males or females according to the same criteria. Because the jacks were considerably larger than the nonjacks, average growth rates of the two groups were reported separately. Estimated average growth rates in FL between ocean entry and capture were higher for fish caught in the August 2000 GLOBEC cruises (1.56-2.22 mm/d) than for fish caught in any other cruises (Table 3). The fish caught in August 2000 were also larger when they entered the ocean (average 170- 178 mm FL) than fish caught in other cruises (averagel54-160 mm FL). Average growth rate of jacks from north of Cape Blanco (2.22 mm/d), was significantly higher (/-test, P<0.05) than growth rates of nonjacks (1.56-1.67 mm/d). Growth rates of nonjacks north and south of Cape Blanco were not significantly different la- test, P<0.05). The combination of large size at ocean entry Brodeur et al.: Distribution, growth, condition, origin, and associations of juvenile salmonids 33 45.0 44.5 44.0 43.5 43.0 42.5 42.0 41.5 Newport Chinook > 0 1 to 5 0 6 to 150 Coho A 1 10 5 A 6 to 150 J Oregon r California 45.0 44.5 44.0 43.5 43.0 42.5 42.0 41.5 125.5 125.0 124.5 124,0 123.5 125.5 Longitude (W) 125.0 124.5 124.0 123.5 Figure 3 Catch distribution for juvenile coho (Oncorhynchus kisutch) and chinook salmon (O. tshawytscha) for the (A) June and (B) August cruise overlaid on surface temperature contours. Plus signs are stations sampled where no salmon were caught. and favorable conditions for growth in the ocean probably contributed to the very high percentage of jack coho salmon in August 2000 in the GLOBEC study area. Estimated average growth rates between ocean entry and capture of juvenile coho salmon were higher in the GLOBEC area than in the plume study area U-tests, P<0.05). For fish caught in June, average growth rate was 1.06 mm/d and 0.63 mm/d in the GLOBEC and plume study areas, respectively. For fish caught in August or September, average growth rate was 1.57-2.22 mm/d in the GLOBEC study area and 1.17 mm/d plume in the study area (Table 3). The higher growth rates of coho salmon caught in the GLOBEC study area suggests that in 2000 conditions for growth were bet- ter there than those in the plume study area farther north off Oregon and Washington. Average instantaneous growth rates in weight were also higher (/-tests, P<0.05) for the fish caught in the June and August 2000 GLOBEC cruises (2.0 and 2.1-2.8% body wt/d, respectively) than for the fish caught in the June and September 2000 plume study cruises (1.2 and 1.7 % body wt/d, respectively; Table 4A). In addition, the average condition index (CI) of juve- nile coho salmon in June was significantly higher (/-test, P=0.03) in the GLOBEC study area (1.12, n=32, SD=0.087) than in the plume study area (1.07, n=245, SD=0.117). Similarly, the average CI of nonjack juvenile coho salmon was higher (/-test, P=0.002) in August in the GLOBEC study area (1.24, n=32, SD=0.096) than in September in the plume study area (1.18, n=132, SD=0.100). Both the high instantaneous growth rates in weight and the high CI of juvenile coho salmon caught in the GLOBEC study area suggest that conditions for growth of coho salmon in this area were very good in 2000. Growth rates estimated from the few CWT fish caught during these cruises (Table 4B) were similar to, and help validate, the growth rates estimated from scales (Table 4A). Average weights at time of ocean entry back-calculated from scales for coho salmon caught in June in the GLOBEC area and in all months in the plume study area (Table 4A) were slightly higher than the average weights of hatchery coho salmon at time of release (Appendix Table 1). For ex- ample, in the plume study area, average back calculated weights at ocean entry ranged from 37.5 g to 42.4 g (Table 4A) — slightly higher than the expected average weights at release of about 32-33 g based on the stock composi- 34 Fishery Bulletin 102(1) Table 2 Summary of mean, standard deviation, and range of FL measured in the field, weight measured in the laboratory, and condition index (CI) of subyearling (age 0.0) and yearling (age 1.0) chinook salmon and yearling (age 1.0) coho salmon caught during the June and August cruises north (N) and south (S) of Cape Blanco (latitude 42.837°). Precocious coho salmon are indicated with a "J". Field FL (mm) Laboratory weight (g) C.I. (wtx 105/ FL3 ) n Mean SD Range Mean SD Range Mean SD Chinook (age 0.0) June (N) 1 121 — — 18 — — 1.04 — August (N) 1 172 — — 70 — — 1.37 — August (S) 125 134 12 109-175 28 9 12-70 1.10 0.08 Chinook (age 1.0) June (N) 27 229 42 144-280 178 91 33-306 1.32 0.10 June(S) 1 174 — — 67 — — 1.28 — August (N) 54 229 26 187-318 164 72 80-468 1.32 0.09 August (S) 35 231 35 190-349 176 94 80-535 1.32 0.07 Coho (age 1.0) June (N) 30 161 33 122-276 56 51 19-292 1.13 0.08 June (S) 2 172 0 172-172 49 1 48-49 0.95 0.01 August (N-J) 24 365 31 310-415 690 209 375-1198 1.38 0.12 August (N) 24 285 51 210-385 326 188 97-766 1.26 0.10 August (S) 8 293 33 239-334 308 103 157-433 1.19 0.05 Table 3 Catch, percentage of the catch that was marked, estimated percentage of hatchery origin, size of scale sample, FL at ocean entry (OE) back calculated from scales, FL at capture, and estimated growth rate in FL while in the ocean for juvenile coho salmon caught during the 2000 GLOBEC and Columbia River plume studies. All length data are from the scale sample only. An ocean entry date of 15 May was used when calculating growth rate in FL. Cruise Catch (n) Marked Estimated % Scale sample hatchery origin (n) Back- calculated FL at OE (mm) mean (SD) FL at capture (mm) Growth rate (mm/d) mean (SD) mean(SD) GLOBEC June 2000 32 32% 33% 11 155 (29.0) 177(42.3) 1.06(1.01) Aug 2000 North of C.Blanco Jacks 24 71% 74% 19 170(22.8) 370(28.1) 2.22 (0.35) Nonjacks 24 46% 48% 9 178(21.6) 309(46.1) 1.67 (0.51) South of C. Blanco Nonjacks 8 38% 39% 6 178(13.0) 303 (29.3) 1.56 (0.22) Plume study May 2000 165 68% 76-80%; 79 157(16.5) 166(17.7) 0.97(1.15) Jun 2000 245 76% 90% 97 160(14.5) 185(23.4) 0.63 (0.53) Sep 2000 132 65% 75% 76 154(19.0) 305 (24.9) 1.17(0.23) ' No genetic stock analysis was available. The higher estimate assumes the same stock composition as in June, hatchery fish were from the Columbia River. the lower estimate assumes that all Brodeur et al.: Distribution, growth, condition, origin, and associations of |uvenile salmonids 35 A 0.004 0002 0 -0.002 -0.004 □ Wet Wt (Somatic Growth) to as -0.006 "D ■ Dry Wl (Energetic Growth) CO 1) 0.02 -, o B 0.01 - —L— H^H -0.01 - -0.02 - -0.03 - -0.04 - -0.05 - l -0.06 - -0.07 - Cape Blanco Cape Blanco North South Figure 4 Wet and dry weight residuals ( + 1 standard error) for (A) yearling chinook (On- corhynchus tshawytscha) and (B) juvenile coho salmon (O. kisutch) collected North and South of Cape Blanco. Weight residuals are derived from the linear relationship between fork length and wet or dry weight (log-transformed data) of juvenile salmon captured in June and August. tion of these catches (Teel et al., 2003) and the release weights (Appendix Table 1). Similarly, the back-calculated weight at ocean entry in June in the GLOBEC area (45.5 g) was slightly higher than the expected average weight at hatchery release (about 41 gl based on the stock compo- sition (Table 5) and the average release weights. These fairly small differences between back-calculated size at ocean entry and average size at release could be due to growth during downstream migration, selectively higher mortality of small smolts, or a bias in the back-calculation procedure. However, the average back-calculated weights at time of ocean entry offish caught in August in the GLOBEC study area (60-69 g) were over two standard deviations above the average weights of hatchery fish released from the Oregon coast or northern California — the main contributors to this catch (Appendix Table 1). These were obviously atypical coho salmon, and the very high proportion of jacks (preco- 36 Fishery Bulletin 102(1) Table 4 (A) Weights at ocean entry I OE ) back-calculated from scales, weights at capture and estimated instantaneous rates of growth while in the ocean iGl for juvenile coho salmon caught during the 2000 GLOBEC and Col umbia River plume studies. An ocean entry date of 15 May was used when calculating growth rate. (B) Similar data for CWT fish. Growth rates of the CWT coho salmon were estimated for the periods between hatchery release and capture in the ocean. A Cruise ;; Back-calc. Wt. at OE (g) Weight at capture (g) G mean (SD) mean(SD) mean (SD) GLOBEC June 2000 11 45.5 (26.8) 78.0(76.4) 0.020(0.015) Aug 2000 North of C. Blanco Jacks 19 68.9(27.2) 719.7(200.0) 0.028 (0.005) Nonjacks 9 59.5 (26.3) 419.2(177.2) 0.023 (0.006) South of C. Blanco Nonjacks 6 60.3(12.8) 336.2 (96.2) 0.021 (0.002) Plume study May 2000 79 39.4 (10.8) 47.9(14.6) 0.020(0.024) Jun 2000 97 42.4(12.5) 71.9(33.3) 0.012(0.009) Sep 2000 75 37.5(13.7) 347.2(158.3) 0.017(0.003) B Cruise n Wt. at release (g) Wt. at capture (g) G mean (SD) mean (SD) mean (SD) GLOBEC Jun 2000 4 44.4(1.3) 86.6 (30.9) 0.018(0.005) Aug 2000 3 35.6 (9.8) 395.7(215.0) 0.024(0.003) Plume study Jun 2000 11 28.3(4.5) 66.1(32.3) 0.012(0.005) Sep 2000 10 33.4(10.91 392.4(283.3) 0.018(0.002) cious, sexually developed males) among the fish was prob- ably a consequence of their very large size at ocean entry and their high rates of growth in the ocean. Freshwater origins of juvenile salmonids Allozyme data were collected from samples of 247 chinook salmon, 88 coho salmon, and 58 steelhead. Genetic mixed stock analyses indicated that chinook salmon in June were predominately (54%, SD=0.18) from rivers and hatcheries along the mid Oregon coast, an area immediately north of Cape Blanco (Table 5, Fig. 5). In August, chinook salmon were largely from rivers that enter the sea south of Cape Blanco. Fish from the Sacramento and San Joaquin rivers in northern California were estimated to comprise 90% (SD=0.07) of the chinook salmon sampled in August north of Cape Blanco. The largest concentration of chinook salmon we sampled was south of Cape Blanco in August, and these fish were mostly from rivers in southern Oregon (539(, SD=0.10) and the Sacramento and San Joaquin rivers (20%, SD=0.05). Chinook salmon from the Colum- bia River Basin were also present, but were estimated to comprise only 18% (SD=0.15) of the June sample and 8% (SD=0.05) of the August sample north of Cape Blanco. Recoveries of hatchery chinook salmon bearing coded-wire tags (CWT) provided direct evidence of stock origins for ten fish, all taken in trawls north of Cape Blanco (Table 5). These data reveal that hatchery fish released from the Umpqua River on the central Oregon coast (;;=6), Columbia River Basin («=3) and Sacramento River (« = 1) contributed to our sample of chinook salmon. The propor- tion of CWT fish from the Umpqua River in our August catch north of Cape Blanco (8%) indicated that the con- tribution of mid Oregon coastal fish was underestimated in the genetic analysis likely because of the small size of the mixture sample. Genetic estimates of coho salmon indicated that most fish originated from coastal Oregon rivers north of Cape Blanco (479S , SD=0.10) and from the Columbia River (13%, SD=0.08 ) (Table 5 ). However, a substantial proportion (40r/i , SD=0.09) of coho salmon were from coastal rivers south of Cape Blanco, a region that includes spawning populations in the Rogue and Klamath rivers. Eight coho salmon in our sample contained CWTs and showed that fish from Brodeur et al.: Distribution, growth, condition, origin, and associations of juvenile salmonids 37 Table 5 Estimated percentage stock compositions, samples sizes, and recoveries of coded wire tags (CWTs) for chinook and coho salmon and steelhead sampled in trawl surveys along the Oregon and California coasts in 2000 Some of tht major baseline stocks are given for coastal stock groups. None of the steelhead sampled contained coded wire tags. June (rc=35) August (?!=157) August (n=55) Entire South of North of Study Area Cape Blanco Cape Blanco Chinook salmon stock group Est. SD CWT Est. SD CWT Est. SD CWT Columbia and Snake Rivers 18 0.15 2 3 0.03 8 0.05 1 North Oregon coast (Nehalem, Trask, Alsea, and Siuslaw Rivers) 0 0.00 0 0.00 0 0.00 Mid Oregon coast (Umpqua, Coquille, Sixes, and Elk Rivers) 54 0.18 3 3 0.03 1 0.02 3 South Oregon coast (Rogue. Chetco, and Winchuck Rivers) 26 0.16 53 0.10 0 0.00 Klamath and Trinity Rivers 0 0.00 14 0.07 0 0.00 North California Coast (Mad, Eel, and Mattole Rivers) 2 0.05 7 0.07 1 0.04 Sacramento and San Joaquin Rivers 0 0.00 20 0.05 90 0.07 1 June and August (rc=88) Coho salmon stock group Entire study area Est. SD CWT Columbia River 13 0.08 2 North and Mid Oregon coast (Nehalem, Siletz, Alsea, Umpqua, and Coos Rivers) 47 0.10 5 Rogue and Klamath Rivers 40 0.09 1 North California Coast (Mad, Russian, Little, and Scott Rivers) 0 0.00 June and August (n=58) Steelhead trout stock group Entire study area Est. SD Columbia and Snake Rivers 0 0.00 North and Mid Oregon coast (Nehalem, Siletz, Alsea, Umpqua, Coos, and Coquille Rivers) 1 0.03 South Oregon coast (Elk, Rogue, Chetco, and Winchuck Rivers) 53 0.08 Smith, Klamath, and Trinity Rivers 0 0.00 North California Coast (Mad, Eel, and Ten Mile Rivers) 10 0.05 Sacramento and San Joaquin Rivers 14 0.05 Central and South California Coast (San Lorenzo River and Scott, Pauma, and Gaviota Creeks 3 0.02 Unknown 19 — hatcheries in the Umpqua River (n=5), Rogue River (n=l), and Columbia River (n=2) were in our study area. Genetic analysis of steelhead samples showed that a large proportion were from the Rogue River and nearby coastal streams (53%, SD=0.08). Steelhead from the Sacra- mento and San Joaquin rivers (14%, SD=0.05) and north- ern California coastal rivers (10%, SD=0.05) were also present. Estimates for steelhead originating from rivers north of Cape Blanco and from south of the San Francisco Bay were near zero. Approximately 19% of the steelhead mixture was not allocated to any source population, sug- gesting that our baseline data for the species is incomplete. No steelhead in our collections contained CWTs. Species associations of juvenile salmonids and other species From cluster analysis of species based on station assem- blages (Fig. 6), MRPP of both sample periods showed strong within-group agreement (P<0.0001) at the first level (two groups); all subsequent groups had sequentially higher levels of within-group agreement. As a result, the cutoff level was determined by balancing a lower percent infor- mation remaining (<30%) in the model while retaining bio- logically meaningful groups. For June this cutoff resided at the second level (three groups) and for August, at the third level (four groups ). For the June cruise, all salmonids includ- 38 Fishery Bulletin 102(1) 1 A I 127° i 122" 1 117"W — 50°N Vancouver "~-~~ Island ^fc--- B.C. - Pacific Ocean Olympic Peninsula Puge! ^ Sound , r" r£*\ •/ - 46" Columbia R -5sk^ "X Columbia R L wA J Snake R _ — 42" N -38" ^ Newport Cape Blanco / ,-.-, / • Yj Crescent City vC7 Eel R. /\\ I s" Umpqua Rogue R CU / o> r /, 3 ) A 3 ( L- o \ ;o V i R. 1 OR V ID CA 3arvJu 1 1 1 0 1 00 200 km I B 1 1 127- 122- 1 117"W June -so-N entire study area .^^ - © f|°oo ~46' August north of Cape Blanco ° oo 7\ -4-' W i August south of #•'-. i Cape Blanco i # • 0 100 200 km 1 1 •• 1 1 — 50* N c — r 132" 127° 122° '46'June and August entire study area — 42" O • N J_ _L J. Figure 5 (A) Map of study area and location of GLOBEC sampling (hatching). (B) Stock compositions of chinook salmon (Oncorhynchus tshawytscha). Stock groups are North of Columbia River (grey), Columbia River Basin (green), north Oregon coast (pink), mid Oregon coast (yellow), south Oregon coast (dark blue), Klamath River Basin (black), north California coast (light blue), and Central Valley (red). (C) Stock compositions of coho salmon (O. kisutch). Stock groups are Columbia River (green), mid and north Oregon coast (dark pink), Rogue and Klamath rivers (blue), and north California coast (orange). Brodeur et al.: Distribution, growth, condition, origin, and associations of juvenile salmonids 39 June 100 Information remaining (%) 75 50 25 H 1 1 1 H Coho Chinook (a) Wolfeel Chinook (j) Lmgcod Steelhead Sablefish Market squid Whitebait smelt Pacific herring Surf smelt Darkblotched rockfish — , Yellowtail rockfish — T~ Rex sole — Speckled sanddab — i August 100 r- Information remaining (%) 75 50 25 H 1 1 1 h i Coho (a) Coho (j) Chinook (a) Chinook (j) Surf smelt Steelhead Medusafish Pacific saury Wolfeel Osmeriid (j) Blue shark Northern anchovy Rex sole Chub mackerel Pacific sardine Jack mackerel Figure 6 Cluster species groupings by cruise. The dashed lines indicate the cutoff levels for each cluster group. See Table 1 for scientific names. i> ing steelhead were classified within the same grouping that included several pelagic juvenile taxa, including wolf-eel, lingcod, and sablefish (Fig. 61. Two other clusters that were not associated with juvenile salmon included a southern inshore group consisting of market squid. Pacific herring, and two species of smelt and an offshore northern group consisting primarily of juvenile rockfish and rex sole. For the August cruise, all salmonid juveniles and adults again clustered together in one large group with surf smelt and medusafish ( Fig. 6 ). The remaining three groups were much smaller and consisted primarily of offshore pelagic species. Cluster analysis of stations based on species assem- blages, and subsequent examination of the cutoff level us- ing MRPP, resulted in three groupings from both sample periods (Fig. 7). MRPP revealed strong within-group agreement for all levels (P<0.0001); however, delineation at three groups was based on maintaining lower percent in- formation remaining (<30%) and still having a meaningful level of resolution. There was some measure of geographic separation among the three groups (Fig. 7). In June, group A was predominantly inshore and mostly in the southern half of the sampling area, group B was found mainly in the middle shelf region and was more northern, and group C was found predominantly offshore. In August, group A consisted of only three stations, all south of Cape Blanco, whereas groups B and C both spanned the entire shelf and offshore region and had no particular north-south affin- ity (Fig. 7). ISA of the groups from both sampling periods showed that only groups A and C had indicator species (Tables 6 and 7), whereas the intermediate groups had none. Ordination analyses and environmental correlates NMS ordination of the June sampling period (Fig. 8A) revealed most of the variance in the data: axes 1 and 40 Fishery Bulletin 102(1) June 2000 AAAAAn] a 44.5- □ A * rw 44.0- cPa A a n nrriAAA d 43.5- n nriAA'fY/ • 43.0- A \ D AA \ ( □□ ao/ duster Groupings 42.5- ' Group A O , Group B A aA Group C [ 42.0- OnA , , , i i , , , , 1 42.0- August 2000 rr D| &A A 125.5 125.0 124.5 124.0 1235 125.5 125.0 124.5 124.0 123.5 Longitude (W) Figure 7 Map showing locations of cluster station groupings by cruise. Table 6 Indicator species analysis showing indicator values for dominant pelagic nekton captu mean, standard deviations (SD), and P- values for each cluster grouping. Cluster Group mined to be indicators of that group. •ed in pelagic trawls during June 2000 and B did not have any species that were deter- Group Species Observed indicator value (IV) Indicator value IV from randomized groups P-value Mean SD A chinook (age 0.0 1 61.0 15.7 6.54 <0.001 A lingcod 26.1 12.6 5.67 0.024 A Pacific herring 71.7 12.8 5.88 <0.001 A surf smelt 86.5 11.8 5.59 <0.001 A whitebait smelt 31.5 10.4 5.55 0.007 A market squid 50.8 15.0 6.20 <0.001 C darkblotched rockfish 66.8 1 5 8 6.31 <0.001 C rex sole 46.0 15.0 6.24 0.002 C sablefish 31.1 16.2 6.32 0.035 C speckled sanddab 52.5 13.4 5.94 0.001 C yellowtail rockfish 98.8 19.0 6.30 <0.001 3 represented 31% and 237f, respectively (stress=16.3). Temperature, depth and salinity best explained the ordi- n;it ion of stations, representing a cross shelf gradient from nearshore high levels of salinity to increasing temperature and depth offshore. Ordination of August stations (Fig. 8B) represented 42' i of the variance in the data, and 23% of the variance was loaded on axis 2 and 19% on axis 3 (stress=19.4). As with June, salinity increased toward the coast and temperature and depth increased off the shelf. The groups derived from the cluster analysis tended to group together in multivariate space, with the exception of group B in the June cruise (triangles in Fig. 8A). Brodeur et al.: Distribution, growth, condition, origin, and associations of |uvenile salmonids 41 Table 7 Indicator Species Analysis showing indicator values for dominant pelagic nekton captured in pelagic trawls during August 2000 and mean, standard deviations (SD), and P-values for each cluster grouping. Cluster Group B did not have any species that were determined to be indicators of that group. Group Species Observed indicator value (IV) Indicator value IV from randomized groups P-value Mean SD A chinook (age 1.0) 76.5 21.3 11.18 0.004 A A chinook (age 0.0) surf smelt 80.4 97.9 22.1 12.4 11.62 8.21 0.003 <0.001 C chub mackerel 33.3 12.8 8.88 0.021 C jack mackerel 73.7 23.0 11.86 0.006 Table 8 Results of statistical tests for habitat associations between juvenile salmon and environmental or station variables from each cruise in 2000. Fish marked by zeros indicate subyearlings and those marked with one indicate yearlings. Shown are the P-levels for 5000 randomizations of the cumulative frequency of the habitat variable and the proportion of the standardized salmon catch associated with each habitat observation. Results are based on the Cramer von-Mises test statistic determined from binned data for depth and neuston biomass. Significance values <0.05 are shown in boldface. Cruise Jun Aug Taxon and age Surface temp. Surface salinity 1-m chlorophyll Bottom depth Neuston biomass chinook (age 1.0) 0.30 0.60 0.13 0.18 0.13 coho (age 1.0) 0.33 0.48 0.21 0.17 0.31 chinook (age 0.0) 0.36 0.25 0.13 0.35 0.42 chinook (age 1.0) 0.04 <0.01 <0.01 0.02 0.29 coho (age 1.0) 0.68 0.04 0.07 0.02 0.45 There were few instances where the habitat associations of juvenile salmon were significantly different from the distribution of environmental variables sampled (Table 8). None of the variables were significant for yearling chinook and coho salmon in the June sampling (no subyearling salmon were caught during that cruise). In August, all the variables except neuston biomass were significant for yearling chinook salmon. These fish were collected at cooler temperatures, higher salinities, higher chlorophyll-o con- centrations, and at shallower depths than have been typi- cally recorded (Fig. 9). Coho salmonjuveniles were found in higher salinities and shallower depths than at the sampled habitat (Fig. 9). These results correlated with the capture of juvenile chinook salmon and to a lesser with extent coho salmon at nearshore stations in the upwelling zone. Discussion Understanding the mechanisms underlying the dynamics of multispecies communities is one of the biggest challenges in ecology. Most communities contain many interacting spe- cies, each of which is likely to be affected by multiple biotic and abiotic factors. In order to effectively characterize a system, we need to differentiate variability resulting from both temporal and spatial factors. Our observations took place during two time periods of about 20 days each and thus were not synoptic "snapshots" of the system. Indeed, during our June sampling, conditions changed markedly from the beginning to the end of the cruise because of the arrival of an anomalous major southwest storm ( Batch- elder et al., 2002), which likely completely altered the hydrography and biology of the system. Thus, short-term temporal variability may obscure patterns observed over the spatial scale of our sampling. The pelagic nekton community sampled during these cruises was not that different from what had previously been shown for purse seine and trawling collections off the coast of Oregon and Washington ( Brodeur and Pearcy, 1986; Emmett and Brodeur, 2000; Brodeur et al., 2003). The early summer nekton community was dominated by coastal forage fishes such as smelts and Pacific herring, but also comprised juveniles of many rockfish, sculpin, and flatfish species. These winter-spring spawning species eventually settle out to demersal habitats sometime in summer (Shenker, 1988; Doyle, 1992), which may in part explain the paucity of these taxa in the August cruise. In contrast, the August nekton community consisted of large, 42 Fishery Bulletin 102(1) highly migratory species such as Pacific sardines, jack mackerel, and chub mackerel. Pacific sardine, which was almost completely absent from the system in the 1980s, has undergone a substantial resurgence and is now one of the most abundant species off the coast in summer (Brodeur et al., 2000; Emmett and Brodeur, 2000; McFarlane and Beamish, 2001). It should be noted, however, that some of the differences between cruises could be accounted for by the inclusion of substantially more offshore stations during A; aa REXS SPSD cr A A A 3 A U STHD t A MASO o WBSM DBRF °-,YTRF D cP SABF A Temperature Depth , Salinityn n LGCD - <% PHER »* D d1 CHIN1 COHO A A * 1 Axisl (r2=0.31) CO CO X < B A A * A * REXS A D STHD A COHOA A D a coftoj D A a D -Depth a Salinity D Temperature CHINO 0 BLSH OSMJ ° CO CD PSAR CHIN1 O o a o ^OEL a °0 CO o Axis 2 (r2=0.23) Figure 8 Nonmetric multidimensional scaling (NMS) ordination plot of stations and nekton species with environmental parameters from June (A) and August (B) 2000 GLOBEC cruises. Station symbols denote: onshore tO>. mid-shelf !▲). and slope (D) groupings; Species abbreviations denote the following taxa: CHIN 0 (chinook, age 0), CHIN 1 (chinook, age al.ll, STHD (steelhead trout). SUSM (surf smelt), PSAU (Pacific saury), WOEL (wolf-eel juvenile), OSM J (osmerid juvenile), REXS (rex sole, larval i, MEDF (medusafish ), PSAR (Pacific sardine), .JAMA (jack mack- erel), CHMA (chub mackerel), NANC (northern anchovy). BLSH (blue shark). the second cruise. Our results from the community analy- ses suggest that juvenile salmon tend to co-occur with each other and with a variety of other pelagic nekton, including adult salmon, and that this spatial overlap varies tempo- rally. Brodeur et al. (2003), in analyzing community struc- ture based on previous pelagic sampling data off Oregon and Washington, arrived at similar results. In both studies, the geographic boundaries of the pelagic assemblages often overlap and are not as distinct as demersal assemblages. However, the pelagic environment is much more spatially and temporally heterogeneous than the demersal environ- ment, and many of the species examined in our study are highly mobile and are likely to respond quickly to changing conditions. Research is presently underway to examine the trophic interactions among salmonids and with other sym- patric nekton species in order to determine what ecological relationships (e.g. predation, competition), if any, are occur- ring in this system. From the results of our sampling, we concluded that ju- venile salmonids, with the possible exception of steelhead, occupy the cool, high salinity, inshore upwelling regions off the southern Oregon coast. However, particularly for the June cruise, many of the coho and chinook salmon juveniles collected may have recently entered the ocean with little time to disperse offshore, so that the capture location may not reflect true habitat preferences. Moreover, the vertical dimensions of our trawl also precluded us from sampling the nearshore, subtidal regions where some subyearling chinook may reside shortly after entering the ocean. Salmon and steelhead differed considerably in stock com- position. The pattern for coho salmon was similar to that of chinook salmon in that fish from sources both north and south of Cape Blanco contributed to our catches. However, steelhead from rivers north of Cape Blanco were absent, presumably having migrated offshore and north shortly after entering the sea, as shown by Pearcy et al. (1990). Although our stock composition estimates for steelhead should be viewed with caution because of an incomplete ge- netic baseline and a relatively small number of samples, our findings support Pearcy et al.'s suggestion that steelhead from rivers south of Cape Blanco have a unique marine distribution and reside throughout the summer in the up- welling zone off northern California and southern Oregon. Our study revealed seasonal shifts in the abundance and stock composition of juvenile salmonids. Although salmo- nids comprised small portions of the vertebrate catches of both the June and August cruises, juvenile chinook salmon, coho salmon, and steelhead were much more abundant later in the summer, likely indicating that fish moving into our study area are from shoreline or riverine habitats. The greater abundance of chinook salmon in late summer can be explained in part by the northern migration offish that originated in rivers south of our study area. Chinook salmon from the Sacramento and San Joaquin rivers in California's Central Valley comprised substantial propor- tions in the August catches both south (20%) and in nth i 90' i ) of Cape Blanco. In contrast, the few chinook salmon caught in June were mostly (549r ) from streams that en- ter the sea immediately north of Cape Blanco such as the Umpqua, Coquille, Sixes, and Elk rivers. Chinook salmon Brodeur et al.: Distribution, growth, condition, origin, and associations of juvenile salmonids 43 E o •Chinook 1 0 Coho 1 .0 -Habitat 12 14 Water temperature (C) j ■*" 09 - ~~, r. ..---' OR - , * 0 07 - / ■" . .. 06 - r- ' 05 - r' 04 - 03 ■ - - -Chinook 1 0 02 - f J" Coho 1 0 n 1 - 1 V Habitat n - 10 15 Chla concentration 1 09 08 07 06 0.5 04 03 0.2 0 1 0 31.50 J i - - -Chinook 1 0 X ' Coho 1.0 Habitat Y 1 ^ } # > ,' 4 ja _ _ _ J 3250 3300 Salinity (PSU) •Chinook 1 0 -Coho 1.0 -Habitat 100 150 200 Water depth (m) Figure 9 Cumulative distribution curves for salmon and environmental or station variables. Only the August variables that showed at least one significant difference are included. See Table 8 for results of the statistical tests. from these rivers are known to primarily migrate north of our study area along the coast (Nicholas and Hankin, 1988). By August, fish from these stocks were nearly absent from our samples. Oregon rivers south of Cape Blanco, an area that includes the Rogue, Chetco, and Winchuck riv- ers, produce chinook salmon with a more southerly pattern of ocean migration (Nicholas and Hankin, 1988; Myers et al., 1998). Chinook salmon from these rivers were found throughout the summer and contributed 53% to our largest catches of chinook salmon along transects south of Cape Blanco in August. Results from our 2000 GLOBEC cruises identified Cape Blanco as an important breakpoint in salmonid life-his- tory variation. Stock distributions of both juvenile salmon and steelhead indicated that different migration patterns of fish originating from southern and northern rivers are evident during their early marine phase. Our August sur- vey also revealed sharp contrasts in life-history type and freshwater origin between the juvenile chinook salmon population in the marine area north of Cape Blanco and that to the south. Chinook salmon captured north of Cape Blanco were nearly all yearlings and largely from the Sac- ramento River drainage. Subyearlings predominated in our catches south of Cape Blanco and included a much larger proportion offish from coastal streams in southern Oregon and northern California. Comparisons of our results with similar studies conduct- ed further north show differences in salmonid migrations on a somewhat broader geographic scale. In several years of sampling during the summers of 1981 through 1985 off the central Oregon to northern Washington coast, most juvenile chinook salmon bearing CWTs were from Columbia River hatcheries (Pearcy and Fisher, 1990; Fisher and Pearcy, 1995). Only one tagged chinook salmon from a river south of Cape Blanco (Klamath River) was captured. Pearcy and Fisher also found that juvenile coho salmon were largely from the Columbia River and that smaller contributions were from coastal rivers north of Cape Blanco. Their find- ings have been corroborated by more recent surveys in the same region (Emmett and Brodeur, 2000) using genetic 44 Fishery Bulletin 102(1) data (Teel et al., 2003). Samples from subsequent cruises will be used to examine the persistence of such fine- and broad-scale geographic structure in the juvenile migrations of salmonid stocks. A major source of error in our estimates of growth rates of juvenile coho salmon back-calculated from scales was uncertainty of when individual fish entered the ocean. We used a single date of ocean entry for all fish (15 May), but individual fish, of course, entered the ocean at different times over the course of a month or more. Consequently, coefficients of variation were relatively large (84—119% and 75-120% of mean growth rate in FL and weight, respec- tively) for fish caught in May and June, when errors in es- timated growth periods likely were large in relation to the actual growth periods. Conversely, coefficients of variation were relatively small ( 14-30% and 10-26% of growth rate in FL and weight, respectively) for fish caught in August or September, when errors in estimated growth periods likely were small in relation to the actual growth periods. (Note the decrease in standard deviation of mean growth rates with month of capture in Tables 3 and 4A). Growth rates of CWT coho salmon between hatchery release and capture in the ocean (Table 4B) were very similar to the growth rates of unmarked salmon estimated from scales for the same months and areas. In addition, the growth rates of the former group ( CWT coho salmon ) helped to validate the growth rates of the latter group (Table 4A). Significant differences in growth and condition of ju- venile coho salmon indicate that different oceanographic environments exist north and south of Cape Blanco. The length of the fish indicated that substantial growth oc- curred in juvenile coho salmon during the study period. As- sessment of other growth features (condition) revealed that juvenile coho salmon grew better north of Cape Blanco. Because we included measurement of condition in both the June and August period in the evaluation, changes in stock composition, described earlier, may be partly responsible for this observation. Although genetic stock composition was different between months, month of sampling was not a significant factor, suggesting that stock composition is not likely a significant factor affecting the difference in condition (a performance metric) of juvenile salmon north and south of Cape Blanco. Several lines of evidence further support the hypothesis that areas north of Cape Blanco benefit juvenile yearling chinook and coho salmon. There were greater numbers of juvenile yearling chinook and coho salmon to the north of Cape Blanco. Although our overall sampling effort was greater north of Cape Blanco, in the mesoscale portion of our survey designed to assess general distribution patterns, more yearling chinook and coho salmon were captured north of Cape Blanco. Secondly, when we evaluated the growth rate of juvenile coho salmon in the GLOBEC region compared to juveniles captured off northern Oregon and Washington, juveniles from the GLOBEC region grew much better. The similar tracking of resource (distribution and abundance) and performance (measured in terms of either somatic and energetic growth or growth rate) metrics for juvenile yearling chinook salmon and coho salmon ninth of Cape Blanco suggests that habitat quality in this region was better. The results of this study help define the biogeo- graphical zones for salmon growth and establish regional- based management strategies for depleted salmon stocks. Acknowledgments We thank the captain and crew of the FV Sea Eagle for their expert help in conducting the trawling operations under sometimes adverse weather conditions. We are grateful to Jackie Popp-Noskov, Paul Bentley, Marcia House, and Becky Baldwin for assistance in field sampling. Donald Van Doornik and David Kuligowski collected the genetic data. We thank Anne Marshall for the use of unpublished chinook salmon allele frequency data. Stephen Smith and Alex De Robertis helped with the statistical analy- sis. Earlier versions of this manuscript were improved by the helpful comments of two anonymous journal reviewers. Research was conducted as part of the U.S. GLOBEC program and was jointly funded by the National Science Foundation (Grant no. OCE-0002855) and the National Oceanic and Atmospheric Administra- tion (NOAA). We also acknowledge the Bonneville Power Administration for funding the plume study. Literature cited Aebersold, P. B., G. A. Winans, D. J. Teel. G. B. 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Weitkamp, L., and K. Neely. 2002. Coho salmon [Oncorhynchus kisutch I ocean migration patterns: insight from marine coded-wire tag recoveries. Can. J. Fish. Aquat. Sci. 59: 1 100- 1 1 1 5. Weitkamp, L. A., T. C. Wainright, G. J. Bryant, G. B. Milner, D. J. Teel, T G. Kope, and R. S. Waples. 1995. Status review of coho salmon from Washington. Oregon, and California. NOAA Tech. Memo. NMFS- NWFSC-24, 258 p. Appendix Table 1 Summary of releases of coho salmon smolts in 2000 by region. This summary of releases of all hatchery coho salmon smolts by region was calculated from data in the Pacific States Marine Fisheries Commission Regional Mark Information System (http://www.rmis.org/ [accessed 5 April 2003]) and in USFWS 2001 (see Footnote 2 in the general text). No. of release groups ToHl fish Release weight (gl released Marked mean I SD ) All British Columbia 250 13,612,715 71.4', 19.6(5.7) Washington: St. Juan de Fuca, Puget Sound, Skagit River, Nooksack River, etc. 83 15,316,299 86 r, 29.1 (19.7) Washington: North of Columbia River to Cape Flattery 63 7,630,257 76 7', 31.6(5.3) Columbia River 140 29,879,137 89.09i 32.0^ 1 Oregon Coast north of Cape Blanco 14 809,962 95.69! 41.6(7.41 Southern Oregon and Northern California: Rogue, Klamath, and Trinity Rivers 5 745.060 99.8^' 42.1 (4.4) ' 100% of the fish released from Klamath and Trinity Rivers were clipped on the maxillary. 47 Abstract— Between June 1995 and May 1996 seven rookeries in the Gulf of Cali- fornia were visited four times in order to collect scat samples for studying spa- tial and seasonal variability California sea lion prey. The rookeries studied were San Pedro Martir, San Esteban. El Rasito, Los Machos, Los Cantiles. Isla Granito, and Isla Lobos. The 1273 scat samples collected yielded 4995 otoliths (95.3%) and 247 (4.7%) cepha- lopod beaks. Fish were found in 97.4% of scat samples collected, cephalopods in 11.2%, and crustaceans in 12.7%. We identified 92 prey taxa to the species level, 11 to genus level, and 10 to family level, of which the most important were Pacific cutlassfish (Trichiuruslepturus), Pacific sardine (Sardinops caeruleus), plainfin midshipman (Porichthys spp. ), myctophid no. 1, northern anchovy (Engraulis mordax). Pacific mackerel (Scomber- japonicus), anchoveta (Ceten- graulis mysticetus), and jack mackerel (Trachurus symmetricus). Significant differences were found among rooker- ies in the occurrence of all main prey (P<0.04), except for myctophid no. 1 (P>0.05). Temporally, significant dif- ferences were found in the occurrence of Pacific cutlassfish, Pacific sardine, plainfin midshipman, northern an- chovy, and Pacific mackerel (P<0.05). but not in jack mackerel lx2=2.94, df=3, P=0.40 1, myctophid no. l(;r= 1.67, df= 3, P=0.64 ), or lanternfishes ( x2=2.08, df=3, P=0.56). Differences were observed in the diet and in trophic diversity among seasons and rookeries. More evident was the variation in diet in relation to availability of Pacific sardine. Spatial and temporal variation in the diet of the California sea lion (Zalophus californianus) in the Gulf of California, Mexico Francisco J. Garcia-Rodriguez David Aurioles-Gamboa Centra Interdisciplinary de Ciencias Mannas-lnstituto Politecnico Nacional Departamento de Biologia Manna y Pesquerias Apdo. Postal 592 La Paz, Ba|a California Sur, Mexico E-mail address (for F J. Garcia-Rodriguez) fjgrodriifflcibnor.mx Manuscript approved for publication 9 October 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull. 102:47-62 (2004). The population of the California sea lion (Zalophus californianus), in the Gulf of California numbers approxi- mately 23,000 individuals, 82% of which inhabit the northern region of the gulf above latitude 28° (Aurioles- Gamboa and Zavala-Gonzalez, 1994). In this region are found the most important reproductive areas and the highest pup production of the Gulf. Aurioles-Gamboa and Zavala-Gonzalez (1994) suggested that the high con- centration of animals in this region is related to high abundance of pelagic fish such as Pacific sardine (Sardinops caeruleus) (also known as South Ameri- can pilchard, FAO), Pacific mackerel (Scomber japonicus). Pacific thread herring (Opisthonema libertate), and anchoveta (Cetengraulis mysticetus) (Cisneros-Mata et al., 19871; Cisneros- Mata et al., 19912; Cisneros-Mata et al., 19973). Despite the importance of the north- ern gulf region, feeding studies of the California sea lion at Gulf of California rookeries have been few and have been conducted at different time periods. Researchers have studied sea lion diet in Los Islotes (Aurioles-Gamboa et al., 1984; Garcia-Rodriguez, 1995), Los Cantiles (Isla Angel de la Guarda), Isla Granito (Sanchez-Arias, 1992; Bautista- Vega, 2000), and Isla Racito (Orta-Davi- la, 1988). These studies have shown that sea lions consume a variety of prey and that differences exist between the diet of sea lions found at different rookeries within the Gulf of California. At Los Islotes, the most important prey were cusk eel (Aulopus bajacali), bigeye bass (Pronotogrammus eos), threadfin bass (Pronotogrammus multifasciatus), and splitail bass (Hemanthias sp.) (Aurioles- Gamboa et al, 1984; Garcia-Rodriguez. 1995). At Los Cantiles and Isla Granito important prey were lanternfish (Dia- phus sp.), northern anchovy (Engraulis mordax). Pacific cutlassfish (Trichiurus nitens), shoulderspot (Caelorinchus scaphopsis), and Pacific whiting (Mer- luccius productus) (Sanchez-Arias, 1992; Bautista-Vega, 2000), whereas at Isla Racito, important prey were Pacific sardine (Sardinops caeruleus). Pacific mackerel (Scomber japonicus), grunt (Haemulopsis spp.), rockfish (Sebastes 1 Cisneros-Mata, M. A.. J. P. Santos-Molina, J. A. DeAnda M.,A. Sanchez-Palafox, and J. J. Estrada. 1987. Pesqueria de sardina en el noroeste de Mexico ( 1985/86 ). Informe Tecnico, 79 p. Centro Regional de Inves- tigaciones Pesqueras de Guaymas. INP. SEPESCA. Calle 20 No. 605 Sur Col. La Cantera. Guaymas, Son. CP. 85400. 2 Cisneros-Mata, M. A., M. O. Nevarez- Martinez, G. Montemayor-Lopez, J. P. Santos-Molina, and R. Morales- Azpeitia. 1991. Pesqueria de sardina en el Golfo de California de 1988/89-1989/90. Informe Tecnico. 80 p. Centro Regional de Investigaciones Pesqueras de Guaymas. INP. SEPESCA. Calle 20 No. 605 Sur Col. La Cantera. Guaymas, Son. CP. 85400. 3 Cisneros-Mata, M. A., M. O. Nevarez- Martinez, M. A. Martinez-Zavala, M. L. Anguiano-Carranza, J. P. Santos-Molina, A. R. Godinez-Cota, and G. Montemayor- Lopez. 1997. Diagnosis de la pesqueria de pelagicos menores del Golfo de Califor- nia de 1991/92 a 1995/96. Informe Tecnico, 59 p. Centro Regional de Investigaciones Pesqueras de Guaymas. INP. SEMARNAP. Calle 20 No. 605 Sur Col. La Cantera. Guavmas, Son. CP. 85400. 48 Fishery Bulletin 102(1) spp. ), and Pacific whiting (Merluccius spp. ) (Orta-Davila, 1988). Some California sea lion prey are important fisheries resources in Mexico. The Pacific sar- dine, for example, is the target of a fishery be- gun in 1967 and which, along with the northern anchovy, contributed to most of the volume of the catch (200,870 t during the 1995-96 season) obtained in the Gulf (Cisneros-Mata et al.3). The central and northern regions of the Gulf of California harbor the greatest abundance of sea lions and schooling fishes, such as the sar- dine and anchovy. Because of this, knowledge of sea lion feeding habits and their temporal and spatial variability is relevant to understanding the potential interaction between sea lions and fisheries in the area (Orta-Davila, 1988; San- chez-Arias, 1992; Bautista-Vega, 2000). In this article, we present the results of concurrent diet studies conducted at various rookeries and haulout areas of sea lions in the northern rookeries of the Gulf of California to examine the prey consumed, and spatial and temporal variability in their diet. Materials and methods 32° 28° 24° 20° 16° 12° Scat samples of California sea lions were collected at Isla San Pedro Martir (SPM, 28°24'00"N, 112°25'3"W), Isla San Esteban (EST, 28°42'00"N, 112°36'00"W), Isla Rasito (RAS, 28°49'30"N, 112°59'30"W), Isla Granito (GRA, 29°34'30"N, 113°32'15"W), Isla Lobos (LOB, 30°02'30"N, 114°. 28'30"W), and at two colonies of Isla Angel de la Guarda known as Los Machos (MAC, 29°20'00"N, 113°30'00"W), and Los Cantiles (CAN, 29°32'00"N, 113°29'00"W, Fig. 1). The total number of California sea lions in these seven rookeries was approximately 15,000 animals (that were hauled out) of which about 12.2% inhabit San Pedro Martir. 34.7% San Esteban, 2.8% El Rasito, 10.0% Los Machos, 8.7%. Los Cantiles, 11.0% Isla Granito, and 20.6% Isla Lobos (Aurioles-Gamboa and Zavala-Gonzalez, 1994). All the animals were spread out along the shoreline of each island, except at Isla Angel de la Guarda, where they were clustered within two areas: Los Cantiles, on the eastern shoreline and Los Machos on the western shoreline. Scat samples were obtained at reproductive and non- reproductive haulout areas between June 1995 and May 1996. At El Rasito, sampling was done only at one reproduc- tive area; fresh and dried samples were collected (Fig. 2). If for any reason a scat was not collected (because it was found in pieces or in poor condition), it was destroyed and the site was cleared to avoid collection during subsequent trips. All fresh and dried samples collected were pooled to represent each sampling period. We assumed that the diet information corresponded to a time period close to the col- lection trip, but some dried scats could have been deposited shortly after the last collection. Pacific Ocean 122° 118° 114° 110° 106° Figure 1 Map of Baja California showing location of California sea lion rook- eries that were studied in the Gulf of California. Scats were stored in plastic bottles and then dried shortly thereafter to prevent decomposition offish otoliths and other hard parts (which were used in subsequent prey identification) until the scats could be processed at a later date. The samples were processed by soaking in a weak biodegradable detergent solution for 1 to 7 days before being sifted through nested sieves of 2. 00-, 1.18-. and 0.5-mm mesh size. Fish bones and scales, eye lenses of fish and squid, otoliths, cephalopod beaks, and crustacean fragments were extracted from the samples. Cephalopod beaks were stored in 70% ethanol, and the other items were dried and stored in vials. Sagittal otoliths and cephalopod beaks were used to identify teleost fish and cephalopods, re- spectively. Identifications were made by using photographs and diagrams from Clarke (1962), Fitch ( 1966), Fitch and Brownell (1968), and Wolff (1984), as well as voucher specimen material from the 1) Center Interdiseiplinario de Marinas Ciencias (CICIMAR), 2) Instituto Tecnologico y de Estudios Superiores de Monterrey, Guaymas, 3) Los Angeles County Museum of Natural History, California, and 4) Centro de Investigacion Cientifica y de Educacion Superior de Ensenada (CICESE). Baja California, Mexico. Prey species identifed to family level were coded by using Garcia-Rodriquez and Aurioles-Gamboa: Spatial and temporal variation in the diet of Zalophus californianus 49 San Pedro Martir (SPM) 28° 24'- HA San Esteban (EST) 112°40' 112=38' 1 12=36' 112=34' 112=32' J L 28=44- 28=42' El Rasito (RAS) Angel de la Guarda 28=49' 113=40' 113=30' 113=20' 113=10' 113=00' I I i i i 29=30'- r^\ «— Los Cantiles (CAN) / (RAyHA) 29° 20'- A\ ^-. 29° 10- Los Machos (MAC)\ ^ (RA y HA) ^v \ M Isla Granito (GRA) Isla Lobos (LOB) 113' 34' 113° 33' i 29° 35'- s RA RA ha *\y 29=34'- 30=03'. 114=29' 1 114= 28 I | RA K HA - Figure 2 Location of sites where samples of California sea lion scats were collected at each island. RA = reproductive area; HA = haulout area. the family name plus a sequential number. Otoliths from prey species that were not identified to species, genus, or family level were coded with "fish species" plus a number. Three indices were used to describe the diet of sea lions. Percent number (PN) represents the percentage of the number of individuals for each prey taxon over the total number of individuals found in all scat samples. Percent of occurrence (PO) represents the percentage of scats hav- ing a given prey taxon and indicates the percentage of the population that is consuming a particular prey species. The third index, index of importance (IIMP) incorporates PN and PO and is defined as IIMP, 'T ^ u X (1) where xt = number of individuals of taxon z' in scatj; X = total number of individuals from all taxa found in scat J; and U = total number of samples with prey. The IIMP, developed for scat analysis (Garcfa-Rodriguez, 1999), was used to determine the importance of prey species, their spatial and temporal variation in the diet. 50 Fishery Bulletin 102(1) diversity of prey estimates, and measures of similarity among rookeries. Crustaceans were not incorporated into the IIMP index because it was not possible to quantify the number of individuals in the samples. We used the IIMP Index because it is less sensitive to changes in the number of prey in an individual scat com- pared to PN. For example, if a scat contains a single prey taxon, the IIMP does not change regardless of the number of individuals of that taxon, in that scat. However, as one increases the number of individuals of a given prey taxon in the scat, the PN will also increase for that prey. The IIMP allows each scat to contribute an equal amount of information, whereas PN can be dominated by a few scats with a large number of a single prey-taxon otoliths. In this manner the IIMP is similar to the split-sample frequency of ocurrence (SSFO) index, developed by Olesiuk (1993), where each scat is treated as a sampling unit and does not assume, as does PN, that the distribution of prey hard parts between scats is uniform. However, with the SSFO index, each prey taxon in a given scat is given an equal weight for that scat. If only one species occurs in a sample, its occurrence is scored as 1, if two species occur, each oc- currence is scored as 0.5, and so forth (Olesiuk, 1993). The IIMP index incorporates more information than the SSFO index, regardless of the number of individuals of each taxon in the scat.4 Percent number (PN) was used only to show differences between broad prey groups (fishes and cephalopods) and PO was used to review the temporal and spatial changes from each main prey (those with average IIMP of at least 10% at any rookery). For all estimations, a single otolith (right or left) or single cephalopod beak (upper or lower) represented one individual prey. We tested the hypothesis that the occurrence of the main prey was independent of the rookery and of the date collection using contingency tables and an estimator of chi-square (x~) (Cortes, 1997). Total length of the otoliths (mm) and the linear equation obtained by Alvarado-Castillo5 were used to estimate the length of the Pacific sardine (total length mm=7. 41+147. 24xotolith length mm); r=0.85, n=2740). Insufficient data did not allow estimating the size of speci- mens from May. All estimated lengths were transformed us- ing loglO, followed by an ANOVA among sampling periods. The size of Pacific sardine consumed by California sea lion was compared to those caught in the commercial fishery. We chose to estimate the size of Pacific sardines because of the broad information available concerning age and growth and other aspects about the fishery and because we found many sardine otoliths in good condition. Spatial and temporal correlation in composition of diet was compared by using the Spearman rank correlation co- 4 Garcfa-Rodriguez, F. J., and J. De la Cruz-Agiiero. In prep. An index to measure the specie prey importance of California sea lion ^Zalophus californianus) based on scat samples. 'Alvarado-Castillo, R. Unpubl. data. Departamento de Biologia y Pesquerias, Centro Interdisciplinary de Ciencias Marinas. Avenida IPN S/N Col. Palo Playa de Santa Rita, La Paz, Baja California Sur, Mexico 23070. efficient (Rs) (Fritz. 1974). Pairs of IIMP values were used for each prey taxon in a pair of sampling events (rookeries or sampling dates) to examine the correlation among them. This analysis was limited to prey that had an IIMP value of 10% or more. Cluster analysis of average IIMP data for the seven rookeries was used to assess the similarity of the diet among rookeries. The dendrogram for the cluster analysis was based on relative Euclidean distances and unweighted pair-grouping methods (UPGMA) strategy (Ludwig and Reynolds, 1988). We included only prey that, for at least one occasion, had IIMP values >10%. Trophic diversity was evaluated by using diversity curves (Hurtubia, 1973) developed from IIMP index data. For each date and colony, the cumulative diversity was calculated for increasing numbers of sequentially numbered (but we as- sumed randomly deposited and collected) scat samples. The diversity curves also allowed us to evaluate sample size (Hurtubia, 1973; Hoffman. 1978; Magurran, 1988, Cortes, 1997) by identifying the point at which the curve flattens. The diversity was estimated by using the Shannon index: H' -^P,\nPr (2) where H' = trophic diversity; S = total number of prey taxa; and Pl = IIMPr and represents the relative abundance of taxon i obtained from each scat and pooled from scat 1 up to the total number of scats collected. The values of trophic diversity were then plotted against the number of pooled scats. Results Identification of prey The 1273 scat samples collected during June 1995 through May 1996 (Table 1) yielded fish remains in 97.4% of the samples, cephalopod remains in 11.2%, and crustacean remains in 12.7%. Fish and cephalopods represented 95.39; and 4.7%, respectively, of the 5242 individual prey (excluding crustaceans). The occurrence and number of these prey groups changed temporally and spatially (Fig. 3). We identified 92 prey taxa to the species level, 11 to the genus level, and 10 to family level from 851 scats (Table 2). Remaining scats had damaged prey structures in a condition that prevented us from identifying species to the genus or family level. We found nine main prey with IIMP average values a 10% (Table 3): the Pacific cutlassfish {THchiurus lepturus), the Pacific sardine (Sardinops eaeruleus), the plainfin mid- shipman (Porichthys spp.), myctophid no. 1, the northern anchovy iEngraulis mordax), Pacific mackerel {Scomber japonicus), the anchoveta (Cetengraulis mysticetus), jack mackerel iTrachurus symmetricus), and the lanternfish (unid. myctophid). Garcia-Rodriquez and Aurioles-Gamboa: Spatial and temporal variation in the diet of Zalophus californianus 51 Table 1 Number of scats collected at each rookery for each sampling period. June 1995 San Pedro Martir (SPM) SanEsteban(EST) ElRasito(RAS) Los Cantiles (CAN) IslaGranito(GRAl Los Machos (MAC) IslaLobos(LOB) Total 22 50 11 20 24 39 72 238 September 1995 January 1996 33 66 56 58 20 32 139 404 91 58 47 41 36 72 433 Mav 1996 29 67 25 35 19 0 23 198 Total 172 274 150 160 104 107 306 1273 Spatial and temporal variability of the main prey The importance (IIMP) of the Pacific cutlassfish was greater in Los Cantiles (28.4%), Isla Lobos (20.8%), and Isla Granito (48.5%) than at other sites (Fig. 4). The Pacific sardine was the dominant prey at Los Machos and to the south. There was a significant correlation across the sea- sons between Los Machos and El Rasito (r=0.998. P=0.04), but not between Los Machos and Isla Granito U-0.602, P=0.59). The IIMP of sardine was also correlated between El Rasito and San Esteban (r=0.95, P=0.04). The plainfin midshipman did not show a clear pattern, but its presence in the diet increased northeastward from Isla Angel de la Guarda. Lanternfishes, especially myctophid no. 1, were the dominant prey at San Pedro Martir, San Esteban, and El Rasito. The presence of Pacific mackerel was positively correlated with the presence of the Pacific sardine. The anchoveta was only found at Isla Lobos, and jack mackerel at El Rasito, San Pedro Martir, and Isla Granito. The changes in the PO of the main prey coincided with the variations of the IIMP. The occurrence of Pacific cut- lassfish. Pacific sardine, plainfin midshipman, northern anchovy, Pacific mackerel, and jack mackerel was signifi- cantly different (P<0.04) among rookeries. Myctophid no. 1 showed no significant difference in ocurrence 10% (Table 3) for a given collection. The Spearman rank correlation coefficient of IIMP between any pair of rookeries during June, September, January, and May was not significant (P>0.08). There was no positive correla- tion among any pair of sampling periods for any rookery (P>0.14), except between January and May at San Pedro Martir (Ps=0.64, P<0.05) and El Rasito (Ps=0.66, P<0.05) and between January and June as well as between Janu- ary and May at Isla Lobos (Rs=0.56, P=0.05; and Ps=0.59, P=0.05. respectively). The similarity in diet was related to the distance between rookeries. A clustering for the seven rookeries was obtained from these 25 prey taxa (Fig 6). We arbitrarily used a "cut- off" distance of 0.3 and 0.4 on the dendrogram as reference points for identifying clusters. The group obtained by us- ing the first distance (0.3) showed four feeding areas: one located in the south ( area I; San Pedro Martir, San Esteban, and El Rasito), another in Canal de Ballenas (area II: Los Machos) and two in the north (area III: Los Cantiles and Isla Lobos; and area IV: Isla Granito). When the second distance (0.4) was used, the seven rookeries grouped into two clusters: 1) the southern region and Canal de Ballenas, and 2) the region north of Angel de la Guarda. Spatial and temporal variability in trophic diversity Temporal variability in trophic diversity was evident between the rookeries (Fig. 7). In general, two patterns could be differentiated: one in which the diversity varied little throughout the year and the other in which diversity was high in January and low in September. The rookeries San Pedro Martir and Isla Lobos showed the first pattern and Los Machos and Isla Granito (and to a lesser extent, San Esteban and El Rasito) showed the second pattern. In September, when diversity was low, the dominant prey at 52 Fishery Bulletin 102(1) 100 80 60 40 20 0 100 T 80 60 40 20 0.- Percent number D Fishes ■ Cephalopods JUNE 1995- MAY 1996 SPM EST RAS MAC CAN GRA LOB □ Fishes ■ Cephalopods JUNE 1995 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 ■ 20 0 SPM EST RAS MAC CAN GRA LOB ■ Fishes ■ Cephalopods SEPTEMBER 1995 SPM EST RAS MAC CAN GRA LOB □ Fishes ■ Cephalopods JANUARY 1996 SPM EST RAS MAC CAN GRA LOB □ Fishes ■ Cephalopods MAY 1996 SPM EST RAS MAC CAN GRA LOB 100 80 1 60 40 1 20 0 100' 80 60 40' 20' 0. Percent occurrence Q Fishes ■ Cephalopods □ Crustaceans JUNE 1995- MAY 1996 M XI Jl SPM EST RAS MAC CAN GRA LOB □ Fishes ■ Cephalopods □ Crustaceans JUNE 1995 n n ^3*. SPM EST RAS MAC CAN GRA LOB □ Fishes ■ Cephalopods D Crustaceans SEPTEMBER 1995 n Jl n 100, 80 60 1 40 1 20 0 SPM EST RAS MAC CAN GRA LOB O Fishes H Cephalopods D Crustaceans JANUARY 1996 SPM EST RAS MAC CAN GRA LOB D Fishes I Cephalopods D Crustaceans MAY 1996 n^Q SPM EST RAS MAC CAN GRA LOB Figure 3 Percent number (PNi and percent occurrence (POl index values for fishes, cephalopods, and crustaceans found in samples of California sea lion scats collected at seven rookeries in the Gulf of California, Mexico, for all sampling periods combined and for each sampling period. San Esteban, El Rasito, and Los Machos was Pacific sar- dine, whereas at Isla Granito, it was Pacific cutlassfish (Fig. 4 1. The curves obtained for Los Cantiles showed a clear pattern of diversity only in September, although the trend in the January curve would suggest a higher diver- sity in January than in September. Garcia-Rodriquez and Aurioles-Gamboa: Spatial and temporal variation in the diet of Zalophus califomianus 53 Table 2 Prey of California sea lion identified from scat samples collected at Isla San Pedro Martir, Isla San Esteban, Isla El Rasito, Los Cantiles, Isla Granito, Los Machos and Isla Lobos from June 1995 through May 1996. n ind. = = number of individuals in the sample; PN = percent number; n occurr = number of occurrences PO = percentage of occurrence; IIMP = index of importance. Scientific name Common name n Ind. PN n Occurr. PO IIMP Trichiurus lepturus Pacific cutlassfish 306 5.837 128 15.041 16.392 Sardinops caeruleus Pacific sardine 358 6.829 88 10.341 10.020 Porichthys spp. midshipman 456 8.699 95 11.163 9.297 Myctophid no. 1 lanternfish 714 13.621 119 13.984 7.901 Engraulis mordax northern anchovy 430 8.203 43 5.053 5.199 Scomber japonicus Pacific mackerel 103 1.965 42 4.935 3.403 Cetengraulis mysticetus anchoveta 410 7.821 15 1.763 2.404 Loliolopsis diomedeae squid 77 1.469 35 4.113 2.399 Trachurus symmetricus jack mackerel 111 2.118 41 4.818 2.273 Merluccius spp. Pacific whiting 55 1.049 25 2.938 2.206 Pontinus spp. scorpionfish 61 1.164 26 3.055 1.842 Enoploteuthid no. 1 squid 95 1.812 23 2.703 1.754 Caelorinchus scaphopsis shoulderspot 65 1.240 25 2.938 1.728 Octopus sp. no. 1 octopus 24 0.458 17 1.998 1.614 Sebastes macdonaldi Mexican rockfish 42 0.801 18 2.115 1.496 Citharichthys sp no. 1 sanddab 120 2.289 23 2.703 1.220 Fish species no. 1 — 49 0.935 25 2.938 1.153 Haemulopsis leuciscus white grunt 176 3.357 21 2.468 1.093 Peprilus snyderi salema butterfish 163 3.110 33 3.878 1.025 Prionotus spp. searobin 12 0.229 9 1.058 0.855 Prionotus stephanophrys lumptail searobin 53 1.011 14 1.645 0.814 Argentina sialis Pacific argentine 19 0.362 13 1.528 0.754 Fish species no. 2 — 55 1.049 27 3.173 0.737 Hemanthias peruanus splittail bass 60 1.145 22 2.585 0.602 Fish species no. 3 — 9 0.172 6 0.705 0.592 Micropogomas ectenes slender croaker 13 0.248 9 1.058 0.547 Lepophidium spp. cusk-eel 9 0.172 3 0.353 0.532 Fish species no. 4 — 10 0.191 3 0.353 0.511 Sebastes exsul buccanner rockfish 15 0.286 10 1.175 0.505 Cranchiid no. 2 Squid 20 0.382 12 1.410 0.501 Haemulon flaviguttatum yellowspotted grunt 11 0.210 3 0.353 0.468 Sela r cru men oph th aim us bigeye scad 24 0.458 12 1.410 0.431 Fish species no. 5 — 33 0.630 19 2.233 0.384 Paralabrax sp. no. 1 sea bass 9 0.172 5 0.588 0.373 Synodus sp. no. 3 lizardfish 10 0.191 3 0.353 0.341 Lepophidium prorates prowspine cusk-eel 5 0.095 4 0.470 0.335 Fish species no. 6 — 9 0.172 5 0.588 0.324 Synodus sp. no. 1 lizardfish 25 0.477 10 1.175 0.324 Octopus sp, no. 2 octopus 8 0.153 7 0.823 0.308 Gonatus berryi squid 5 0.095 5 0.588 0.274 Mugil cephalus striped mullet 1 0.019 1 0.118 0.265 Paranthias colonus Pacific creole-fish 1 0.019 1 0.118 0.265 Batistes polylepis finescale triggerfish 13 0.248 4 0.470 0.245 Physiculus nematopus charcoal mora 30 0.572 12 1.410 0.244 Hemanthias spp. sea bass 9 0.172 6 0.705 0.234 Fish species no. 7 — 10 0.191 8 0.940 0.233 Uroconger varidens conger eel 8 0.153 5 0.588 0.189 Larimus spp. drum 8 0.153 6 0.705 0.174 Apogon retrosella barspot cardinalfish 5 0.095 4 0.470 0.173 Dosidicus gigas squid 8 0.153 5 0.588 0.171 continued 54 Fishery Bulletin 102(1) Table 2 (continued) Scientific name Common name n Ind. PN n Occurr. PO IIMP Merluccius productus Pacific whiting 1 0.019 1 0.118 0.167 Fish species no. 8 — 2 0.038 2 0.235 0.159 Synodus sp. no. 2 lizardfish 12 0.229 5 0.588 0.132 Scorpaena sonorae Sonora scorpionfish 2 0.038 1 0.118 0.130 Eucinostomus spp. mojarra 13 0.248 5 0.588 0.129 Fish species no. 9 — 3 0.057 3 0.353 0.127 Cynoscion reticulatus striped weakfish 23 0.439 7 0.823 0.124 Fish species no. 10 — 10 0.191 1 0.118 0.122 Caulolatilus affinis bighead tilefish 4 0.076 3 0.353 0.114 Paralabrax auroguttatus goldspotted sand bass 18 0.343 4 0.470 0.110 Fish species no. 11 — 3 0.057 2 0.235 0.102 Cranchiid no. 5 squid 1 0.019 1 0.118 0.097 Bodianus diplotaenia mexican hogfish 1 0.019 1 0.118 0.087 Prionotus sp. no. 1 searonbin 2 0.038 2 0.235 0.087 Strongylura exilis California needlefish 1 0.019 1 0.118 0.083 Synodus spp. lizardfish 6 0114 5 0.588 0.146 Fish species no. 12 — 3 0.057 3 0.353 0.074 Fish species no. 13 — 2 0.038 1 0.118 0.065 Fish species no. 14 — 3 0.057 1 0.118 0.060 Fish species no. 15 — 2 0.038 1 0.118 0.058 Fish species no. 16 2 0.038 2 0.235 0.056 Porichthys sp. 1 midshipman 1 0.019 1 0.118 0.052 Fish species no. 17 — 5 0.095 3 0.353 0.049 Calamus brachysomus Pacific porgy 5 0.095 2 0.235 0.043 Fish species no. 18 — 1 0.019 1 0.118 0.042 Fish species no. 19 — 5 0.095 2 0.235 0.041 Ophididae no. 1 — 1 0.019 1 0.118 0.040 Fish species no. 20 — 5 0.095 3 0.353 0.039 Sebastes sinesis blackmouth rockfish 2 0.038 1 0.118 0.039 Symphurus spp. tonguefish 3 0.057 1 0.118 0.038 Fish species no. 21 — 2 0.038 1 0.118 0.036 Pronotogrammus multifasciatus threadfin bass 8 0.153 2 0.235 0.029 Fish species no. 22 — 2 0.038 2 0.235 0.027 Fish species no. 23 — 2 0.038 1 0.118 0.021 Orthopristis reddingi Bronze-striped grunt 16 0.305 1 0.118 0.020 Fish species no. 24 — 2 0.038 1 0.118 0.020 Fish species no. 25 — 1 0.019 1 0.118 0.016 Cranchiidae no. 4 squid 2 0.038 2 0.235 0.014 Fish species no. 26 — 2 0.038 2 0.235 0.014 Histioteuthis heteropsis squid 0.019 1 0.118 0.014 Scorpaenidae no. 1 — 0.019 1 0.118 0.011 Fish species no. 27 — 0.057 2 0.235 0.011 Fish species no. 28 — 0.019 1 0.118 0.010 Fish species no. 29 — 0.019 1 0.118 0.008 Cranchiidae no. 3 squid 0.019 1 0.118 0.006 Bollmannia spp. goby 0.019 1 0.118 0.006 Fish species no. 30 — 0.019 1 0.118 0.005 Cranchiidae no. 1 squid 0.019 1 0.118 0.004 Paralabrax maculatofasciatus spotted sand bass 0.019 1 0.118 0.003 Ophidian scrippsae basketweave cusk-eel 0.019 1 0.118 0.003 Physiculus spp. cod. codling, mora 2 0.038 1 0.118 0.003 Ophididae no. 2 — 4 0.076 1 0.118 0.002 Unid. Carangidae jacks 8 0.153 3 0.353 0.141 continued Garcia-Rodriquez and Aurioles-Gamboa: Spatial and temporal variation in the diet of Za/ophus californianus 55 Table 2 (continued) Scientific name Common name n Ind. PN n Occurr. PO IIMP Unid. Engraulidae anchovies 1 0.019 1 0.118 0.248 Unid. Haemulidae grunts 13 0.248 11 1.293 0.509 Unid. Labridae wrasses 1 0.019 1 0.118 0.005 Unid. Mycthophidae lanternifishes 216 4.121 71 8.343 4.895 Unid. Ophididae cusk-eel 2 0.038 1 0.118 0.098 Unid. Scianidae drums 13 0.248 9 1.058 0.643 Unid. Scorpaenidae scorpionfishes 30 0.572 18 2.115 1.078 Unid. Serranidae sea bass 13 0.248 6 0.705 0.176 Unid. Triglidae searobins 1 0.019 1 0.118 0.002 Unid. fishes 39 0.744 16 1.880 1.819 Unid. cephalopod 4 0.076 4 0.470 0.373 Unid. fishes (very eroded ) 381 7.268 231 27.145 Remains of cephalopods 14 1.645 Remains of crustaceans 162 19.036 Discussion Stomach acids attack otoliths, affecting their size and number and consequently the estimate of prey occurrence and importance. Erosion of otoliths during digestion has been analyzed in studies of pinnipeds in captivity. Bowen (2000) reviewed nine studies that estimated the propor- tion of otoliths recovered in scat samples to obtain a prey-number correction factor (NCF). He found that NCF is greater than 1.0 because many prey species are not recovered in the scat samples. Additionally, the erosion level can be significantly different among prey species (Bowen, 2000) because of differences in the shape and microstructure of otoliths. Therefore, estimates of biomass based on scat analysis should be carefully interpreted because the consumption of some prey species can be under- or overestimated. Correction factors are needed to compensate for differential erosion for the prey species of each pinniped. In this study the most important prey of California sea lions were pelagic fish with small, thin, and fragile otoliths (Nolf, 1993). The lanternfish also have small otoliths — perhaps smaller than those of any other prey taxa found in the scats. Their true importance in California sea lion feeding may be underestimated because of erosion caused by stomach acids (Da Silva and Neilson, 1985; Murie and Lavigne, 1985; Jobling and Breiby, 1986; Jobling, 1987; Toll- it et al., 1997). Similarly, the presence of cephalopods may have been underestimated because their jaws are composed of chitin, which is harder to digest, and frequently are re- gurgitated (Pitcher, 1980; Hawes, 1983). However, the high resistence to digestion of cephalopod beaks allows recovery of them in good shape. Thus they are a good choice to use in such diet analyses (Lowry and Carretta, 1999). A numerical index of prey species importance may over- or underestimate the dominance of prey species in the diet because it does not consider the weight of the prey. For IIMP, a numerical index that assumes a similar weight for all prey species, the true importance of the individual large prey in the diet can be underestimated and the importance of individual small prey can be overestimated. This prob- lem is also present when the PO, PN, and the SSFO index are used because these are all based only upon the number and occurrence of otoliths and cephalopods beaks. As when using PN. and the SSFO, the IIMP does not work for species that cannot be enumerated, such as crustaceans. Given the tendencies of the trophic diversity curves, the sample size was suitable in almost all cases. However, at San Pedro Martir a few more samples would have been useful to fully depict the diet. At Los Cantiles, except during September 1995, the samplings should have been more intense because the flattened portion of the diversity curves are not clear. The information, therefore, that comes from those samples could be biased. However, the number of scats that we analyzed contained a high percentage of the consumed species, especially the main prey. The results of this study indicate that the California sea lion consumed mainly fish and some crustaceans and cephalopods. According to the PN index, fish were more important than cephalopods in the diet of sea lions. In ad- dition, fish were more frequent (PO) than crustacean and cephalopods. Crustaceans were represented in a similar manner in scats from all rookeries. Cephalopods, however, were more important at San Pedro Martir and San Esteban, prob- ably because they are more common towards the southern gulf. Species of the suborder Oegopsida, which includes oceanic species (Roper and Young, 1975), were most com- monly found in scats from these rookeries. Orta-Davila (1988) and Sanchez-Arias (1992) have also noted the low consumption of cephalopods at the northern rookeries. Fish were the most diverse and commonly eaten prey. In contrast to cephalopods, fish were slightly less important in the southern region. The availability and abundance of the various prey resources influenced the diet of the sea lions in the Gulf 56 Fishery Bulletin 102(1) Table 3 Prey of California sea lions having IIMP index values alO^ in at leas t one sampling period for seven rookeries in the Gulf of Cali- fornia, Mexico Blank indicate that species were not recorded in diet; ' — " means unavailable data. Prey species June 1995 September 1995 January 1996 May 1996 Average San Pedro Engraulis mordax 29.7 2.1 0.5 8.1 Marti r myctophid no. 1 29.0 10.5 9.0 20.5 17.3 Porichthys spp. 11.2 2.0 6.8 15.5 8.9 Prionotus stephanophrys 0.6 3.3 3.3 10.9 4.5 enopleoteuthid no.l 27.3 0.8 7.0 Sebastes macdonaldi 10.4 2.6 Haeumulopsis leuciscus 16.7 6.0 5.7 San Esteban Trichiurus lepturus 24.9 3.4 3.0 7.8 Sardinops caeruleus 10.0 34.1 4.2 12.1 unid. Myctophidae 13.79 3.4 4.3 10.9 8.1 myctophid no. 1 2.8 11.8 8.9 18.8 10.6 enopleoteuthid no. 1 16.9 4.2 Sebastes macdonaldi 2.1 9.7 1.4 3.3 fish species no. 1 1.7 11.0 3.2 El Rasito Porichthys spp. 26.2 4.0 2.3 8.1 unid. Myctophidae 16.4 1.5 8.1 16.4 10.6 Scomber japonicus 13.8 3.2 3.7 2.5 5.8 Pontinus spp. 11.5 5.1 4.1 10.9 7.9 Octopus sp. no. 1 11.5 2.9 7.7 5.5 myctophid no. 1 6.6 5.1 21.4 6.8 10.0 Sardinops caeruleus 1.6 40.1 0.9 7.3 12.5 Trachurus symmetricus 22.0 5.0 23.4 12.6 Caelorinchus scaphopsis 3.6 13.5 10.5 6.9 Los Machos Sardinops caeruleus 21.0 64.1 16.8 — 34.0 Scomber japonicus 19.0 10.9 — 10.0 Merluccius spp. 15.4 8.2 — 7.9 Trichiurus lepturus 11.7 5.4 — 5.7 Sebastes macdonaldi 1.8 11.3 — 4.4 Los Cantiles Porichthys spp. 66.7 15.5 20.6 Trichiurus lepturus 22.2 38.2 53.1 28.4 Engraulis mordax 3.7 0.4 14.3 4.6 myctophid no. 1 17.6 4.8 5.6 Sardinops caeruleus 6.8 19.0 6.5 fish species no. 3 0.9 14.3 3.8 unid. fishes 0.9 19.0 5.0 unid. Scianidae 14.3 3.6 Lepophidium spp. 14. 3.5 Lo/iolopsis diomedcav 21.1 5.3 Isla Granito Engraulis mordax 49.3 7.8 14.3 Trichiurus lepturus 22.0 70.1 2.0 100.0 48.5 unid. myctophidae 1.7 1.1 12.6 3.9 Sardinops caeruleus 0.9 18.7 4.9 Porichthys spp. 0.5 18.2 4.6 5.8 Citharichthys sp. no. 1 21.7 5.4 Isla Lobos Cetengraulis mysticetus 32.7 0.1 6.8 27.8 16.9 Trichiurus lepturus 25.2 27.7 15.8 14.3 20.8 Porichthys spp. 9.0 10.3 23.2 35.5 19.5 Loliolopsis diomedeae 4.9 2.2 11.6 3.5 5.6 Peprilus snyderi 23.5 5.2 7.2 Garcia-Rodriquez and Aurioles-Gamboa: Spatial and temporal variation in the diet of Zalophus califomianus 57 100 80 60 SPM EST RAS MAC CAN GRA LOB 11111 Ml H l l i 1 ^_ ^B Trichiurus lepturus 20 « hJI L^l h^^^m. I a 5 fe'i a i fell a i fell Si ? fell & i fell & 5 fe'i & i | n 10 * SI 3 $ t SH to n Sl=5 to t Sin (0 t Sin to n 5 1 => to -> S 100 80 1 1 1 1 1 1 40 20 \_m i ■ J - _« : _ i Sardinops caeruleus ■7 Q. ;r >- 1 Z Q_ Z >- 1 2 Q. z >- 1 z 0- Z >-'Z 0- Z >;! Z 0- Z >r'Z 0- z ^ 3 W * S't W 3 5 ' => co =5 5 ' =^ w> ^ 5I=3 « ^ 5l=i W ^ 5' =5 w -> 5 100, 80 1 I 1 1 1 1 1 I 1 1 I 1 60 40 I ! - Porichthys spp 20 - | ■ P- ' - ' — ^ iMI;ilil;iill;ili l;l ft i £;5 M 1;5 8l 1 100 80 60 40 20 Myctophidae no. 1 Z Cl Z >'Z 0- Z >"'z Q- Z V'z Q- Z i ' Z CL Z >' Z 0- Z b'z CL Z £ D 111 < (0 -» S -> CO ->5 CD C_> c a o Q. 1 CD CD ro c 100 80 60 40 20 Myctophidae z cl z >-'z n z v'zcl z v'z cl z >'z 0- z b'z cl z i'z cl z > i 8 1 1;1 8 1 i;l 8 1 S;i 8 1 I;3 8 1 3;3 8 1 I;r 8 1 i 100 80 60 Scomber japonicus o 20 __)■ Q_ z o. z v'z 0- z >-'z Q. z bz Q- z i'z Q. z >: ' z 0. z >: ' z Q- z >; TWn5TU)nS=50)-)S-iV)->S->WnS->W-)S->W-iS 100 80 60 40 20 ■ ■_ Engraulis mordax Z 0. Z >-'z 0- Z >-'z D- z >-'z 0. Z i'z Q- z £ ' Z 0- Z £: Z 0. Z >: t CO n S =) CO * S ' =j 0) n So CO n S =3 CO n S =5 CO n S =i CO =3 5 100 80 40 20 ■ -m Cetengraulis mysticetus i & i fell a s s!i a ? si? & s fell a i fell a i fell a ? | T CO n 5 n CO * S n CO n St » n S n (0 n St 0) n 5 ( => to -, S 100 80 40 20 ^_ _L ^_ _L _^ -L Trachurus symmetricus Z 0- Z >".Z tL Z >,Z D- Z >-.Z 0- Z >ZtZ 0- Z ^,Z 0- Z £,Z Q- Z 5j D S tf -t'D QJ < D Ul < to * S|=i CO t S,=5 to n S,n to t S,=i to n Srn W n S spm : est : ras : mac : can : GRA : LOB Figure 4 Index of importance (IIMP) for nine prey species identified from samples of California at seven rookeries in the Gulf of California, Mexico, during June and September 1995 sea lions scats collected . and January and May 1996. of California. The distribution pattern of Pacific sardine closely agrees with its importance in the sea lions diet. The Pacific sardine occurred in high concentrations around Angel de la Guarda and Isla Tiburon during the summer and along the coast of southern Sonora during the winter, where spawning occurs (Cisneros-Mata et al.3). Sardines 58 Fishery Bulletin 102(1] were consumed in the Canal de Ballenas region during the summer (September), when they are very abundant. Larger size Pacific sardines were consumed by sea lions most frequently during the summer when adult sardines occur more frequently in the Canal de Ballenas. As adult sardine left Canal de Ballenas ( Cisneros-Mata et al., 1997 ), the proportion of young individuals in the diet of sea lions increased. The fish eaten by sea lions were apparently smaller than those captured by the commercial fisher- ies. The average estimated size of the sardines consumed was 150.4 mm, whereas the average size of commercially caught fish during the 1995-96 season was 162.4 mm (Cis- neros-Mata et al.3). This 7% difference in size may have been caused by an underestimation of otolith size because of digestive erosion ( Jobling and Breiby, 1986). If this is so, then the size of Pacific sardines consumed by sea lions is similar to the size of those captured by the fishery. Isla Lobos was the only rookery where Pacific sardine was not consumed. This finding differs from those of Cisneros- Mata et al.3 which show the Pacific sardines present as far north as Isla Lobos. However, their study period was during the 1991-92 El Nino episode, whereas our study occurred during normal oceanographic conditios in 1995-96. Less is known about the spatial and temporal availability of other important prey. As with commercial captures (Arvizu-Martinez, 1987), Pacific mackerel occurred together with Pacific sardine. Similar varia- tions in occurrence for both species have been noticed from stomach content analyses of the giant squid (Dosidicus gigas) (Ehrhardt, 1991). Lanternfishes were abundant north of Isla Angel de la Guarda (Robison, 1972); however they were not im- portant in the diet of the California sea lion in this region. Their greater importance in the diet at southern rookeries was probably due to the absence of more preferred prey such as Pacific sardine, Pacific cutlass- fish, or anchoveta. The consump- tion of northern anchovy tended to be less important towards Canal de Ballenas, where Pacific sardine reached its maximum importance. The low spatial overlap of these two species has also been noted in other studies. The anchoveta was present only at Isla Lobos. This is an estuarine-lagoon species, typical of coastal lagoons of northern Sinaloa and Sonora (Castro-Aguirre et al., 1995). The presence of this prey in Isla Lobos is possibly due to the sandy coast (Walker, 1960), which is similar to that of the Sinaloa-Sonora coast. The diet of California sea lions differed among rooker- ies, probably due to differences in feeding sites and prey availability. Antonelis et al. (1990) studied the foraging characteristics of the northern fur seal (Callorhinus ur- sinus) and the California sea lion at San Miguel Island and found differences between foraging areas among 0.15 200-i 180- 160- 140- | 120- •£_ 100- £ 80- _J 60- 40- 20- 0 n=121 JUN95 SEP95 JAN95 Figure 5 Size of Pacific sardine iSardinops caeruleus) estimated from otoliths found in California sea lions scats collected in Isla San Esteban, El Rasito, Granito, Los Cantiles, and Los Machos. One standard deviation is indicated from each mean. 0.2 0.25 0.3 0.35 0.4 0.45 Figure 6 Dendrogram of cluster analysis of seven rookeries determined with Euclidean dis- tance (computed from the IIMP of the 25 prey that had on at least one occasion a value >10%) and the UPGMA (unweighted pair-grouping methods) strategy. The vertical lines represent the points of references to delimit the groups. species. The northern fur seal was found most frequently foraging in oceanic water within 72.4 km from the island, whereas Califorinia sea lions forgaged more often in the shallower neritic zone, within 54.2 km from the island. Different foraging distances in California sea lions from San Miguel Island were found by Melin and DeLong ( 1999). During the nonbreeding season a higher percent- age of foraging locations occurred at distances less than 100 km, whereas during the breeding season most of the foraging locations occurred at distances greater than 100 km. These differences are probably due to the in- creased California sea lion population in San Miguel; this increase in population forces sea lions to exploit new areas as a density-dependent response to population Garcia-Rodriquez and Aurioles-Gamboa: Spatial and temporal variation in the diet of Zalophus californianus 59 SPM ■Jun95 »Sep96 ■4 I I I I I I I I I I I I I I I I I I I I I I t I 0 2 4 6 8 10 G M 16 18 2022242628303234 36 384042'! EST 0 2 4 6 8 10 12 2022 24 26283032 34 36 384042444648 50 RAS 0 2 4 6 8 10 12 14 16 18 202224262830323436384042444648 50 3 50 ■ MAC 3 00 . * ■ " * 2 50 . t „ / 2 00 . 1 50 . */ i - - - Jan t 00 . 0 50 . 1 t 1 1 1 1 1 1 1 1 * I I l l l l I I l ■t-f H- ■h-i 0 2 A 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Sample size CAN I'l I I I i I i i i I I I I I I I I I i I I I I l l 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 GRA I'l i i i i i i i i i i i i i i i i i i i 0246610121416 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 3 50 - LOB 3 00 . 2 50 . _, _. - -J—v 2 00 . r v J C^y 1 50 . 100 . . A /• - - - Jan96 0.50 ■ •_i/v - - May9 /, i -t-t- Mill i i i i i i i 0246810121416 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 40 50 Sample size Figure 7 Trophic diversity curves for California sea lions determined from scat samples collected at seven rookeries in the Gulf of California, Mexico. SPM = San Pedro Martir; EST = San Esteban; RAS = Isla Rasito; MAC = Los Machos; CAN = Los Cantiles; GRA = Isla Granito; LOB = Isla Lobos. 60 Fishery Bulletin 102(1) growth. Although, these differences could also be due to variability in the distribution of prey (Melin and DeLong, 1999), as suggested by Antonelis and Fiscus (1980), forag- ing areas might change with season and annual variations in prey availability and abundance. Foraging areas in the Gulf of California could lie closer to rookeries than those recorded for San Miguel Island sea lions because the diet was different among rookeries in spite of the shorter distance between them (54.2 km). At Los Islotes, Baja California Sur, adult females fed within 20 km of the colony (Duran-Lizarraga. 1998). Kooyman and Trillmich (1986a, 1986b) reported similar data in sea lion colonies of the Galapagos Islands. In the northern region of the Gulf of California, feeding range could be shorter than that at Los Islotes because of the higher concentration of food at high nutrient concentrations (phosphate, nitrate, nitrite, and silicate) in Canal de Ballenas that is associated with strong tidal mixing (Alvarez-Borrego, 1983). Four foraging zones were discerned from dietary differ- ences in sea lions from the seven rookeries studied. Zone I, which included San Pedro Martir. San Esteban. and El Rasito, was characterized by the consumption of lantern- fish; zone II, which included Los Machos was characterized by the consumption of Pacific sardine and Pacific mackerel; zone III, which included Isla Granito, by the consumption of Pacific cutlassfish and the northern anchovy; and zone IV, Los Cantiles and Isla Lobos, was characterized by the consumption of the plainfin midshipman and the Pacific cutlassfish. These four zones may indicate differences in habits used by sea lions or may indicate different oceano- graphic conditions exploited by sea lions. The eastern coast of the Gulf of California displays high photosyn- thetic pigment concentrations, associated with upwelling induced by winds from the northwest in the winter. These conditions may make Canal de Ballenas one of the most important for the distribution of Pacific sardine during the summer. Trophic diversity varied spatially and temporally. San Pedro Martir and Isla lobos sea lions seem to depend on a more stable feeding areas compared to sea lions at rook- eries on Isla Granito and Los Machos, where changes in diversity of consumed species indicated that sea lions feed on fewer species during certain times of the year. Similar results in relation to the changes in diversity were also noticed in the rookeries of the Channel Islands and Faral- lon Islands, California (Bailey and Ainley, 1982; Antonelis et al., 1984; Lowry et al., 1990; Lowry et al., 1991 ). Perhaps the tendency to have the highest values of diversity and little seasonal variation at San Pedro Martir is the result of this rookery being located in a zone of transition between two biogeographical areas. This geographical position con- fers greater environmental heterogeneity and greater ecological diversity (Walker, 1960). California sea lions in the upper region of the Gulf of California obtain the main portion of their diet from a relatively small number of species. The decrease in abun- dance of any of these food resources can seriously affect the population, particularly at Isla Granito and Los Machos because sea lions from these rookeries depend on a few species. Acknowledgments We wish to thank Secretaria de Marina, Armada de Mexico, for its great support during the field activities, and the Consejo Nacional de Ciencia y Tecnologia (CONACYT) for funding this study under grant number 26430-N. The Secretaria de Medio Ambiente, Recursos Naturales y Pesca (SEMARNAP) provided permits for field work (DOO.-700- (2)01104 and DOO.-700(2).-1917). We would like to thank Robert Lavenberg and Jeff Siegel for allowing us the use of otoliths from the collection at the Natural Museum His- tory of Los Angeles County and also Lawrence Barnes for his logistical support during the stay of first author at Los Angeles; we also thank Manuel Nava for allowing us the use of otoliths from the collection in Tecnologico de Monterrey, Campus Guaymas. We are also grateful to Unai Markaida for his assistance in prey identification based on the examination of cephalopods beaks. 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Universidad Autonoma de Baja California. Ensenada, B.C. Pitcher, K. W. 1980. Stomach contents and feces as indicators of harbour seal, Phoca vitulina, foods in the Gulf of Alaska. Fish. Bull. 78:797-798. 62 Fishery Bulletin 102(1 Robison. B. H. 1972. Distribution of the midwater fishes of the Gulf of California. Copeia (19721:449-61. Roper. C. F. E., and R. E. Young. 1975. Vertical distribution of pelagic cephalopods. Smith- sonian Contribution to Zoology 209(51 1:31. Sanchez-Arias, M. 1992. Contribucion al conocimiento de los habitos alimen- tarios del lobo marino de California Zalophus califomianus en las Islas Angel de la Guarda y Granito, Golfo de Cali- fornia, Mexico. Tesis de Licenciatura, 63 p. Universidad Nacional Autonoma de Mexico. Mexico, D.F. Tollit, D. J., M. J. Steward, P. M. Thompson. G. J. Pierce, M. B. Santos, and S. Hughes. 1997. Species and size differences in the digestion of oto- liths and beaks: implications for estimates of pinniped diet composition. Can. J. Fish. Aquat. Sci. 54:105-119. Walker, B. W. 1960. The distribution and affinities of the marine fish fauna of the Gulf of California. System. Zool. 9(3):123-133. Wolff, G A. 1984. Identification and estimation of size from the beaks of 18 species of cephalopods from the Pacific Ocean. NOAA Tech. Rep. NMFS 17, 49 p. 63 Abstract— Recruitment of bay anchovy {Anchoa mitchilli) in Chesapeake is related to variability in hydrologi- cal conditions and to abundance and spatial distribution of spawning stock biomass (SSB I. Midwater-trawl surveys conducted for six years, over the entire 320-km length of the bay, provided information on anchovy SSB, annual spatial patterns of recruitment, and their relationships to variability in the estuarine environment. SSB of anchovy varied sixfold in 1995-2000; it alone explained little variability in young-of-the-year (YOY) recruitment level in October, which varied ninefold. Recruitments were low in 1995 and 1996 (47 and 31xl09) but higher in 1997-2000 (100 to 265 xlO9). During the recruitment process the YOY popu- lation migrated upbay before a subse- quent fall-winter downbay migration. The extent of the downbay migration by maturing recruits was greatest in years of high freshwater input to the bay. Mean dissolved oxygen (DO) was more important than freshwater input in controlling distribution of SSB and shifts in SSB location between April- May (prespawningl and June-August (spawning) periods. Recruitments of bay anchovy were higher when mean DO was lowest in the downbay region during the spawning season. It is hypothesized that anchovy recruit- ment level is inversely related to mean DO concentration because low DO is associated with high plankton produc- tivity in Chesapeake Bay. Additionally, low DO conditions may confine most bay anchovy spawners to the downbay region, where production of larvae and juveniles is enhanced. A modified Ricker stock-recruitment model indicated den- sity-compensatory recruitment with respect to SSB and demonstrated the importance of spring-summer DO levels and spatial distribution of SSB as con- trollers of bay anchovy recruitment. Recruitment and spawning-stock biomass distribution of bay anchovy (Anchoa mitchilli) in Chesapeake Bay* Sukgeun Jung Edward D. Houde University of Maryland Center for Environmental Science Chesapeake Biological Laboratory 1 Williams St., P.O. Box 38 Solomons, Maryland 20688 E-mail address (for S Jung): iung@cbl.umces.edu Manuscript approved for publication 30 September 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull. 102:63-77 (20041. Recruitment for marine fishes is vari- able and is regulated or controlled by a combination of density-dependent and density-independent processes. It has been hypothesized that density-inde- pendent processes dominate from the egg to larval stages whereas density- dependent control by predation may be more important in the juvenile stage (Sissenwine, 1984; Houde, 1987). Den- sity-dependent processes may be stock dependent, regulated by adult abun- dances, or dependent on abundances of the early-life stages (Ricker, 1975). In estuarine systems, where hydrological conditions (e.g. dissolved oxygen, tem- perature, and circulation) vary widely, the roles of density-independent physi- cal factors on fish recruitments may be dominant, making it difficult, but still important, to partition density- dependent and density-independent processes, particularly for short-lived small pelagic fishes such as anchovies and sardines. Bay anchovy {Anchoa mitchilli) (En- graulidae) is a coastal species distrib- uted broadly in the western Atlantic from Maine to Mexico. This small fish is the most abundant and ubiquitous fish in Chesapeake Bay, the largest estu- ary on the east coast of North America (Houde and Zastrow, 1991; Able and Fahay, 1998). It is not fished, yet there is evidence that recruitment is variable (Newberger and Houde, 1995). It feeds on zooplankton — primarily copepods and other small Crustacea — and is a major prey of piscivores, including several eco- nomically important fishes (Baird and Ulanowicz, 1989; Luo and Brandt, 1993; Hartman and Brandt, 1995). Male and female bay anchovy in Chesapeake Bay mature at 40^15 mm fork length (44-50 mm total length) at about 10 months of age, and peak spawning occurs in July (Zastrow et al.. 1991). Most eggs are produced by age-1 individuals (Luo and Musick, 1991; Zastrow et al., 1991). Bay anchovy may survive to age 3+ and reach approximately 100 mm length and 5 g wet weight ( Newberger and Houde, 1995; Wang and Houde, 1995). Newberger and Houde (1995) noted large differences in annual survey abundances of bay anchovy that appar- ently resulted from variability in an- nual recruitments. In Chesapeake Bay, abundance, growth, and mortality rates of bay anchovy eggs and larvae vary temporally and spatially (Dorsey et al, 1996; MacGregor and Houde, 1996; Rilling and Houde, 1999a, 1999b). Indi- vidual-based models were developed to test the hypothesis that recruitment of bay anchovy is determined by variable growth and mortality during early-life stages that are regulated by density-de- pendent processes (Wang et al., 1997; Cowan et al., 1999; Rose et al„ 1999). In previous research, there was little knowledge of levels of spawning stock biomass or density-independent envi- ronmental factors that may control re- cruitment through their effects on spa- tial and temporal variability in growth and mortality of prerecruit anchovy. * Contribution 3696 of the University of Maryland Center for Environmental Sci- ence, Chesapeake Biological Laboratory, Solomons, MD 20688. 64 Fishery Bulletin 102(1) 39°N 38°N ^vt^w N 37°N gi quehanna Upper — Middle Lower Atlantic Ocean 77°W 76°W Figure 1 Chesapeake Bay and stations sampled by the midwater trawl in the 1995-2000 surveys. Horizontal lines indicate boundaries of three designated regions. We evaluated environmental factors, spatial distribution of spawning stock biomass (SSB), and possible ontogenetic migrations of prerecruits (Dovel, 1971; Loos and Perry. 1991; Wang and Houde, 1995; Kimura et al, 2000) with respect to bay anchovy recruitment variability. Our objec- tives were 1) to estimate annual and regional variability in bay anchovy recruitment. 2) to evaluate effects of hy- drological conditions (mainly, freshwater input, and dis- solved oxygen concentration) on stage-specific distribution, ontogenetic migration, and recruitment, and 3) to identify mechanisms and describe patterns or trends in the bay anchovy recruitment process. Data were obtained in a six-year, multidisciplinary research program conducted throughout Chesapeake Bay. Materials and methods Study area Chesapeake Bay is a coastal plain estuary of partially mixed fresh water and sea water. Its 320-km mainstem varies in width from about 6 to 50 km (Fig. 1 ). The Bay is shallow; less than 10' r of its area is >18 m deep and approximately 50' i is <6 m deep. More than 809& of the freshwater entering the bay is from tributaries on its northern and western sides (Chesapeake Bay Program1 ). Salinity grades from near-full seawater at the mouth of the bay to freshwater near its head. Water temperatures reach 28-30°C in mid summer, and fall to 1^°C in late winter (Murdy et al, 1997 ). Despite shallow depth, the bay usually has a strongly developed pycnocline, and has seasonally strong vertical gradients in temperature, salinity, and dissolved oxygen. Surveys Trawl surveys were conducted three times annually over the entire bay (April-May, June-August, and October). 1995-2000 (Table l.Fig. 1). Midwater-trawl (MWT) fish col- lections2 were made on transects in three regions: the lower bay (37°05'N-37°55'N), middle bay (37°55'N-38°45'N I, and upper bay (38°45'N-39°25'N). As defined, the lower bay contains 51% of water volume, the middle bay 32^ .and the upper bay 17^ (Fig. 1). The number of midwater trawl sta- Chesapeake Bay Program. 2000. Chesapeake Bay: Introduc- tion to an ecosystem. U.S. Environmental Protection Agency, publ. EPA 903-R-00-001. 30 p. EPA. 410 Severn Ave, Suite 109, Annapolis. MD 21403. Trophic interactions in estuarine systems, midwater trawl sur- vey. University of Maryland Center for Environmental Sci- ence, Chesapeake Biological Laboratory, http://www.ch.esa peake.org/ ties/mwt laccessed 15 October 20031. Jung and Houde: Recruitment and spawning-stock biomass distribution of Anchoa mitchilli 65 Table 1 Cruise dates, mean standard errors for temperatures (°C) individual cruises. salinities (psul, and dissolved oxygen (mg/L). ir years, seasons, and regions of Chesapeake Bay, tegrated from surface to bottom, and pooled 1995-2000. CV = coefficient of variation for annual means. Temperature SE Salinity SE Oxygen SE Cruise date ( depart 28 Apr 95 ure) 13.88 0.11 15.01 0.42 8.53 0.1.3 23 Jul 95 28.13 0.12 15.48 0.44 6.50 0.14 28 Oct 95 17.26 0.12 17.39 0.45 7.59 0.14 28 Apr 96 13.87 0.10 10.84 0.36 10.21 0.11 17 Jul 96 24.66 0.11 11.80 0.41 7.43 0.13 22 Oct 96 16.10 0.10 11.26 0.36 8.55 0.11 20 Apr 97 10.93 0.13 11.41 0.50 10.01 0.16 11 Jul 97 25.28 0.13 13.59 0.51 7.10 0.16 29 Oct 97 14.64 0.13 18.19 0.51 8.01 0.16 11 Apr 98 12.26 0.12 8.90 0.44 9.95 0.14 04 Aug 98 26.15 0.12 12.89 0.46 7.01 0.15 19 Oct 98 18.60 0.13 16.64 0.49 8.64 0.15 19 Apr 99 11.97 0.13 13.51 0.49 10.04 0.16 26 Jun 99 23.52 0.15 16.02 0.56 5.75 0.18 23 Oct 99 16.30 0.14 17.38 0.53 8.87 0.17 29 Apr 00 12.95 0.17 12.51 0.64 8.98 0.20 25 Jul 00 24.26 0.14 14.06 0.53 5.17 0.17 17 Oct 00 17.89 0.15 16.73 0.56 7.63 0.18 Year 1995 19.76 0.07 15.96 0.25 7.54 0.08 1996 18.21 0.06 11.30 0.22 8.73 0.07 1997 16.95 0.08 14.40 0.29 8.37 0.09 1998 19.00 0.07 12.81 0.27 8.53 0.08 1999 17.26 0.08 15.64 0.30 8.22 0.10 2000 18.36 0.09 14.43 0.33 7.26 0.11 CV 5.8% 12.5% 1.2% Season April-May 12.64 0.05 12.03 0.20 9.62 0.06 June-August 25.33 0.05 13.97 0.20 6.49 0.06 October 16.80 0.05 16.27 0.20 8.22 0.06 Region of bay Lower 18.40 0.04 21.19 0.16 8.15 0.05 Middle 18.33 0.05 14.06 0.19 8.33 0.06 Upper 18.04 0.06 7.02 0.23 7.85 0.07 tions per survey ranged from 24 to 52 (six-year total=597). Additional baywide surveys (August 1997 and September 1998) and partial surveys (June 1997, July 1998, and July 1999) also provided data (total stations =146). An 18-m2 mouth-opening midwater trawl (MWT), with 3-mm codend mesh was deployed from the stern of the 37-m research vessel Cape Henlopen. All trawling was conducted at night. Standardized tows of 20-min dura- tion were conducted and the trawl was deployed at graded depth intervals from surface to bottom ( 2 minutes at each depth interval ) in order to provide a sample of fish from the entire water column. Fish catches (or subsamples) were counted, measured (to the nearest 1.0 mm), and weighed on deck immediately after a tow. Abundance and biomass of bay anchovy recruits and spawners We separated bay anchovy catches into YOY and spawn- ers based on total length (TL). The minimum length of bay anchovy retained by the MWT was 21 mm TL, which we also defined as the minimum TL for recruited YOY bay 66 Fishery Bulletin 102(1) anchovy. Modal lengths of young-of-the-year (YOY) bay anchovy cohorts were determined from length-frequency distributions in MWT catches and a modal analysis (Bhat- tacharya, 1967; King, 1995). Based on the modal analysis of summer and fall survey data, the maximum TL of YOY bay anchovy and, therefore, the minimum TL of spawners, were estimated (Table 2). Length-dependent gear selectivity for bay anchovy was adjusted by comparing catches of the MWT and a 2-m2 Tucker trawl with catches from 707-iim meshes at the same stations during a September 1998 baywide survey. The length-specific MWT:Tucker-trawl catch ratios (N^^j/ iVj^catch per unit of effort MWT 4- catch per volume of water Tucker trawl) for anchovies 21-70 mm TL indicated that both gears fished with a consistent selectivity for bay anchovy of 30-48 mm TL, and with a slight decrease in NTT for 48-70 mm TL. However, the values ofNMWTINTT were lower by factors of 1-7 for 21-30 mm TL fish, indicating that small anchovies were collected less efficiently by the MWT. We concluded that length classes of anchovies >30 mm TL were equally vulnerable to the MWT and those >48 mm TL were less vulnerable to the Tucker trawl. Accord- ingly, we adjusted MWT catches of ^30 mm TL anchovy by multiplying them by a weighting factor estimated from the regression of values of iVMH,T/./V.r7. for 21-30 mm TL bay anchovy. ( Weighting factor) = -0.59 TL + 19.08, (r2=0.96) where TL = total length. The weighting factor equals 1.0 for anchovy >30 mm TL because MWT selectivity is constant for anchovy >30 mm TL. To estimate water sampled in a 20-min MWT tow, and where D« dn = nmwt/ vmwt = ( 1/s ' x Nt/Vtt MWT — ^ x ^-^ MWT TT x TT ♦ bay N, MWT N 77' the concentration of 31-48 mm TL anchovy at a station (i.e. number/m3); the number of 31-48 mm TL bay anchovy collected per 20-min MWT tow at a station; VMWT = the effective water volume sampled by a 20-min MWT tow (m:!); the number of 3 1-48 mm TL bay anchovy col- lected by the 2-m- Tucker trawl at the same station; vulnerability to the Tucker trawl (s=l if all bay anchovies in water volume, V^, are col- lected); and VTT is the volume filtered by the Tucker trawl (m3) estimated from a flowme- ter in its mouth. The mean of AfWHT/./V7T for 30-48 mm TL bay anchovy during the September 1998 survey indicated that V'WUT = 4961 m\ if 30-48 mm TL bay anchovy did not significantly avoid the mouth of the 2-m2 Tucker trawl (i.e. s=l). Assum- ing s=l (i.e. VMVVT=4961 m3), we estimated "relative" bay- Table 2 Estimated maximum total lengths of young-of-the-year bay anchov y (mm ) from Chesapeake Bay, based on analy- sis of length-frequency distributions. Year Date Length (mm) 1995 23 Jul 28 Oct 52 69 1996 17 Jul 22 Oct 57 68 1997 11 Jul 2 Aug 29 Oct 30 56 66 1998 4 Aug 7 Sep 19 Oct 50 62 69 1999 26 Jun 23 Oct 30 65 2000 25 Jul 17 Oct 52 67 wide abundance and biomass of YOY and spawners for the 18 surveys from 1995 to 2000. To coarsely estimate a typical value of s. "absolute" bay- wide spawner biomasses in June— August were estimated for 1995-2000 according to an egg production method (Parker, 1985; Rilling and Houde, 1999a). Bay anchovy eggs had been collected in a 1-m2 Tucker trawl during the same surveys and provided estimates of egg abundance. The coverage of stations and sampling design for the Tucker trawl was comparable to that of the MWT, but the Tucker trawl was deployed during both day and night. We presumed that all eggs collected between 00:00 and 20:00 h had been spawned near a midnight peak 1 00:00 h) (Za- strow et al., 1991) and decreased in abundance at a mean instantaneous mortality (reported for bay anchovy eggs in Chesapeake Bay as M = 0.066/h; Dorsey et al., 1996). Based on the estimated number of eggs spawned at 00:00 h for each station, the regional mean weight of individual spawners (defined by the minimum TL in Table 2) in MWT catches, and the reported fecundity-weight relationship for females (Zastrow et al., 1991), we were able to coarsely estimate "absolute" baywide spawner biomass. We as- sumed that the spawning fraction of adult females per day- was essentially 1.0 (i.e. all adult females participated in spawning, Zastrow et al., 1991) and the fecundity-weight relationship was constant over years. Comparison of the baywide estimates of spawner bio- mass in June-August based on the egg production method ("absolute" biomass) with estimates based on the MWT catch-per-unit-of-effort ("relative" biomass) indicated that. on average, for 1995 to 2000, s is equal to 0.20. Therefore, the mean effective water volume fished by a 20-min MWT tow was 4961x0.20 = 989 m3. Jung and Houde: Recruitment and spawning-stock biomass distribution of Anchoa mitchilli 67 Because NmvT of bay anchovy was highly variable, even at stations on the same sampling transect, and a mixed model (SAS version 6.12, SAS Inst. Inc., Cary, NC) includ- ing spatial covariance ( variogram ) did not significantly im- prove precision in annual, seasonal, and regional means or differences of NMWT, a stratified sampling design ( Steel and Torrie, 1980), i.e. stratum = region, was adopted. Based on the mean effective water volume (=sxVMWJ, ), we estimated regional "absolute" abundance and biomass (number and wet weight) and related standard errors of the linear com- bination by regional subvolumes (Samuels, 1989) of bay anchovy >21 mm TL for all MWT surveys from 1995 to 2000 by multiplying regional mean MWT catch by Vr/989, where Vr represents the water volume (m3) in each bay region (Cronin, 1971): N!olal=(N^Vl+Nn V,„ + N. Vj/(sxVMWT)xVlotal SEN=ScNjVr/n,+V*/nn +v:?/n„ where Nlotal v„ vm, vu SEX ScN = baywide absolute abundance; mean values of NmvT for the lower (1), middle (m), and upper (u) bay; bay subvolumes for the lower (1), middle (m), and upper (u) bay (from Cronin, 1971), V, = 26.7 x 109 m3, Vm= 16.8 x 109 m3, V„ = 8.7 x 109 m3, V„„„, = V, + Vm + V„ =52.1 x 109 m3; standard error of Nlolal; number of midwater trawl stations for the lower (1), middle (m), and upper (u) bay; pooled standard deviation of NMWT = square root of mean squares within groups in analysis of variance table = t/< SS, + SSm + SS„ ) / ( n,lMl -3i, where SS,, SSm, SStl = sum of squares of NMWT for the lower (1), middle (m), and upper (u) bay, and "total = nl + nm + nu- Environmental factors Depth profiles of temperature, salinity, and dissolved oxygen ( DO ) concentration were determined from conduc- tivity-temperature-depth ( CTD ) casts at sampling stations. DO data were adjusted by calibrating against Winkler titration data from water samples collected in Niskin bot- tles deployed with the CTD cast. However, DO data from the CTD could not be adjusted for the 1999 summer and all calendar year 2000 cruises because Winkler titrations were not conducted. To estimate regional means for the water column, we averaged temperature, salinity, and DO values by integrating the observed values with respect to depth, after dividing the water column into "above pycnocline" and "subpycnocline" layers. Ontogenetic migration We analyzed length-frequency distributions along the south-north axis of the bay (i.e. by latitude) to delineate possible ontogenetic migrations of YOY and adult bay anchovy. To parameterize the distribution of YOY and adult abundance and biomass, we estimated the biomass- weighted mean latitudes of occurrence for each length class (3-mm interval). lb.i = 2_,BkjLk/2jBtl, where LB , = biomass-weighted mean latitude of a length class, /; Lk = latitude of the station, k; and B = biomass (g, wet weight) per 20-min tow. We devised a metric to parameterize the location of bay anchovy SSB. We assumed that the baseline boundary for SSB distribution during the spring was at the mouth of the bay (37°00'N). Then, the upbay difference between biomass-weighted mean latitude of SSB (in decimal units) in Jun-August and the baseline for SSB during the spring lAL i was calculated: SL biomass-weighted mean latitude of SSB in June - August -37.00. Recruitment model As an exploratory step, a correlation analysis was under- taken to examine the relationships between bay anchovy SSB, migration patterns, and recruitment levels with respect to regional and depth-layer-specific mean tempera- ture, mean salinity, mean DO, their gradients, and monthly mean freshwater flow from the Susquehanna River. Cross- correlations revealed that SSB migration pattern {AD, regional mean DO concentrations, and October YOY recruitment level were closely correlated. Regional mean DO concentration provided the best fit to YOY recruitment level in October when baywide SSB also was included as an explanatory variable in multiple regressions. However, because there is uncertainty in the uncalibrated DO measurements in 1999 and 2000. we did not use regional mean DO in our recruitment model. Instead, we developed a modified Ricker-type stock-recruitment model (Ricker, 1975) that included AL as an explanatory variable: Rx = a S exp (-/3j S - /i, AL) + e (modified Ricker model ) where R, recruitment level = October YOY abun- dance in each year ( 1995-2000); y; a, l\ and p.-, = regression coefficients; S = estimated baywide SSB (male-i- female) in metric tons for April-May; and £ = the error term. In this model, if AL is held constant, Rs. is maximum at S = l//3j. Although no abiotic factor was included explicitly in the model, AL is strongly correlated with regional mean DO and serves as a proxy for it. For the modified Ricker model, collinearity, and jackknife influence diagnostic tools were 68 Fishery Bulletin 102(1) Table 3 Seasonal mean freshwater flow entering Chesapeake chesbay/RIMP/adaps.html. Bay ft' Dm the Susqu ehanna River ( m3/s ). Data source : http://va. water. usgs.gov/ Period 1995 1996 1997 1998 1999 2000 Jan-Mar 1289 2495 1474 2563 1325 1379 Apr-Jun 728 1702 920 1625 791 1627 Jul-Sep 238 768 239 334 294 393 Oct-Dec 923 2230 746 194 642 504 Annual mean 795 1799 845 1179 763 976 applied to evaluate reliability of the regression model (Belsley et al„ 1980; SAS, 1989). Results Environmental factors Stream flows from the Susquehanna River (Table 3) varied annually and seasonally. Freshwater stream flows were higher in 1996 and 1998 than in other years. Baywide mean values of water temperature, salinity, and DO concentration, averaged from surface to bottom, varied annually, seasonally, and regionally (Table 1 ). Annually, mean temperature was highest in 1995 and lowest in 1997. Mean salinity was highest in 1995 and lowest in 1996. Mean DO concentration was highest in 1996 and lowest in 2000. Regionally, salinity was more variable than temperature and DO concentration. Seasonally, temperature and DO concentration were more variable than salinity. Tem- perature was highest in the June-August period, the spawning season of bay anchovy. Seasonally, salinity increased progressively from April-May to October. Mean DO concentration was consistently lowest in June-August. Trends in abundance and recruitment Estimates of bay anchovy abundance reported in our study are for the entire mainstem of Chesapeake Bay. The estimated recruitment levels (baywide abundance of YOY bay anchovy >30 mm TL in October) varied ninefold and were low in 1995 and 1996 (47.5 ±16.6 and 30.6 ±8.6xl09 individuals) but much higher in 1997-2000 (99.6 ±12.4 to 264.8 ±32.6xl09). Baywide estimates of bay anchovy biomass for individuals >30 mm TL increased from April to October in each year (Table 4). October baywide biomass varied sevenfold from 27.1 ±5.5 x 103 to 192.9 ±20.4 x 103 tons and was highest in 1998 and lowest in 1996. Estimated spawning stock biomass (SSB) in April-May was lowest in 1995 (3.3 ±1.1 x 103 tons), and highest in 1997 (20.1 ±5.3 x 103 tons), indicating sixfold variability. SSB in June-August was lowest in 1996 (2.4 ±0.2 x 103 tons), and highest in 1997 (21.1 ±2.3 x 103 tons). The SSBs in April-May and June-August did not show any obvious relationship to YOY abundance (recruitment) in October. Ontogenetic migration The length-specific mean locations (latitudes of occur- rence ) of bay anchovy revealed an apparent ontogenetic migration. Small juveniles of bay anchovy tended to move upbay and were located primarily upbay until they were approximately 45 mm TL, after which they began to move downbay (Fig. 2). In April-May, age-1 bay anchovy <60 mm TL, consisting of individuals recruited from the previous year, varied annually in their mean latitude of occurrence, whereas large (sage 1, a60 mm TL) bay anchovy had relatively stable locations near the boundary between the lower and middle bay regions, centered at latitude 37°40'N (Fig. 2A). Compared to April-May, age-l+ bay anchovy in June-August were more variable in their annual mean locations, but both YOY and adult bay anchovy tended to occur upbay of latitude 38°00'N, except in year 2000 (Fig. 2B). In 1997 and 1999, when annual mean temperatures were lowest (Table 1), YOY bay anchovy were too small to be sampled by the MWT in June-August and are not represented in Figure 2B. In October, mean latitudes of occurrence (Fig. 2C) indicated a consistent distribution pattern and an apparent ontogenetic migration by YOY anchovy. The most probable explanation for the observed latitudinal distributions was that small YOY bay anchovy tended to move upbay initially, but then downbay at about 45 mm TL. Distribution of age-l-t- individuals in October was variable. The SSB of bay anchovy (excludes YOY) from 1995 to 2000 was centered near 38°00'N in April-August except in June-August of 1995 and 1996, when the SSB was centered farther upbay (Fig. 3A). In 2000, the migration pattern differed from other years. Spawning bay anchovy in 2000 were located farther downbay in July than in April (Fig. 3A). The April-May location of prespawning SSB was mostly explained by the mean flow of the Susquehanna River from June of the previous year to February of the current year (r2=0.94, P=0.0012; Fig. 3B ). But, in June-Au- gust, the mean location of spawning fish was more strongly and significantly related to the subpycnocline-layer mean Jung and Houde: Recruitment and spawning-stock biomass distribution of Anchoa mitchilli 69 Table 4 Baywide abundance and biomass estimates for bay anchovy >30 mm TL (young-of-the-year + adult). SE = = standard error. Year Period Abundance I xlO9) Biomass xlO3 metric tons) Estimate SE Estimate SE 1995 April-May 2.1 0.7 3.3 1.1 June-August 57.8 28.1 32.6 17.5 October 47.5 16.6 51.9 21.0 1996 April-May 4.9 1.1 8.9 2.0 June-August 5.3 1.6 3.7 1.3 October 30.6 8.6 27.1 5.5 1997 April-May 11.8 3.3 20.1 5.3 June-August 9.4 2.3 21.1 5.0 October 99.6 12.4 85.6 10.8 1998 April-May 3.5 0.7 6.1 1.3 June-August 14.4 4.5 17.0 7.9 October 264.8 32.6 192.9 20.4 1999 April-May 6.9 1.4 10.6 2.2 June-August 5.5 1.2 10.6 2.4 October 124.5 28.3 115.3 25.0 2000 April-May 6.2 4.1 13.0 6.6 June-August 144.6 51.2 56.0 17.0 October 169.1 43.7 152.9 40.0 DO during that same period in the middle bay (/•-=(). 75, P=0.02;Fig. 3C). Correlations Correlation analyses suggested that regional mean DO concentrations are the most important environmental correlate associated with spatial distribution of SSB and recruitment processes of bay anchovy. The mean locations (latitudes of occurrence), abundances, and biomasses for YOY and adult bay anchovy were analyzed with respect to environmental variables (Table 5). Recruitment levels (YOY abundance) in October were consistently inversely correlated with DO concentrations in the lower and middle bay in June-August (/-=-0.13 to -0.89). Biomass- weighted mean latitude of SSB (age 1+) in April-May was consistently and positively correlated with regional salini- ties in April-May (r=0.30 to 0.88). On the other hand, in June-August, surface-layer mean salinity in the lower Bay and subpycnocline-layer mean DO in the lower and middle bay were significantly and positively correlated with mean latitude of SSB or AL (r=0.82 to 0.91). Baywide SSB in April-May and June-August tended to be negatively cor- related with water temperature in April-May (r=-0.45 to -0.90). Recruitment model Although SSB alone did not correlate significantly with recruitment level, mean DO in June-August was signifi- cantly related to the mean latitude of SSB in June-August (or AL) and bay anchovy recruitment level in October (Figs. 3C and 4). AL was selected as the explanatory variable, rather than DO, because DO data were uncalibrated in 1999 and 2000. The correlation observed between AL and DO ( Fig 3C ) suggested that AL can serve as a proxy for DO in the stock-recruitment model. Including AL and SSB for April-May in a modified Ricker model provided a good fit to bay anchovy recruitment levels observed from 1995 to 2000 (Fig. 5). The model is Rv = 365 S exp (-0.19 S 1.35 AL) (modified Ricker model). In the model, if AL is held constant, predicted recruitment level of bay anchovy is maximum when baywide SSB in April-May is approximately 5.3 x 103 tons. Collinearity and influence diagnostic statistics did not indicate collinearity between the two independent variables (S and AL), or that an observation in any year had a dominating influence on parameter estimates. Discussion Complex environmental processes and biological interac- tions control bay anchovy recruitment in Chesapeake Bay. Dissolved oxygen (DO), freshwater flow, salinity, and tem- perature acting on prerecruits and adults are important factors affecting bay anchovy distribution and levels of recruitment. Spawning stock size also is related to recruit- 70 Fishery Bulletin 102(1) ment level. Our results have demonstrated that there is a strong spatial component in the recruitment dynamics of bay anchovy. Although fish recruitment processes his- torically have been difficult to understand, our six-year, spatially extensive research has provided new insights into processes that control bay anchovy recruitment. Ontogenetic migration pattern It is apparent that ontogenetic migration plays a role in the spatial and temporal patterns in abundance, biomass, and production of bay anchovy. There are several lines of evidence. Rilling and Houde (1999a), in a baywide analy- sis, reported that mean density of eggs and larvae in June and July 1993 was very high in the lower Chesapeake Bay compared to more upbay sites. Dovel (1971) and Loos and Perry (1991) reported possible upbay or upriver migra- tion of bay anchovy larvae and juveniles in the mainstem and tributaries of the Bay. Recent otolith microchemical analyses have strongly supported the hypothesis that an upbay ontogenetic migration by small YOY anchovy (>25 mm, late larvae and small juveniles) occurs (Kimura et al., 2000). In the middle Hudson River estuary (Schultz April-May 39°00' £ 38°00 37°00 39°00 38°00 37°00' 30 40 50 60 70 80 90 100 TL (mm) 1995 1996 1997 1998 1999 2000 Figure 2 Abundance-weighted mean latitude of occurrence of bay anchovy (Am hoa mitchilli) in Chesapeake Bay, 1995-2000. et al., 2000) and Chesapeake Bay (North and Houde, in press), selective tidal-stream transport was suggested as a mechanism for up-estuary movements of bay anchovy larvae. Our conceptual model of the bay anchovy life cycle includes migration patterns in the bay based on available knowledge and evidence (Fig. 6). It is uncertain what benefits YOY bay anchovy derives from upbay migration in summer and whether the migra- tion is passive or active before a subsequent reverse migra- tion in the fall. To explain upbay movements of estuarine fishes, Dovel ( 1971 ) proposed that there is a "critical zone" of low salinity and high prey production in the upper bay, which is important as a nursery for bay anchovy and other fish species. In late spring and early summer, age-1 and age-l+ bay anchovy mature and move upbay while spawning, although the year 2000, when mean freshwater streamflow during the previous fall-winter was lowest, was an exception. Recruited YOY bay anchovy apparently over- winter primarily, but not entirely, downbay until spring. There remains a possibility of significant immigration to the bay by adult bay anchovy in some years from the coastal ocean or tidal tributaries of the bay. Without such immigration, baywide adult abundance would decrease continuously during the April-October period through natural mortality However, in two years of our six-year study, 1995 and 1998, estimated adult abundance in- creased substantially from April to July, and in 1999 adult abundance increased from June to October, implying significant immigration to the bay in those years (Jung, 2002). Recruitment control and regulation The modified Ricker recruitment model that included SSB and AL as explanatory variables provided a good fit to bay anchovy recruitments. Although the model fitted well, there were only six years of data, and the underlying mechanisms explaining relationships between the distribution and level of SSB, hydro- logical conditions, and density-dependent regulatory processes in recruitment of bay anchovy are not yet clear. Nevertheless, correlations and the recruitment model clearly indicated a density-dependent effect of SSB level and also implicated environmental factors (at the mesoscale) that are related to mean DO concen- tration, latitudinal distribution of SSB (AL), and the recruitment level of bay anchovy (Fig. 4). The modified Ricker model for bay anchovy < Fig. 5) indicates a density-compensatory stock-recruitment relationship (Ricker, 1975). although we do not know at what life stages density-dependent processes are most important. Without accounting for the control- ling effect of AL and mean DO on a regional scale, the density-dependence might have gone undetected (Fig. 4 1. Recent individual-based models suggest that density-dependent processes during early-life stages could stabilize bay anchovy recruitments (Wang et al., 1997; Cowan et al., 1999; Rose et al, 1999). At the small scales of several meters modeled by Wang et al. (1997) and Cowan et al. (1999), larval-stage feeding Jung and Houde: Recruitment and spawning-stock biomass distribution of Anchoa mitchilli 71 Zl 39°00' 38°00' CO CO CO 37°00' April-May June-August 1995 1996 1997 1998 1999 2000 2000 38°00' 1999^ 1=38.30 - 0.00087 X r-=0.94(/)=0.0012) 37045' 1995 --4?^- 1998 B 1996_ 37°30' i , 300 < 39°00' c C CO CO c 38°00' 400 500 600 700 Mean river flow from June to Feb (m3/sec) 1995 1996^ 1999 1998 37°00' 2000 1997 Y= 35.78 + 0.53 A' r2=0.75(p=0.02) 3.0 3.5 4.0 4.5 5.0 Dissolved oxygen (mg/L) 5.5 Figure 3 Mean location (latitude) of adult bay anchovy {Anchoa mitchilli) spawn- ing stock biomass (SSB) in Chesapeake Bay. (A) Mean latitude and standard deviation in April-May and in June-August. The upper verti- cal bar represents mean + standard deviation for June-August, and the lower vertical bar represents mean-standard deviation for April-May, I B l Mean latitude in April-May and mean Susquehanna River flow from June of the previous year to February of the current year. (C) Mean lati- tude in June-August and mean dissolved oxygen in the subpycnocline layer of the middle bay in June-August. processes were important and high adult SSB could pro- duce abundant first-feeding larvae with subsequent den- sity-dependent food competition. In Tampa Bay, Florida, Peebles et al. ( 1996) hypothesized that bay anchovy's size- specific fecundity is directly related to prey availability for adults. Modeled results of Rose et al. (1999) suggested that density-dependent growth of bay anchovy larvae and juveniles in Chesapeake Bay would lead to density-depen- dent survival of these stages. Hunter and Kimbrell (1980) and Alheit (1987) proposed that cannibalism by adults on eggs and larvae provides a degree of density-dependent regulation in anchovies of the genus Engraulis. Analyses of feeding by adult bay anchovy did not indicate that pe- lagic fish eggs were a significant part of bay anchovy diet (Vazquez-Rojas, 1989; Klebasko, 1991), although no specific study of cannibalism has been undertaken. We propose three hypotheses that may explain the rela- tionships among regional DO concentration, the latitudi- nal shift in SSB distribution during the spawning season (AL), and recruitment levels of bay anchovy in October. The 72 Fishery Bulletin 102(1) hypotheses are the following: 1) averaged DO concentra- tion is inversely related to levels of plankton productivity in a region and high plankton productivity favors high re- cruitments of planktivorous bay anchovy; 2 ) low dissolved oxygen concentrations can restrict spatial distribution of bay anchovy SSB to the lower bay insuring high egg and Table 5 Cross-correlation coefficients for bay anchovy distribution and abundance with respect to region- and layer-specific means of tem- perature, salinity, and dissolved oxygen from 1995 to 2000. Mean latitude is biomass-weighted mean latitude of occurrence of bay anchovy. Abundance and biomass are baywide total estimates. AL = (mean latitude in June-August) -37.00. Abbreviations are as follows: SAL = salinity, TEM = water temperature, OXY = dissolved oxygen; the fourth and fifth digits: 04 = April-May, 07 = June-August; the sixth character: L = lower bay, M = middle bay, U = upper bay; The last character: S = layer above the pycnocline. B = layer below the pycnocline. * = significant at a = 0.05. Young-of-the-year Adult Mean latitude Abundance Mean latitude Biomass April-May June-August (orAL) October October April-May June-August SAL04LS 0.29 -0.43 0.74 0.26 -0.17 -0.52 SAL04MS 0.45 -0.63 0.30 0.71 -0.41 -0.22 SAL04US 0.27 -0.60 0.42 0.53 -0.18 -0.02 SAL04LB -0.24 0.01 0.88* -0.16 -0.14 -0.31 SAL04MB 0.08 -0.17 0.59 0.33 -0.39 -0.05 SAL04UB 0.29 -0.61 0.45 0.46 -0.03 0.05 SAL07LS 0.83* -0.75 0.91* -0.46 SAL07MS -0.12 0.06 0.14 0.31 SAL07US 0.06 -0.03 -0.04 -0.33 SAL07LB 0.70 -0.75 0.64 -0.11 SAL07MB -0.41 0.60 -0.31 0.19 SAL07UB 0.15 -0.20 0.01 -0.42 TEM04LS 0.16 -0.25 -0.03 0.65 -0.90* -0.48 TEM04MS 0.50 -0.46 0.14 0.65 -0.71 -0.85* TEM04US 0.53 -0.32 -0.36 0.52 -0.56 -0.85* TEM04LB 0.29 -0.49 0.19 0.71 -0.72 -0.45 TEM04MB 0.22 -0.42 0.39 0.47 -0.55 -0.62 TEM04UB 0.40 -0.26 -0.39 0.48 -0.60 -0.77 TEM07LS -0.49 -0.04 0.11 0.45 TEM07MS -0.16 -0.21 0.47 0.14 TEM07US -0.29 -0.08 0.39 0.38 TEM07LB -0.68 0.24 -0.11 0.38 TEM07MB -0.24 -0.10 0.37 -0.04 TEM07UB -0.45 0.16 0.2] 0.46 OXY04LS 0.63 -0.22 -0.80 0.39 -0.10 -0.30 OXY04MS -0.27 0.56 0.23 -0.81 0.55 -0.04 OXY04US -0.43 0.41 -0.30 -0.30 0.30 0.88* OXY04LB 0.93** -0.68 -0.59 0.63 0.04 -0.38 OXY04MB 0.47 -0.35 -0.31 -0.09 0.70 -0.12 OXY04UB -0.57 0.65 -0.32 -0.46 0.21 0.78 OXY07LS 0.18 -0.30 0.29 0.32 OXY07MS 0.01 -0.13 0.29 0.56 OXY07US 0.23 -0.32 0.50 0.10 OXY07LB 0.67 -0.48 0.82* -0.28 OXY07MB 0.72 (l,SH 0.87* -0.04 OXY07TJB 0.01 0.16 0.21 0.37 Jung and Houde: Recruitment and spawning-stock biomass distribution of Anchoa mitchilli 73 larval production there; and 3) density-depensatory predator satiation occurs when concentrations of bay anchovy larvae and juveniles at the mesoscale ( 10-100 km ) are high in relation to satiation potential of preda- tors, which favors larval production and high anchovy recruitments. First, averaged DO level in the bay or its regions may be an indicator of ecosystem metabolism and sec- ondary production. DO level in the subeuphotic layer is an indicator of respiration and secondary produc- tion by planktonic and benthic communities (Kemp and Boynton, 1980; Kemp et al., 1992). Recruitment levels of bay anchovy increased substantially in 1997 and in subsequent years. We speculate that enhanced detrital production potentially increased zooplankton prey abundances in the subsequent year and that asso- ciated elevated levels of respiration by detrital micro- organisms and zooplankton contributed to low mean DO. Increased zooplankton prey abundances, in turn, may have promoted production of larval and juvenile bay anchovy in 1997 and 1998. Thus, increased prey availability, associated with low mean DO concentra- tion, could have enhanced recruitment (Fig. 4). The second hypothesis proposes that spatial restric- tion of SSB by low DO is a factor controlling bay anchovy recruitment. Based on our results, hypoxic conditions in the bay appear to define the distribution and potential for upbay migration of bay anchovy SSB (Fig. 3C). In years 300 1998 7= -88 .V+ 5 10 C 200 o 1 00 cr r-=0.79P=0.01S 2000 ^~"-\1999 ^^19,97 ~"\J995 1W(, 0 3.0 3.5 4.0 4.5 5.0 Dissolved oxygen (mg/L) Figure 4 Relationship between mean dissolved oxygen below the pycno- cline in the middle Chesapeake Bay during the June-August period and recruitment level of bay anchovy in October, r2 = coefficient of determination. when the baywide subpycnocline mean DO level was low, spawning bay anchovy tended to be most concentrated in the lower bay (Table 5, Fig. 3, A and C), possibly because hypoxia in deeper waters of the mid-bay region discouraged upbay migration. The region selected by adult anchovy as the predominant spawning area and its variability played R = 365 Sexpf-O.l0- S - 1.354Z.) r2-- 2.0 0 Figure 5 Stock-recruitment model (modified Ricker model). R = baywide number of recruits in October (xlO9). AL = location of bay anchovy iA?iclioa mitchilli) spawning stock biomass in June-August in relation to the baseline latitude at the mouth of the bay, 37°00'N. S = baywide spawning stock biomass (SSB xlO3 metric tons for April-May 1. Balloon symbols are observed data from 1995 to 2000. 74 Fishery Bulletin 102(1) a strong role in controlling YOY recruitment levels. The four highest recruitment years in our series had the lowest mean subpycnocline DO levels and had distribution pat- terns of SSB that differed little between the prespawning April-May and spawning June-August periods (Fig. 4). Al- though we do not fully understand how DO, and possibly hypoxic conditions, affect migratory behavior and distribu- tion patterns of bay anchovy, hypoxia in Chesapeake Bay has been demonstrated in other research to affect spatial and temporal patterns of fish abundance, including bay anchovy (Breitburg, 1992; Keister et al., 2000). Our third hypothesis proposes that predation is an im- portant regulator of fish recruitment in early-life stages (Sissenwine, 1984; Bailey and Houde, 1989). We hypoth- esize that abundant and spatially concentrated larval or juvenile anchovy, as observed in the lower bay, could promote early-life survival by satiating predators, even if some predators migrate to areas where larval and juvenile anchovy are abundant. At mesoscale distances of 10-100 km, distribution of predators (e.g. YOY and age-1 weakfish [Cynoscion regalis] ) may be important. If the maximum number of prey that can be eaten by predators is reason- ably constant, the effect of predation can be density-depen- satory (Hilborn and Walters, 1992), i.e. predation mortality rate decreases as prey density increases. In support of the third hypothesis, a correspondence analysis on fish species assemblages by year, season, re- gion, and life stage (Jung and Houde, 2003) indicated that distributions and abundances of YOY weakfish, a major predator of bay anchovy in Chesapeake Bay (Hartman and Brandt, 1995), and YOY bay anchovy were closely as- sociated spatially, seasonally, and annually in our six-year study. The major spawning area of bay anchovy is spatially restricted. If predator migration to the area is limited, then as the supply of larvae and juveniles increases, it may satu- rate predator demand, the condition necessary for depensa- tion to be important. It may seem contradictory to propose that density-com- pensation with respect to SSB (the negative sign of j\) and density-depensation with respect to AL (the second or third hypothesis ) can act simultaneously during larval and juvenile stages. Under this circumstance, the number of surviving postlarval anchovies is hypothesized to decrease because of food limitation when larval abundance is high, reducing subsequent predation-related mortality rate on postlarvae and small juveniles. Low abundance of anchovy early-life stages will lead to the opposite effect (Fig. 7). The proposed opposing responses of the early-larval and late- larval-juvenile stages are explained by differences in the spatial scales of distribution and densities of life stages of bay anchovy (Fig. 7). The spatial scale of processes that affect distributions of late-stage larvae and juveniles is large compared to that for early-stage larvae because of the increased dispersal and swimming ability of juveniles. Comparing early-larval and late-larval-juvenile stages of bay anchovy, we propose that effects of prey concentration (the first hypothesis) and SSB level (density-compensa- tion) act primarily on the dynamics of early-larval stages, whereas predation mortality and the inhibitory effects of low DO (density-depensation; the second and third hy- Nursery Ground (3) Fall YOY recruits, adults Late-stage larvae, juveniles, some adults Eggs and larvae Overwintering Recruited anchovy Adult Immigration from tributaries'? Major Spawning Mature adults. / eggs, larvae ground (1) Spring Adult Immigration from ocean? Figure 6 Conceptual model representing bay anchovy (Anchoa mitchilli) life cycle and ontogenetic migration within Chesapeake Bay, and possible immigration of adults from tributaries and coastal ocean. potheses) are more important regulators and controllers, respectively, during late-larval and juvenile stages. The three hypotheses that relate DO, SSB distribution, and recruitment of bay anchovy are not mutually exclusive. If low mean DO level is an indicator of enhanced prey pro- duction and availability to larvae and juveniles, increased prey productivity in the lower bay could enhance bay anchovy recruitment potential by supplying enough zoo- plankton prey to spawning adults, larvae, and juveniles. At the same time, low mean DO in the mid-Bay could confine most spawning bay anchovy to the lower bay. thus increas- ing spawning and larval production there, and possibly enhancing survival of juveniles by predator satiation. Ul- timately, other hypotheses may provide better explanations of the relationships between regional mean DO. latitudinal shifts in distribution of spawners, abundances of spawners. and recruitment of bay anchovy. For example, abundant gelatinous organisms, such as the scyphomedusa (Chn'sa- ora quinquecirra) and the lobate ctenophore \Mnemiopsis leidyi), can be important predators on early-stage anchovy and competitors with juveniles and adults (Purcell et al., Jung and Houde: Recruitment and spawning-stock biomass distribution of Anchoa mitchil/i 75 Early-stage and larvae Density-compensatory Prey is smaller Small scale (1 m-10 km) Densiy of early-stage larvae (1 m-10 m scale) Late-stage larvae and juveniles Density-depensatory Predator is bigger Mesoscale(IO-lOOkm) Recruits Ontogenetic migration Densiy of late-stage larvae (10-100 km scale) SSB Figure 7 Hypotheses and conceptual model of the bay anchovy {Anchoa mitchilli) recruitment process in Chesapeake Bay. The density-compensatory process acts at a small spatial scale during the early- larval stages, whereas the density-depensatory process acts at a broader spatial scale during late-stage larval and juvenile stages. The ontogenetic migration is controlled by dissolved oxygen levels and other hydrological factors. 1994), but their potential role with respect to bay anchovy recruitment could not be defined in our study. For the present, it is clear that most spawning occurs in the lower and mid Chesapeake Bay, from which larval and juvenile anchovies disperse upbay. We hypothesize that food avail- ability is the major factor controlling production of bay anchovy early-larval stages whereas predation becomes more important during late-larval and juvenile stages. Our results and hypotheses implicate density-related pro- cesses, operating at different spatial scales, as regulators of recruitment of bay anchovy in Chesapeake Bay. Acknowledgments We thank S. Leach, E. North, J. Hagy, C. Rilling, J. Cleve- land, A. Madden, D. O'Brien, B. Pearson, D. Craige, T. Auth, and the able crew of RV Cape Henlopen for assistance in field surveys. T. Miller and E. 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Predation mortality of bay anchovy Anchoa mitchilli eggs and larvae due to scyphomedusae and ctenophores in Chesapeake Bay. Mar. Ecol. Prog. Ser. 1 14 :47-58. Ricker, W. E. 1975. Computation and interpretation of biological statis- tics of fish population. Bull. Fish. Res. Board Can. 191: 1-382. Rilling, G. C, and E. D. Houde. 1999a. Regional and temporal variability in distribution and abundance of bay anchovy (Anchoa mitchilli i eggs, larvae, and adult biomass in the Chesapeake Bay. Estuaries 22: 1096-1109. 1999b. Regional and temporal variability in growth and mortality of bay anchovy. Anchoa mitchilli. larvae in Chesa- peake Bay. Fish. Bull. 97:555-569. Rose, K. A.. J. H. Cowan, M. E. Clark. E. D. Houde. and S. B. Wang. 1999. An individual-based model of bay anchovy population dynamics in the mesohaline region of Chesapeake Bay. Mar. Ecol. Prog. Ser. 185:113-132. SAS Institute Inc. 1989. SAS/STAT user's guide, version 6, 4th ed., 1686 p. SAS Institute Inc.. Gary, NC. Jung and Houde: Recruitment and spawning-stock biomass distribution of Anchoa mitchilli 77 Samuels, M. L. 1989. Statistics for the life sciences, p. 409-42. Prentice- Hall. Inc., Upper Saddle River, NJ. Schultz. E. T., R. K. Cowen, K. M. M. Lwiza. and A. M. Gospodarek. 2000. Explaining advection: Do larval bay anchovy lAnchoa mitchilli) show selective tidal-stream transport? ICES J. Mar. Sci. 57:360-371. Sissenwine, M. P. 1984. Why do fish populations vary? In Exploitation of marine communities (R. M. May, ed.l, p. 59-94. Springer- Verlad, Berlin. Smith, E. M., and W. M. Kemp. 2001. Size structure and the production/respiration balance in a coastal plankton community. Limnol. Oceanogr. 46: 473-485. Steel, R. G. D., and J. H. Torrie. 1980. Principles and procedures of statistics. A biometrical approach. 2nd ed., 633 p. McGraw-Hill Inc. New York, NY. Vazquez-Rojas, A. V. 1989. Energetics, trophic relationships and chemical compo- sition of bay anchovy, Anchoa mitchilli in the Chesapeake Bay. M.S. thesis, 166 p. Univ. Maryland, College Park, MD. Wang, S. B., J. H. Cowan, K. A. Rose, and E. D. Houde. 1997. Individual-based modelling of recruitment variability and biomass production of bay anchovy in mid-Chesapeake Bay. J. Fish Biol. 51 (suppl. A):121-134. Wang, S. B., and E. D. Houde. 1995. Distribution, relative abundance, biomass and produc- tion of bay anchovy Anchoa mitchilli in the Chesapeake Bay. Mar. Ecol. Prog. Ser. 121:27-38. Zastrow, C. E., E. D. Houde, and L. G. Morin. 1991. Spawning, fecundity, hatch-date frequency and young- of-the-year growth of bay anchovy Anchoa mitchilli in mid- Chesapeake Bay. Mar. Ecol. Prog. Ser. 73:161-171. 78 Abstract— Increasing interest in the use of stock enhancement as a man- agement tool necessitates a better understanding of the relative costs and benefits of alternative release strate- gies. We present a relatively simple model coupling ecology and economic costs to make inferences about optimal release scenarios for summer flounder (Paralichthys dentatus), a subject of stock enhancement interest in North Carolina. The model, parameterized from mark-recapture experiments, predicts optimal release scenarios from both survival and economic standpoints for varyious dates-of-release, sizes-at- release, and numbers of fish released. Although most stock enhancement efforts involve the release of relatively small fish, the model suggests that optimal results (maximum survival and minimum costs) will be obtained when relatively large fish (75-80 mm total length! are released early in the nursery season (April). We investigated the sensitivity of model predictions to violations of the assumption of den- sity-independent mortality by includ- ing density-mortality relationships based on weak and strong type-2 and type-3 predator functional responses (resulting in depensatory mortality at elevated densities). Depending on postrelease density, density-mortality relationships included in the model con- siderably affect predicted postrelease survival and economic costs associated with enhancement efforts, but do not alter the release scenario (i.e. combina- tion of release variables ) that produces optimal results. Predicted (from model output) declines in flounder over time most closely match declines observed in replicate field sites when mortality in the model is density-independent or governed by a weak type-3 func- tional response. The model provides an example of a relatively easy-to-develop predictive tool with which to make inferences about the ecological and economic potential of stock enhance- ment of summer flounder and provides a template for model creation for addi- tional species that are subjects of stock enhancement interest, but for which limited empirical data exist. Manuscript approved for publication 17 July 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull. 102:78-93 (2004). Coupling ecology and economy: modeling optimal release scenarios for summer flounder (Paralichthys dentatus) stock enhancement G. Todd Kellison David B. Eggleston Department ol Marine, Earth, and Atmospheric Sciences, North Carolina State University Raleigh, North Carolina 27695-8208 Present address (for G T. Kellison, contact author): National Park Service/ Biscayne National Park 9700 SW 328th St, Homestead, Florida 33033 E-mail address (for G T Kellison) todd_kellison 5 nps gov Commercially important marine fish and invertebrate populations are declining worldwide in response to overexploitation and habitat degrada- tion (Rosenberg et al„ 1993; FAO 1998). This reduction in harvestable fishery resources has stimulated increasing interest in the use of hatchery-reared (HR) animals to enhance wild stocks (Munro and Bell, 1997; Travis et al., 1998; Cowx, 1999; Kent and Draw- bridge, 1999). Unfortunately, many stock enhancement programs proceed before ecological concerns are adequately addressed (Blankenship and Leber, 1996), and without the identification of goals or the evaluation of the success of enhancement efforts (Cowx, 1999). If fishery managers can satisfactorily determine that enhancement efforts will have no ecologically significant negative ramifications, then managers should establish specific, quantifiable goals and objectives of enhancement efforts as part of a responsible approach to stock enhancement (Blankenship and Leber, 1996; Heppell and Crowder, 1998). Once such goals have been established, managers should identify stocking approaches that will lead to the most cost-efficient realization of enhancement goals — a process that can be accomplished with the aid of coupled ecological and economic models. Although numerous (advanced) models (conceptual and species-specific) exist to predict the biological and ecological impact of alternative enhancement scenarios (e.g. Botsford and Hobbs, 1984; Salvanes et al„ 1992; Barbeau and Caswell, 1999; Sutton et al., 2000), there are few models ( of which we are aware) that have attempted to link the biological and ecological results of stock- ing efforts (e.g. addition of biomass to a stocked population) with the economic costs associated with various release scenarios (e.g. Botsford and Hobbs, 1984; Hobbs et al., 1990; Hernandez-Llamas, 1997; Kent and Drawbridge, 1999). Such a link is critical to the responsible use of funding to rebuild or manage fisher- ies, and for the comparison of predicted costs of enhancement versus alternative management techniques. In North Carolina, there has been recent interest in stock enhancement with summer flounder (Paralichthys dentatus) (Waters, 1996; Rickards, 1998; Waters and Mosher, 1999; Burke et al., 2000; Copeland et al. ' ) because of a combination of heavy commercial and recreational exploitation, established techniques for mass hatchery-rearing (Burke et al., 1999), and considerable knowledge of summer flounder life his- tory (Powell and Schwartz, 1977; Burke et al., 1991; Burke, 1995). Nevertheless, there have been no large-scale release experiments ( and subsequent collection of data) by which to make empirical inferences about stock enhancement potential for this species. We present a compartmental model, parameterized from mark-recapture field experiments, Copeland, B. J., J. M. Miller, and E. B. Waters. 1998. The potential for flounder and red drum stock enhancement in North Carolina. Summary of workshop, 30-31 March. 1998, 22 p. ' (Available from North Carolina State Univ, Raleigh. NC 27695.] Kellison and Eggleston: Modeling release scenarios for Paralichthys dentatus 79 Table 1 Range of numbers of summer flounder (Paralichthys dentatus) released (and resulting postrelease densities), sizes-at-release, and dates of release simulated in the model. Number released Postrelease density Size-at-release Dates of release 100-400,000 0.001-4.0 30-80 mm 1 April-15 July that incorporates size of fish released, date-of-release, and number offish released to calculate 1) predicted numbers of survivors and 2 ) economic costs associated with varying re- lease scenarios under density-independent mortality. We in- vestigated the sensitivity of model predictions to violations of the assumption of density-independent mortality because there is abundant evidence that mortality rates, or processes underlying mortality rates (e.g. growth), are affected by den- sity-dependent relationships in the wild ( see, for recent ex- amples. Bucket et al., 1999; Bystroem and Garcia-Berthou. 1999; Jenkins et al, 1999; Kimmerer et al., 2000). We did so by repeating model simulations under varying density- mortality relationships (depensatory in nature at elevated densities ), using experimental evidence from our own field studies and published observations for similar species to parameterize density-mortality relationships. Additionally, we used a scenario in which the density-mortality relation- ship changed over time to make inferences about the effect of more complex density-mortality relationships on postrelease mortality of juvenile summer flounder. Finally, we generated predicted temporal patterns of field densities under vary- ing density-mortality relationships and compared them with observed (in the field) patterns to determine whether model output under the considered density-mortality relationships matched actual patterns in the field. The model provides an example of a relatively easy-to-develop predictive tool with which to make inferences about the ecological and economic potential of stock enhancement with summer flounder and provides a template for model creation for additional species that are subjects of stock enhancement interest, but for which limited empirical data exist. Materials and methods Background In North Carolina, wild summer flounder recruit to shal- low-water estuarine nursery habitats from February to May, after which small juvenile (20-35 mm total length [TL] ) densities range from -0.1 to 1.0 fish/m2 (Burke et al., 1991; Kellison and Taylor2). Juveniles subsequently make an ontogenetic habitat shift to deeper waters ( Powell and Schwartz, 1977), apparently after reaching a total length 2 Kellison, G. T., and J. C. Taylor. 2000. Unpubl. data. De- partment of Marine, Earth, and Atmospheric Sciences, North Carolina State University, Raleigh, NC 27695-8208. of -80 mm (Kellison and Taylor2). By mid-July, densities of juvenile summer flounder in the shallow water nursery habitats are near zero (Kellison and Taylor2). Model pathway Our compartmental model simulated the daily mortality and growth of different-size hatchery-reared (HR) fish released in the field over a 105-day period ( 1 April to 15 July, based on observed field abundances) in a hypotheti- cal release habitat of 10 hectares. The model predicted the percentage of released fish surviving and economic cost- per-survivor under 2730 release scenarios for a specified number offish released (see below). To begin the model, a value of number offish released (NFR) ranging from 100 to 400,000 (Table 1) was chosen (Fig. 1), resulting in postre- lease densities (assuming even postrelease distribution) of 0.001-4.0 fish/m2. These values included a range of densi- ties of juvenile summer flounder observed in wild nursery habitats ( -0-1 fish/m2; mean -0.05 fish/m2; Kellison and Taylor2), but also included unusually high densities (>1 fish/m2) in order to examine how such release strategies would affect model output (we did not examine densities >4 fish/m2 because of a lack of data on fish response to resource limitation likely to occur as densities increased past values for which we had empirical growth data). Each group of NFR was initially assigned a "size-(TL) at-release" of 30 mm (the smallest size-at-release simulated in the model), after which a size-dependent economic cost associ- ated with the release of the 30-mm-TL fish was calculated (see below). The release group was then assigned a mini- mum Julian "day of release" of 92 (corresponding to 1 April, the earliest release date simulated in the model). A range of Julian days of release was included in the model because field-estimated growth rates were dependent on Julian day (Kellison, 2000), and growth rates are potentially impor- tant to the determination of mortality rates (Rice et al. 1993). With this model, we then calculated daily mortality and growth (described below) in the hypothetical release habitat over the number of days at large (DAL), where DAL = 197 (the Julian day corresponding to 15 July) - 92 (Julian release day), and output a number of survivors and a calculated cost- per-survivor (CPS), where CPS = cost associated with release -f predicted number of survivors, 80 Fishery Bulletin 102(1 I Input number released (NR) ' assign size-at-release (SAR) * calculate cost of release (COR) <— Size-at-release N Density- independent Julian day ' assign date of release (DOR) < I ' determine number of survivors (NOS) DAL at the beginning of the day (= «\ initial # of fish or # surviving from previous day) / / 1 da ly mortality ^ da ly growth *M * calculate number of survivors and total length (TL) at the end of the day 1 I I * output - number of survivors - cost per survivor (CPS) \ / Figure 1 Model flowchart. Dashed arrows represent model "backloops" to the indicated compartment where simulations continue with the next value of the arrow-labeled variable. Side graphs indicate the three relationships between density and mortality (number offish consumed) that were considered, and the general relationship between growth and Julian day. for the initial release scenario of fish size = 30 mm TL. Julian day = 92, and an NFR input determined by the mod- eler). The model then looped back to the "date-of-release" step and simulated the release of the 30-mm-TL fish for Julian release days 93-197, outputting a predicted number of survivors and cost-per-survivor for each release date. The model then repeated all previous steps under sequentially larger size-at-release scenarios, looping back to the "size- at-release" step and simulating the release of fish ranging in size from 32-80 mm TL fish in steps of 2 mm TL. The model output was a predicted number of survivors and economic cost-per-survivor for each release day (92-197) for each size-at-release (Fig. 1). Thus, for each input NFR, there were 26 size-at-release possibilities x 105 Julian days of release possibilities, which resulted in 2730 simulations, each of which resulted in a predicted number of survivors and cost-per-survivor for that particular release scenario. For each input NFR, the results from the 2730 simulations were plotted on two response surfaces, with an .v-axis of size-at-release, a y-axis of date-of-release, and a 2-axis of either 1) predicted number of survivors (NOS), or 2) cost- per-survivor ( CPS ), to identify release scenarios resulting in the maximum predicted number of survivors and minimum cost-per-survivor, respectively. The scenarios resulting in the maximum predicted number of survivors and minimum cost-per-survivor were not necessarily identical. Calculation of mortality, growth, survival, and economic costs associated with release During each day at large (DAL), released fish were sub- jected to a density-independent daily mortality rate of 0.02153, derived from postrelease mark-recapture data of HR summer flounder (Kellison et al., 2003b). In deriv- ing this value, mean postrelease densities were used to estimate a total number of survivors from experimental releases. Daily survival was then calculated with the equation Kellison and Eggleston: Modeling release scenarios for Paraltchthys dentatus 81 NFR x SDDAL = NOS, where NFR = number released; SD = daily survival; DAL - days at large (from release date until Julian day 197); and NOS = estimated number of survivors. Daily mortality (MD) was then calculated from the equation Mr 1-Sr At the end of each simulated day, all fish that were alive increased in growth according to the equation GD = -0.0061 x Julian day + 1.2487, which was derived from mark-recapture data (Kellison, 2000), and in which GD is daily growth in millimeters. Fish reaching 80 mm TL during the model (i.e. by 15 July) were considered to make an ontogenetic hab- itat shift to deeper waters. These fish were then subjected to one half year of natural mortality to simulate mortality- related losses from deeper-water habitats (M=0.28; Froese and Pauley, 2001). Remaining fish, now having survived -one year of natural mortality, were considered to be sur- vivors (available to the commercial fishery), which is a con- servative assumption because 1-yr-old summer flounder are only partially recruited to the commercial fishery. All fish not reaching a total length of 80 mm were assumed to perish. To determine size-dependent economic costs offish pro- duction, we used the following regression equation derived for Japanese flounder (Paralichthys olivaceus) by Sproul and Tominaga ( 1992 ) because equivalent economic data for summer flounder were unavailable: CPF = 14.24 + 1.234 x TL, where CPF = the cost per fish in Japanese yen (¥); and TL = the total length of the HR fish. Costs were then converted into US$ by using an exchange rate of 106. 7¥ per 1 US$ (universal currency converter). We feel use of this cost-of-fish-production equation is appro- priate because the Japanese flounder is closely related and similar in life history traits to the summer flounder (Tanakaet al., 1989; Burke etal., 1991 ), resulting in similar optimal rearing practices for hatchery-reared Japanese and summer flounder (Burke et al., 1999), and thus likely simi- lar rearing costs. Additionally, the scale of Japanese floun- der hatchery production is similar to, or greater than, other government subsidized hatchery production programs (e.g. red drum in Texas, cod in Norway [Svasand, 1998] ). Density-mortality relationships We tested the sensitivity of the model results (optimal predicted number of survivors and cost-per-survivor esti- mates under varying NFRs) to violations of the assumption 0.50 -i ~ 0.40 ■ * Type 2 - weak k * Type 2 - strong E 0.30 - * ^—^-^ d Type 3 - weak ra k f ^^^^ ■ Type 3 - strong o t 0.20 ■ o Q. O *# ^^^^ o- 0.10 -j |^»W °°OOnnn„ 0 12 3 4 Density (number of fish/m2) Figure 2 Proportional mortality curves for juvenile summer flounder corre- sponding to weak and strong type-2 and type-3 mortality responses. of density-independent mortality by incorporating varying types and strengths of density-dependent mortality (depen- satory in nature at elevated densities; see below) into the model. As a basis for these sensitivity analyses, we assumed that predation was the driving mechanism underlying the postrelease mortality of HR summer flounder under the densities examined (Kellison et al., 2000; Kellison et al., 2003b). Thus, we made daily mortality rates correspond to either a type-2 or type-3 predator functional response (Holling, 1959; see Lindholm et al., 2001 for example), in which proportional mortality due to predation decreases with increasing density (type-2 response) or increases ini- tially with increasing density, reaches a zenith, and then decreases with increasing density (type-3 response) (Fig. 2). Both type-2 and type-3 responses result in decreasing (depensatory) mortality at elevated prey densities due to predator satiation. We did not include scenarios in which mortality increased at elevated densities (as would be expected when densities reached those likely to result in resource limitation ) because we did not include in the model elevated release densities likely to result in resource limita- tion. We parameterized the daily mortality curves so that each response (type 2 or 3) incorporated the daily mortality rate of 0.02153. These mortality curves contain mortality values that are within ranges reported in the literature for other species of juvenile marine fishes (Bax, 1983; Houde, 1987; Nash, 1998; Rose et al, 1999). To make further infer- ences about the importance of density-dependent mortal- ity to model results, we included a 1) weak and 2) strong form of each functional response (types 2 and 3) (Fig. 2), as well as scenarios in which the response shifted temporally from 3) type 2 to 3, and 4) type 3 to 2 at the midpoint of the nursery season (Julian day 145). We included both the weak and strong forms of the type-2 and type-3 functional responses to determine the extent to which variation in the strength of the functional response would affect model pre- dictions. The strength of the functional response could vary because of annual variation in the presence or abundance of prey or because predators could affect the density-mor- tality relationship (see, for example, Hansen et al., 1998). 82 Fishery Bulletin 102(1) For example, a strong positive (compensatory) density- mortality relationship driven by predators might become weaker in years when predator abundance was lower than average. We included the temporally shifting functional response scenarios to determine the extent to which tem- poral variation in the form of the functional response would affect model predictions. Temporal variation in the form of the functional response might occur because of temporal changes in the predator community, or because of changing predator-prey size dynamics (e.g. Stoner, 1980; Black and Hairston, 1988). For example, as the nursery season for summer flounder progresses, proportionately greater num- bers of juveniles grow to sizes at which they are capable of preying on smaller juveniles (Kellison, personal obs. ). If cannibalistic summer flounder exhibit a different predatory functional response from that of the predator guild commu- nity predominating earlier in the season, then the density- mortality relationship may change seasonally. We replicated all model simulations over each of the six density-mortality relationships (weak and strong types 2 and 3, and shifting patterns [type 2 to 3 and type 3 to 2] ) to determine optimal release scenarios (maximum num- ber of survivors, minimum cost-per-survivor) under each relationship. We then compared results to those obtained under density-independent mortality to make inferences about the importance of density-mortality relationships to model results. Correspondence between predicted and observed temporal abundance patterns Different density-mortality relationships may result in distinct temporal patterns of abundance (e.g. rapid versus more gradual declines in abundance) depending on initial densities. We generated predicted patterns of temporal field abundance of juvenile summer flounder under den- sity-independent mortality and four additional density- mortality relationships (governed by weak and strong type 2 and 3 functional responses) and under varying initial densities (0.1, 0.3, and 0.5 fish/m2) to examine whether the different density-mortality relationships would result in distinct temporal patterns of abundance. We used 1998-99 field data and logarithmic or polynomial regression models to generate curves that best fitted (based on r2 values) observed (from natural nursery sites) temporal declines in abundance under varying initial densities. We compared the best-fit curves to those predicted by the model under density-independent and four additional density-mortal- ity relationships. These comparisons allowed us to make qualitative inferences about which density-mortality relationship* s) resulted in the best match between pre- dicted and observed temporal patterns of abundance. Model assumptions The assumptions of the model are the following: 1 Daily mortality is independent of size. Although there is strong evidence that mortality of fishes in the wild is size-dependent (Lorenzen, 2000 ), particularly in regard to the importance of size to susceptibility to predation (see, for example, Elis and Gibson, 1995; Furuta, 1999; Manderson et al., 1999), we found no evidence (from recaptures of released hatchery-reared fish ) of size- selective daily mortality for juvenile summer flounder ranging in size from -30-80 mm TL in shallow-water nursery areas (Kellison et al., 2003a). Implications for violations of this assumption are addressed in the "Dis- cussion" section. 2 Daily growth is independent of fish density. We based this assumption on field experiments that indicated no growth limitation at densities roughly equal to the maximum densities explored in the model (Kellison et al., 2003b). Similar findings (i.e. no food-limitation or density-dependent growth) have been reported for similar-size plaice in shallow-water nursery habitats (van der Veer and Witte, 1993). 3 Economic cost per fish (CPF) is independent of the number of fish acquired for release (i.e. within the range of numbers offish released in model simulations, there is no decrease in cost per fish as the number of fish acquired from the production hatchery for release increases). This assumption is likely to be valid over changes in numbers of fish released common to stock enhancement programs (Sproul and Tominaga, 1992) but may not be valid as numbers released change over orders of magnitude because of economy of scale (Adams and Pomeroy 1991; Garcia et al., 1999). 4 There is no emigration from the release habitat until fish exhibit an ontogenetic shift in habitat at 80 mm TL. Although pre-ontogenetic habitat shift emigration may not truly be zero, we feel that it is also unlikely that pre- ontogenetic habitat-shift emigration accounts for more than a minimal amount of loss of released fish from the habitat of release, as supported by several points. First, rates of pre-ontogenetic shift emigration in wild juveniles are apparently low (Kellison and Taylor2), suggesting that large-scale spatial migrations may not be part of the behavioral repertoire of early juvenile summer flounder. Second, irregular temporally repli- cated sampling outside of experimental release sites resulted in zero captures of emigrating hatchery-reared fish (Kellison et al., 2003b). Third, emigration rates of closely related HR Japanese flounder {Paralichthys olivaceus) are reported to be very low (Tominaga and Watanabe, 1998). In combination, these points suggest that our zero emigration assumption is appropriate. 5 Fish that do not grow to 80 mm TL during the model period (i.e. by 15 July) do not survive. Although this assumption cannot be examined with our field data, data do show that juvenile summer flounder are absent from shallow-water nursery habitats by mid to late July (Kellison et al.3). Thus, all fish have either perished or made ontogenetic habitat shifts to deeper habitats by this time. Our field observations suggest that the deeper habitats to which larger flounder :t Kellison, G. T., J. C. Taylor, and J. S. Burke. 2000. Unpubl. data. Department of Marine, Earth, and Atmospheric Sciences, North Carolina State Univ., Raleigh, NC 27695-8208. Kellison and Eggleston: Modeling release scenarios for Paralichthys dentatus 83 make ontogenetic habitat shifts are inhabited by relatively high densities of potential predators (e.g. blue crabs, age 1+ flounders, red drum [Sciaenops ocellatus], searobin [Prionotus sp.], and lizardfish [Synodus sp.] ), which may be considerably less abundant in shallow-water habitats. These relatively large and abundant predators would presumably expose small migrating fish to high rates of predation (see, for example, Elis and Gibson, 1995; Furuta, 1999; Manderson et al„ 1999). This assumption is supported by research with the congener Japanese flounder (Paralich- thys olivaceus). Although a range of sizes of hatchery-reared Japanese flounder may survive within relatively shallow nursery habitats, fishes less than 90 mm TL moving into relatively deep waters are poorly rep- resented in subsequent age classes, most likely due to predation-induced mortality (Yamashita et al., 1994; Furuta, 1999). There is no relationship between length of rearing period (time spent in the hatchery environment) and probability of postrelease mortality related to behavioral deficits (Olla et al., 1998). Hatchery-specific selection pressures may result in HR fish that are behaviorally selected to survive in the hatchery and not in the wild (see Olla et al., 1998; Kellison et al., 2000; for discus- sion). We assume that behavioral deficits are not exacerbated with time spent in the hatchery (i.e. behavioral deficits are equal for all sizes-at-release). Results The most important factor affecting the number of survivors (and therefore percent survival) was size-at-release because the greatest numbers and percentages of survi- vors were always produced by releasing the largest fish possible (80 mm TL in the model). Number of survivors decreased with decreas- ing size-at-release and with increasing Julian day of release (Fig. 3A). The cost-per-survivor ( CPS ) was also most affected by size-at-release, such that CPS decreased with increasing size- at-release (Fig. 3B). CPS generally increased with increasing Julian day of release (Fig. 3B), although this effect was less important than the effect of size-at- release. Because mortality was originally assumed to be density-independent, the optimal cost-per-survivor did not vary with the number offish released (Fig. 4), and the relationship between number offish released and number of survivors was linear (Fig. 4), such that the maximum number of survivors were generated from the greatest number offish released (NFR=400,000). 220 20 80 90 220 Figure 3 Response surfaces of iAi number offish survivors (summer flounder I and (Bi cost-per-survivor (CPS) as a function of date of release and size at release at number released (NR) = 5000 (postrelease density=0.05) under density-independent mortality. CPS values greater than $10 were set equal to $10 for ease of presentation. Sensitivity of model predictions to violations of density-independent mortality assumption Model results varied considerably under the various den- sity-mortality relationships (Fig. 5, A and B), indicating the importance of knowledge of the relationship between numbers of fish released (density) and mortality in the wild to predicting optimal release scenarios. Variation in model output was dependent on the type and strength of Fishery Bulletin 102(1) the density-mortality relationship. For example, at postre- lease densities of 0.5 fish/m2 (NFR=50,000), survival of released flounder under density-independent mortality was ~28% higher than that predicted under strong type-3 mortality, but only -2% higher than that predicted under weak type-2 mortality (Fig. 5A). At postrelease densities of 0.001 fish/m2 (NFR=100), survival of released flounder under density-independent mortality was ~41% higher 450000 -I m 400000 • § 350000 • £ 300000 • « 250000 ; ° 200000 ■ E 150000 ■ | 100000 ■ z 50000 ■ — ■ — optimal number of survivors : — o— optimal CPS ^^^" r 1 60 : 1 50 O ■ 1.40 g ■ 1 30 -g -1,20 5 [110 <§ - 1 00 g ■0.90 < ■0 80 - ■ 0.70 C ■ 0-60 W 0 50000 10000 15000 20000 25000 30000 35000 40000 Number released Figure 4 Optimal number of fish survivors and cost-per-survivor as a function of varying numbers of summer flounder released under density-indepen- dent mortality. 0 12 3 4 5 Density (number of fish/m2) Figure 5 l A i Optimal percent survival and iBi optimal cost-per-survival (US$) as a func- tion of postrelease density undci density-independent and varying density- dependent, mortality relationships for summer flounder. than that predicted under strong type-2 mortality, but -2% less than that predicted under strong type-3 mortality ( Fig. 5A). In contrast, when postrelease densities were relatively high, there was less of an impact of density-mortality rela- tionship on postrelease survival and costs associated with stock enhancement. For example, at postrelease densities of three fish/m2 (NFR=300,000), survival of released floun- der differed by less than 4% between density-independent, weak or strong type-2, and weak type-3 mor- tality, although survival under strong type-3 mortality was ~99c less than that predicted under density-independent mortality and -11% less than that predicted under strong type-2 mortality (Fig. 5A). Thus, the model results were most sensitive to violations of the assumption of density-independent mortality at low densities offish released in the field. Type-2 mortality As with density-indepen- dent mortality, the most important factor affecting number of survivors and cost per survivor under type-2 mortality was size-at- release (Fig. 6, A and B). In all simulations, the greatest number of survivors was pro- duced by releasing the largest fish possible. Number of survivors decreased with increas- ing Julian day of release (Fig. 6A). There was a considerable interaction between size- at-release and number of fish released, such that low postrelease densities were subjected to relatively high proportional mortality. Thus, when fish were released in low numbers and at small sizes, the fish were subjected to relatively high proportional mortality rates for long periods of time (while they grew towards the 80-mm-TL ontogenetic shift size) and consequently produced few or no survi- vors (Fig. 6A). Optimal release scenarios under strong type-2 mortality produced substantially lower (>40% in some cases) percent survival (and therefore substantially higher cost-per-survivor) estimates at low to moderate numbers released (NFR= 100-50,000; postrelease density=0.001-0.5 fish/m2) than under density-independent mortality (Fig. 5, A and B). Differences in percent survival estimates (and thus cost-per-survivor estimates) between density-indepen- dent survival and weak or strong type-2 mortality declined to less than 5ri when the numbers released increased to 25,000 (postrelease density=0.25 fish/m2) under weak type-2 mortality and 75.000 (postrelease density=0.75 fish/m2) under strong type-2 mortality (Fig. 5A). Thus, model predictions under density-inde- pendent mortality differed most from predictions under mortality governed by - density-independent -type 2 - weak - type 2 - strong -type 3 - weak ■type 3 - strong density-independent type 2 - weak type 2 - strong type 3 - weak type 3 - strong Kellison and Eggleston: Modeling release scenarios for Paralichthys dentatus 85 B a type-2 predator functional response when postrelease densities were relatively low. Type-3 mortality As in all other simulations, the most important factor affecting number of survivors under type-3 mortality was size- at-release, such that the greatest numbers of survivors were always produced by releasing the largest fish possible (Fig. 7A). Number of survivors decreased with increasing Julian day of release (Fig. 7A). Percent survival was considerably lower (>25% in some cases) under type-3 mortality than under density- independent mortality at moderate to high numbers released (NFR=10, 000-400, 000) (Fig. 5 A). In nearly all simulations, the lowest CPS values were produced by releasing the larg- est fish possible (Fig. 7B). The exceptions to the "large size = optimal CPS" rule occurred when postrelease densities were small (cor- responding to numbers released of 100, 500, and 1000) and the mortality curve was type 3 (weak or strong). In these instances, mortality was sufficiently low at low release densities ( Fig. 7B ) so that the difference in overall sur- vival between small- and large-released fish was small enough to be overridden by the in- creased cost of the larger fish, and the mini- mum CPS was obtained when small (42-44 mm TL) fish were released (e.g. Fig. 7B). At low numbers released (NFR=100-1000), optimal cost-per-survivor was considerably lower (>45% in some cases) under type-3 mortality than under density-independent mortality (Fig. 5A). As NFR increased, CPS under type-3 mortality became greater ( -40^ in some cases) than that achieved under den- sity-independent mortality (Fig. 5B). Temporal shift in functional response from type 2 to type 3, and from type 3 to type 2 The optimal numbers of survivors under varying numbers released were identical, and optimal CPS values nearly identical, when the form of the functional response changed from a type 2 to a type 3, and from a type 3 to a type 2, midway through the juvenile nurs- ery season (Fig. 8, A and B). The differences at low postrelease densities between optimal CPS values under shifting type 2 to type 3 and type 3 to type 2 scenarios (Fig. 8A) occurred because initial mortality under the type-3 functional response was sufficiently low that the difference in overall survival between small- and large-released fish was small enough to be overridden by the increased cost of the larger fish (Fig. 8A). The minimum CPS was obtained when small (42-44 mm TL) fish were released (in all other cases, optimal results were obtained when size-at-release was maximized) (Fig. 8A). The major difference between the two shifting scenarios is that the re/ease Figure 6 Response surfaces of (A) number offish (summer flounder I survivors and (B) cost-per-survivor (CPS) as a function of date of release and size at release at number released (NR) = 5000 (postrelease density=0.05l under a strong type-2 functional response. CPS values greater than $10 were set equal to $10 for ease of presentation. release dates producing optimal results for a given number of fish released varied depending on the direction of the shifting functional response. For example, when the func- tional response shifted from a type 2 to a type 3, a release of 100,000 HR organisms achieved optimal results when release occurred early in the season (Julian day <145) (Fig. 9A). When the functional response shifted from a type 3 to a type 2, a release of 100,000 HR summer floun- der achieved optimal results only when releases occurred later in the season (Julian day >145) (Fig. 9B). When the 86 Fishery Bulletin 102(1) functional response shifted from a type 3 to a type 2, releas- ing 100,000 HR organisms prior to Julian day 146 resulted in markedly decreased survival (and therefore increased CPS ) compared to results obtained from releases after day 146 (e.g. releasing on Julian day 92 resulted in a decrease in number of survivors and an increase in CPS of 22.8% and 29.7%, respectively) (Fig. 9B). Thus, date-of-release had a significant effect on the results (and therefore in determining optimal release strategies) when the relation- ship between density and mortality changed temporally, suggesting that the presence of a temporal shift in the func- 500 £ 400 300 200 E z 100 220 OaV' Size at re/ease Figure 7 Response surfaces of (A) number offish (summer flounder) survivors and (B) cost-per-survivor (CPS) as a function of date of release and size at release at number released (NR) = 500 (postrelease density=0.005) under a strong type-3 functional response. CPS values greater than $10 were set equal to $10 for ease of presentation. tional response of the predator guild would have consider- able effects on the number of survivors and CPS for stock enhancement efforts with juvenile summer flounder. Correspondence between predicted and observed temporal abundance patterns Under the assumption of a type-2 functional response, predicted declines in juvenile summer flounder density over time were rapid when initial density was relatively low (i.e. 0.1 fish/m2) (Fig. 10, A and B). These predictions contrast with those observed in the field, in which declines at relatively low initial densities were gradual (compare Fig. 10A and 10B to Fig. 10F). Under the assumption of a type-3 functional response, predicted declines were rapid when initial density was relatively high (i.e. 0.5 fish/m2) I Fig. 10, C and D). These results generally contrast with those observed in the field, in which declines at relatively high densities were much less rapid than those predicted under a strong type-3 functional response, and somewhat less rapid than those predicted under a weak type-3 functional response (Figs. 10F and 11). Under density-independent mortality, there was little difference in predicted declines in juvenile summer flounder density over time between the three initial density levels (0.1, 0.3, and 0.5 fish/m2); in each case there was a gradual decrease in density over time (Fig. 10E). These results were similar to those observed in the field, although declines at rel- atively high densities in the field were some- what more rapid than those predicted under density-independent mortality ( compare Figs. 10E and 10F). Thus, a density-mortality rela- tionship lying between that generated under density-independence and that generated under the weak type-3 functional response in the model would most closely predict the temporal declines observed in the field. Discussion Implications for stock enhancement of summer flounder Regardless of the relationship between den- sity and mortality, size-at-release was the most important variable in the model affect- ing survival and costs associated with stock enhancement of summer flounder. The model predicts that under all release scenarios, 1) survival will be maximized and 2) costs asso- ciated with stock enhancement (i.e. cost per survivor) will be minimized when HR fish are released at the largest size possible. From a survival standpoint, these results are not Kellison and Eggleston: Modeling release scenarios for Paralichthys dentatus 87 surprising. Larger fish spend fewer days than smaller fish in the wild nursery habitats before making an ontogenetic habitat shift to deeper waters and thus are susceptible to daily natural mortality for fewer numbers of days than are smaller fish. Thus, total mortality of smaller fish is greater than that of larger fish. Additionally, although we chose to make mortality independent of size in the model, abundant literature suggests that natural mortality (especially due to predation ) may decrease with increasing size by mecha- nisms such as enhanced resistance to starvation, decreased vulnerability to predators, and better tolerance of environ- mental extremes (Sogard, 1997; Hurst and Conover, 1998; Lorenzen, 2000). Thus, the difference in predicted survival between 1 ) relatively large and relatively small fish and 2 ) fish released early versus late in the season in our model would be even greater if larger summer flounder suffered lower natural mortality than smaller fish. Furthermore, the daily mortality estimate used in the density-inde- pendent simulations and to parameterize the different types of density-mortality relationships may have been an underestimate of daily mortality (Kellison, 2000). If a greater estimate of daily mortality had been used, the dif- ference in predicted survival between relatively large and relatively small fish in our model would have been further exacerbated because smaller fish spend longer amounts of time in the model growing to the 80-mm-TL ontogenetic shift size. These conclusions are supported by empirical research demonstrating that relatively large released HR fish suffer lower mortality than relatively small HR fish released in the field (e.g. Yamashita et al., 1994; Leber, 1995; Willis et al., 1995; Tominaga and Watanabe, 1998; Svasandetal.,2000). Although the survival predictions of the model (total mortality decreases with increasing size-at-release) are not surprising, the economic (cost-per-survivor) predic- tions were unexpected. The paradigm for stock enhance- ment strategy is that the rearing of relatively large fish for release is cost prohibitive, so that mass releases of relatively small, inexpensive-to-rear fish are a better strategy than the release of larger, expensive-to-rear fish (Kellison, personal obs.). Thus, relatively small juveniles are released in virtually all current stock enhancement programs (e.g. Russell and Rimmer, 1997; Masuda and Tsukamoto, 1998; McEachron et al., 1998; Svasand, 1998; Serafy et al., 1999). Nevertheless, large-scale hatcheries and grow-out facilities are using ever-increasing technol- ogy to minimize the costs associated with the production of relatively large fishes (Sproul and Tominaga, 1992). Thus, for species for which 1) hatcheries are capable of producing relatively large fish at relatively low costs (as is likely for summer flounder), and 2) postrelease survival rates increase with release size, release scenarios utilizing the largest fish possible may maximize the potential (i.e. produce maximum survival at minimum costs ) of stock en- hancement efforts. In these cases, the "small fish maximize stock enhancement potential" paradigm might be replaced with a "large fish maximize potential" paradigm. As a ca- veat, this "large fish" strategy may be limited by spatial limitations of hatcheries in producing large numbers of relatively large fish. Because reared fish generally must 1 40 i */» ^_ 1.20- o 2 1 00- w 0 80- (1> Q. 0 60- If) O 0 40- O Type 2 to 3 Type 3 lo 2 0 20-1— -! , 1 r Postrelease density Figure 8 Optimal lA) economic cost-per-survivor and (B) per- cent survival of released hatchery-reared summer flounder under temporally shifting functional re- sponses of type 2 to type 3 and type 3 to type 2. be kept below critical densities in hatchery environments because of water quality and fish interaction issues (e.g. cannibalism), larger fish necessarily require more space than smaller fish for rearing. If the demand for space to rear large quantities of large fish can be realized, then the model simulations indicate that stock enhancement strat- egies in which size-at-release is maximized will produce the maximum number of survivors. Although not as important as size-at-release, Julian day of release had a significant effect on survival and cost-per- survivor in the model, such that enhancement efforts were always more successful (more survivors, lower costs) when fish were released at the earliest Julian day possible. These results occurred because growth in the model decreased with increasing Julian Day. Although the mechanisms un- derlying this decrease in growth with increasing Julian day are unknown, they may be related to decreased prey avail- ability or metabolic efficiency as temperatures increase with increasing Julian day (Malloy and Targett, 1994a, 1994b; Fujii and Noguchi, 1996; Howson, 2000). Thus, for a given size-at-release, fish released earlier in the season experienced greater growth rates than fish of the same size-at-release released later in the season and therefore reached the 80-mm-TL ontogenetic shift size faster (over a period of fewer days) than fish released later in the season. Thus, fish released earlier in the season were susceptible to natural mortality for fewer days than fish released later in the season and therefore suffered lower total mortality. These results emphasize the importance of knowledge of possible time-dependent growth in the field prior to stock enhancement efforts. Fishery Bulletin 102(1) Is density important? Effects of varying density-mortality relationships Our results suggest that the relationship between density and mortality has the potential to significantly affect opti- mal release scenarios associated with stock enhancement efforts. Because the original simulations were performed under density-independent mortality, the number of survivors originally increased linearly with the number B 1e+5 (/> 8e+4 o > > 6e+4 tfl n m 4e+4 a h 3 2e+4 z 0 80 released, resulting in a density-independent cost-per- survivor. Thus, when mortality is independent of density (over a given range of densities) for a target species for stock enhancement, managers will maximize the number of survivors produced by releasing the greatest number of fish possible within that range for a given size class. When mortality varied with density of released fish, the number of survivors and cost-per-survivor depended on the den- sity-mortality relationship. In some cases, optimal results (maximum survival and minimum cost) differed depending on whether the response variable was number of survivors or cost-per-survivor. Under the assumption of a strong type-3 functional response and under relatively low postrelease densities, survival was optimized (maximized) by releasing the largest fish ( 80 mm TL) possible; however, cost-per-survivor was optimized (mini- mized) by releasing smaller fish (42-44 mm TL). This result occurred because mortality at low postrelease densities was sufficiently low that the difference in total mortality attributed to the longer "susceptibility" period of the smaller fish was insufficient to override the economic advan- tage of releasing smaller fish. Simulations under shifting functional responses (type 2 to type 3 and type 3 to type 2) produced optimal results similar to those obtained when nonshifting type- 2 or type-3 functional responses were employed because densities were generally reduced to such low numbers by the time the shift occurred that the changing density-mortality relationship was inconsequential. Importantly, when functional responses shifted temporally, the predicted number of survivors and economic cost per survivor was at times very dependent on date of release, suggesting that identifying or ruling out shifting functional responses in the wild may be critical to accurate prediction of response vari- ables (survivors and economic costs) associated with stock enhancement. Although we are not aware of reports in the literature of shifting functional responses in the wild, we are also not aware of studies that have tested for such a phenomenon, possibly because of the logisti- cal difficulties inherent in identifying a shifting functional response. Correspondence between predicted and observed temporal abundance patterns Figure 9 (A) Response surface of optimal number of summer flounder survivors as a function of date of release and size at release at number released (NR) = 100,000 (postrelease density=1.0 fish/m2 1 under the assumption of a temporally shifting functional responses from type 2 to type 3. Response surfaces of optimal number of survivors as a function of date of release and size at release at number released (NR) = 100. 000 (postrelease density=1.0 fish/m2 > under the assumption of a temporally shifting functional responses from type 3 to type 2. Predictions of field abundance patterns of juve- nile flounder density over time were noticeably different under density-independent mortality and density-dependent mortality governed by type-2 and type-3 functional responses. For example, our simulations predict that fish den- sity should decrease rapidly under relatively low initial densities if the functional response is type 2, decrease rapidly at relatively high initial densities if the functional response is type 3, and Kellison and Eggleston: Modeling release scenarios for Parahchthys dentatus 89 OS- 04- 0.3 0.2 01 00 E Strong type 2 Strong type 3 150 Dl B Weak type 2 05 0.4 0 3 02 110 130 150 170 190 210 D Weak type 3 Julian day Figure 10 Predicted temporal trends in summer flounder abundance under initial densities of 0.5, 0.3, and 0.1 fish/m2 under the assumption of a functional response that is a (A) strong type 2. IB) weak type 2, (C) strong type 3. i D i weak type 3. and under the assumption of (E) density-independent iDIl mortality. The curves in iFi are best fitted (highest r2 value) to data collected in Duke Beach 1999 (curve a, r2=0.82). Haystacks Marsh 1999 (curve b, r2=0.73), Prytherch Marsh 1999 (curve c. ;-2=0.82), Towne Beach 1999 (curve d, r2=0.91). Radio Beach 1999 (curve e, r2 = 0.27), Duke Beach 1998 (curve f, r2=0.31), and Prytherch 1998 (curve g, r2=0.16) (see Fig. 11 for data). gradually decrease regardless of initial density if mortal- ity is density independent. From examinations of tempo- ral abundance patterns from several nursery sites (see Kellison et al., 2003b, for site descriptions), it is evident that observed declines at relatively low initial densities are similar to predicted declines under both density-inde- pendent mortality and a weak type-3 functional response; whereas observed declines at relatively high initial densi- ties are somewhat less gradual than predicted under den- sity-independent mortality, but somewhat more gradual than predicted under the weak type-3 functional response. These results suggest that model predictions made under the assumption of a weak type-3 response may give rea- sonably accurate but conservative predictions of juvenile summer flounder mortality and economic costs associated with stock enhancement for comparison with alternative management methods. As a caveat, although we found no evidence of size-dependent daily mortality over the range of fish sizes examined in this study, it is very likely that such a relationship exists to some extent (Sogard, 1997; Lorenzen, 2000). Incorporating size-dependent mortality into the model would decrease the slopes of the predicted temporal abundance curves but should not change the conclusion that the observed data lie somewhere between values predicted under density-independent mortality and those governed by a weak type-3 functional response, respectively. Additionally, because the portions of the curves used to delineate between temporal abundances expected under density-independent versus varying den- sity-mortality relationships are from early in the growth season (later parts of the curve converge on very low den- sities) and because nearly all fish in these portions of the curves are at sizes well below that at which ontogenetic emigration occurs, the exclusion of emigration from these simulations should not affect the general conclusions reached. These issues could be clarified with further field trials to investigate the dependence of daily mortality rates on fish size. 90 Fishery Bulletin 102(1) E E Prytherch 1999 Radio 1999 003 ♦ Prytherch 1998 * r* = 0.1575 0 02 ♦ 001 * _. ♦ ♦♦ 95 105 115 125 135 145 155 165 B 03 ♦ Duke 1999 0 2 • ♦ r = 0 8162 01 ♦ 9* ♦ * ♦*4U**TMT *' •*♦ D Haystacks 1999 Towne 1999 r" i 0 9063 ^s^ • ♦ "" "Y — ♦ ♦♦ » '«W. W. LT» 95 115 135 155 175 195 Duke 1998 0 1 ♦ ♦ * r2 = 03113 0.05 • * **> ♦. • ♦ ♦ ~~7 • ♦ ♦ ♦ 95 115 135 155 175 195 Julian day Julian day Figure 11 Temporal density patterns from (A) Duke Beach, 1999; (B) Haystacks Marsh, 1999; (C) Prytherch Marsh, 1999; (D) Towne Beach, 1999; (E) Radio Beach, 1999; (F) Duke Beach, 1998; and (G) Prytherch Marsh 1998. Densities are corrected for gear bias (see Kellison, 2000). Model utility and implications Although model results varied considerably under the various density-mortality relationships, the overall pre- dictions that survival would be maximized and economic costs minimized when relatively large fish were released early in the season were unaffected by the density- mortality relationship. These results suggest that manag- ers may use this model to make inferences about optimal release scenarios even if density-mortality relationships are unknown. Additionally, these results have important implications for the cost efficiency of stock enhancement programs. Managers can use the model to determine the release scenarios under which they can 1) maxi- mize the number of survivors, given a financial limit (e.g. given a budget of x dollars, what release scenario or scenarios will produce the greatest number of survi- vors?), and 2) minimize costs, given a goal of number-of- survivors-produced (e.g. given a goal of producing .v survivors, what release scenario or scenarios will be most cost efficient?). In conclusion, the compartmental model used in this study provides an example of a relatively easy-to-develop predictive tool with which to make inferences about the ecological and economic potential of stock enhancement, in relation to alternative management approaches, to rebuild depleted fisheries. Kellison and Eggleston: Modeling release scenarios for Paraltchthys dentatus 91 Acknowledgments We thank Brian Burke (NCSU) for tutelage in the use of Visual Basic. Mike Denson (South Carolina Department of Natural Resources) and Pete Schuhmann (UNC-Wilm- ington ) greatly contributed to the editing of an earlier ver- sion of this manuscript. Mark Wuenschel, Michael Martin, Brian Degan, Lisa Etherington. and Mikael Currimjoe pro- vided valuable laboratory and field assistance necessary for parameter estimation. This project was partially funded by the University of North Carolina at Wilmington/North Carolina State University Cooperative Ph.D. Program, and a grant from the National Science Foundation (OCE 97- 34472) to D. Eggleston. Literature cited Adams, C. M.. and R. S. Pomeroy. 1991. Scale economies in hard clam aquaculture. J. Shell- fish Res. 10:307-308. Barbeau. M. A., and H. Caswell. 1999. A matrix model for short-term dynamics of seeded populations of sea scallops. Ecol. 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Flounder aquaculture and stock enhancement in North Carolina: issues, opportunities and recommendations. NC Sea Grant Publication, UNC-SG-99-02, 24 p. Sea Grant, Raleigh, NC. Willis, S. A.. W. W Falls, C. W. Dennis, D. E. Roberts, and P. G. Whitechurch. 1995. Assessment of season of release and size-at-release on recapture rates of hatchery-reared red drum. Uses and effects of cultured fishes in aquatic ecosystems. Am. Fish. Soc. Symp. 15:354-365. Yamashita, Y, S. Nagahora, H. Yamada, and D. Kitigawa. 1994. Effects of release size on survival and growth of Japa- nese flounder Paralichthys olivaceus in coastal waters off Iwate Prefecture, northeastern Japan. Mar. Ecol. Prog. Ser. 105:269-276. 94 Abstract— Sex-specific demography and reproductive biology- of stripey bass [Lutjanus carponotatus l I also known as Spanish flag snapper. FAO ) were exam- ined at the Palm and Lizard island groups, Great Barrier Reef ( GBR). Total mortality rates were similar between the sexes. Males had larger L . at both island groups and Lizard Island group fish had larger overall L_,, Female:male sex ratios were 1.3 and 1.1 at the Palm and Lizard island groups, respectively. The former is statistically different from 1, but is unlikely significantly different in a biological sense. Females matured on average at 2 years of age and 190 mm fork length at both loca- tions. Female gonadal lipid body indices peaked from August through October, preceding peak gonadosomatic indices in October, November, and December that were twice as great as in any other month. However, ovarian stag- ing revealed 50^ or more ovaries were ripe from September through February, suggesting a more protracted spawning season and highlighting the different interpretations that can arise between gonad weight and gonad staging meth- ods. Gonadosomatic index increases slightly with body size and larger fish have a longer average spawning season, which suggests that larger fish produce greater relative reproductive output. Lizard Island group females had ovaries nearly twice as large as Palm Island group females at a given body size. However, it is unclear whether this reflects spatial differences akin to those observed in growth or effects of sampling Lizard Island group fish closer to their date of spawning. These results support an existing 250 mm minimum size limit for L. carponotatus on the GBR, as well as the timing of a proposed October through December spawning closure for the fishery. The results also caution against assessing reef-fish stocks without reference to sex-, size-, and location-specific biologi- cal traits. Sex-specific growth and mortality, spawning season, and female maturation of the stripey bass {Lutjanus carponotatus) on the Great Barrier Reef Jacob P. Kritzer School of Marine Biology & Aquaculture and CRC Reef Research Centre-Effects of Line Fishing Project James Cook University Townsville. Queensland 4811, Australia Present address: Department of Biological Sciences University of Windsor 401 Sunset Avenue Windsor, Ontario N9B 3P4, Canada E-mail address kntzenSuwindsorca Manuscript approved for publication 22 July 2003 by Scientific Editor. Manuscript received 22 July 2003 at NMFS Scientific Publications Office. Fish Bull. 102:94-107 (2004). Lutjanid snappers are among the most prominent species comprising the catch of hook-and-line fisheries on tropical reefs worldwide (Dalzell, 1996). A notable exception is the line fishery on Australia's Great Barrier Reef (GBR). There, the finfish catch, and therefore the majority of fisheries research, is dominated by coral trouts of the genus Plectropomus (Mapstone et al.1). However, the GBR finfish harvest is diverse and the catch of many sec- ondary species has risen steadily since the early 1990s (Mapstone et al.1). Furthermore, over the past decade, the GBR fishery has changed with the advent of the lucrative Asian live reef- fish market. At present, only a handful of the many species harvested on the GBR are exported to the live reef-fish market. However, continued expansion of the trade coupled with the depletion of fish stocks in other source nations (Bentley2) has the potential to intro- duce demand for a wider range of spe- cies. Even in the absence of changes in the species composition of live reef-fish exports, increased demand for second- ary species due to changes in either domestic preferences or availability of primary species has the potential to elevate harvest of currently nontarget species (Kritzer, 2003). Effective multispecies management of the GBR fishery will ultimately re- quire understanding the biology of more than simply the primary target species. For example, spawning closures of the fishery have been proposed for nine-day periods around the new moon in Octo- ber, November, and December on the rationale that this will protect spawn- ing activity of a wide range of harvested species (Queensland Fisheries Manage- ment Authority3). Yet, spawning season information for species beyond the com- mon coral trout {P. leopardus ) ( Ferreira, 1995; Samoilys. 1997 ) is nearly nonexis- tent. The GBR fishery is in a fortunate position with respect to management of many species for which exploitation is still at relatively low levels because baseline biological characteristics can be estimated before stock structure is drastically altered by fishing. These da- ta can then be used in both formulating management strategies and monitoring effects of fishing. 1 Mapstone. B. D.. J. P. MacKinlay, and C. R. Davies. 1996. A description of the com- mercial reef line fishery log book data held by the Queensland Fisheries Management Authority. Report to the Queensland Fisheries Management Authority. 480 p. Primary Industries Building, GPO Box 4(i. Brisbane. Queensland 4001. Australia. 2 Bentley. N. 1999. Fishing for solutions: can the live trade in wild groupers and wrasses from Southeast Asia be managed? TRAFFIC Southeast Asia report. 143 p. Unit 9-3A, 3rd Floor. Jalan SS23/11, Taman SEA. 47400 Petaling Java, Selan- gor, Malaysia. 3 Queensland Fisheries Management Auth- ority. 1999. Queensland coral reef fin fish fishery. Draft management plan and regulatory impact statement, 80 p. Pri- mary Industries Building. GPO Box 46, Brisbane, Queensland 4001, Australia. Kritzer: Sex-specific growth and mortality, spawning season, and female maturation of Lut/anus carponotatus 95 One of the most prominent secondary species in the GBR fishery is the stripey bass (Lutjanus carponotatus) (Spanish flag snapper. FAO). In relation to other large predators on the GBR, L. carponotatus is highly abundant on inshore reefs, common on mid-continental shelf reefs, and absent from outer-shelf reefs (Newman and Williams, 1996; Newman et al., 1997; Mapstone et al.4). Although this affinity for inshore reefs has the potential to make the species more susceptible to recreational fishing, the limited available data do not suggest that it is heavily exploited by the recreational fleet (Higgs, 1993) in relation to the commercial fleet (Mapstone et al.1). Lutjanus carponota- tus has a broad-based diet, consuming a wide variety of smaller reef fishes and invertebrates (Connell, 1998). Its role as a predator coupled with its abundance, particularly on inshore reefs, suggests that the species might have an important ecological function on the GBR in addition to its role as a fishery resource. Davies (1995) and Newman et al. (2000) have collected basic demographic data for L. carponotatus on the north- ern and central GBR, respectively. They both reported a pronounced asymptote in the growth trajectory and that most growth occurred over the first three to five years and little subsequent growth over a lifespan that can reach 15 to 20 years. Newman et al. (2000) also reported a heavily male-biased sample and larger body sizes among males. Unlike age and growth data, no information on reproduc- tion of L. carponotatus has been available despite that fact that existing (minimum size limits) and proposed (spawn- ing closures) fisheries regulations are based largely on reproductive traits (Queensland Fisheries Management Authority3). Specific aims of this study were 1) to estimate sex ra- tios and sex-specific schedules of growth and mortality; 2) to estimate age- and size-specific schedules of female maturation; 3) to identify the spawning season; and 4) to determine whether reproductive output is proportional to body size by examining the ovary weight-body weight relationship and the average spawning duration of large and small fish. All traits were estimated at the Palm Island group on the central GBR. Additionally, sex-specific growth and female maturity schedules were also examined at the Lizard Island group on the northern GBR to develop spa- tial comparisons. Materials and methods Field methods Size, age, and reproductive data were obtained for 465 L. carponotatus collected by spear fishing on fringing reef slopes during monthly fishery independent sampling at 4 Mapstone, B. D., A.M. Ayling, and J. H.Choat. 1998. Habitat, cross shelf and regional patterns in the distributions and abun- dances of some coral reef organisms on the northern Great Bar- rier Reef. Great Barrier Reef Marine Park Authority research publication 48, 71 p. GPO Box 1379, Townsville, Queensland 4810, Australia. Pelorus, Orpheus, and Fantome Islands in the Palm Island group on the central GBR ( Fig. 1 ) from April 1997 through March 1998. No sampling took place in January 1998 because of severe flooding in the area. To develop spatial comparisons, samples of 118 and 18 fish were obtained in October 1997 and April 1999, respectively, by spear fishing at the Lizard Island group approximately 400 km north of the Palm Island group (Fig. 1). Fish were collected from depths of 2 to 15 m by teams of two to four scuba divers. Lutjanus carponotatus most commonly inhabits depths less than 15 m (Newman and Williams, 1996); therefore sam- pling efforts encountered the majority of the population. Fish were targeted as encountered, without preference based on size, in order to collect as representative a sample as possible. Fish <150 mm fork length (FL) were rare in the samples because they were infrequently observed on reef slopes (Kritzer, 2002). Therefore, supplemental spear fishing on reef flats targeting smaller fish was conducted at the Palm Island group (n=24) in April and December 1999 and at the Lizard Island group (n=25) in May 1999 to obtain growth data for size classes against which the primary sampling was biased. Total weight (TW, g) and FL (mm) of each specimen were recorded. Ovaries and testes of small lutjanids on the GBR are characterized by a lipid body running along the length of each lobe, akin to that found in tropical acanthurids (Fishelson et al., 1985). Gonads and these associated lipid bodies were removed and preserved in FAAC (formaldehyde 4%, acetic acid 5%, calcium chloride 1.3%). Sagittal otoliths were removed, cleaned, and stored for later analyses. Gonad processing and ovarian staging The lipid body was removed from each ovary or testis after fixation and the weight of the gonad (GW) and lipid body (LW) were measured to the nearest 0.01 g. A gonadoso- matic index (GSI) and lipidsomatic index (LSI; after Lobel, 1989) were calculated for each sample as the percentage of TW represented by GW and LW, respectively. Features of whole fixed ovaries including color, speckling, and surface texture were noted as potential criteria for macroscopic staging after comparison with samples processed histologi- cally. Sex of the April 1999 Lizard Island group samples was determined macroscopically only, and was therefore used in sex-specific growth analyses but not in analysis of maturity. Fish <150 mm FL had undeveloped gonads and sex of these specimens was not determined or assigned a reproductive stage. A subsample of 131 ovaries spanning the range of gonad sizes and external appearances were prepared for histo- logical examination. Samoilys and Roelofs (2000) found that medial gonad sections were adequate for determina- tion of reproductive status. Therefore, a medial section was removed from one gonad lobe, dehydrated, and embedded in paraffin. Embedded ovarian tissues were sectioned at 5 nm and stained with hematoxylin and eosin. Ovaries were staged on the basis of the most advanced oocyte stage pres- ent (West, 1990). Additional features used in histological staging included the presence of brown bodies and atretic 96 Fishery Bulletin 102(1 120° 130° N 4 Australia Great LG Barrier 15° Reef PG Queensland 35° Lizard Island group (LG) J\ Lizard Island <\ V-— V^" ,25km, Palfrey Q . ' ' Island _ Seabird Islet South Island Palm Island group (PG) Pelorus Island Brisk Island )). where Lt = FL at age t\ L^= the mean asymptotic FL; A' = the Brody growth coefficient; and t0 = the age at which fish have theoretical FL of 0. Growth functions were fitted by nonlinear least-squares regression of FL on age by using samples for which sex was determined. Because VBGF parameter estimates can be sensitive to the range of ages and sizes used (see Ferreira and Russ. 1994, for an empirical example), a common t0 equivalent to the .v-intercept of the early growth estimates was used in all models (see "Results" section). Although the sex-specific sample sizes at the Lizard Island group were smaller (n=65 for females; n=62 for males), VBGF parameter estimates achieved high precision at sample sizes between 50 and 100 (Kritzer et al., 2001); therefore the Lizard Island group data were included in the analy- sis. Growth parameters were compared by plotting 959c confidence regions of the parameters K and Lx (Kimura, 1980) for each sex from each location and assessing the degree of overlap. Sex-specific total mortality rates, Z, were estimated by using the age-based catch curve of Ricker (1975) as the slope of a linear regression of natural log-transformed fre- quency on age class. Everhart and Youngs ( 1981 ) proposed that catch curve analysis should exclude age classes with n<5 and Murphy ( 1997) proposed that age structures used in catch curves should be truncated at the first age class with n<5. Alternatively, Kritzer et al. (2001) proposed that a sample should contain an average of at least ten fish per age class irrespective of age class-specific sample sizes. Therefore catch curves were fitted by two different methods for each sex at the Palm Island group. The first catch curve began at the modal age class and stopped before the first age class with n < 5. The second catch curve likewise began at the modal age class but included all age classes that were thereafter represented in the data set. Sex-specific sample sizes for the Lizard Island group were too small by any of these criteria and this location was excluded. Mor- tality estimates for Palm Island group fish were compared between the fitting methods within each sex as well as between sexes by ANCOVA. Reproductive biology Maturation schedules of female fish were estimated for each island group by fitting a logistic model, P, = l/(l + exp(a-W)), where P- = the proportion of mature fish in age or 20-mm size class i; a adjusts the position of the curve along the abscissa; and r determines its steepness. Age- and size-specific maturity functions were used to estimate the mean age, r50, and size, L50, at which 50% of females are mature at each island group. Monthly mean LSI and GSI values of mature Palm Island group fish were plotted separately for males and females to determine seasonal patterns of energy storage and the peak spawning period of L. carponotatus. The pro- portion of specimens at each mature female reproductive stage in each month was also plotted to examine ovarian development patterns throughout the year and the degree of spawning activity occurring outside of peak months. To examine whether relative reproductive output in- creases with body size, GW and GSI for stage-IV ovaries collected during peak spawning months were regressed against TW. Residual plots were used to assess deviation from a linear relationship and to identify three outliers, which were removed from the regression analysis. Regres- sion slopes were compared between the two island groups by ANCOVA. Also, mean GSI values and the proportion of Palm Island group females with stage-IV ovaries during spawning months were compared between females <230 mm FL and those >230 mm FL to examine whether the duration of spawning varies between size classes (nota bene: 230 mm FL is approximately the mean size of mature Palm Island group females and splits each month's sample approximately in half). Results Ovarian staging Five female reproductive stages were identified through histological analysis (Table 1) and were based largely on the scheme of Samoilys and Roelofs (2000). Ovarian stages I (immature) and II (resting mature) have similar oocyte stages. These can be distinguished by the presence of brown bodies or atretic oocytes, which are typically prod- ucts of prior spawning (e.g. Ha and Kinzie, 1996; Adams et al., 2000) and are usually absent from stage-I ovaries. However, these structures will not necessarily persist in ovaries that have spawned, and in fact were rare among the samples; therefore identification of immature females was based primarily on structural organization of the ovary. Stage-I ovaries typically have a thin ovarian wall and more compacted oocytes, whereas ovaries that have previously spawned tend to have a thicker ovarian wall and a more disorganized arrangement of oocytes (Table 1). Also, there were distinct size differences between stage-I ovaries and other stages. The mean GW of stage-I ovaries was approximately one-third that of stage-II ovaries, and mean GSI was approximately one-half of that at stage II (Table 1), and the distribution of body sizes offish at stage I had much lower minimum, maximum, and modal size 98 Fishery Bulletin 102(1) Table 1 Description of histological and macroscopic features (after fixation in a formaldehyde, acetic acid, calcium chloride solution I of ovarian developmental stages of Lutjanus carponotatus. Stage definitions and descriptions are largely a modification of the scheme proposed by Samoilys and Roelofs (2000). Mean ovary weight (GW) and gonosomatic index (GSI) for the larger Palm Island group sample are provided. Stage Histological features Macroscopic features Inactive I Immature II Resting Active III Ripening IVa Ripe IVb Running ripe Relatively thin ovarian wall; lamellae well packed; only darkly purple staining previ- tellogenic oocyte stages (oogonia and peri- nucleolar stages) present. Relatively thick ovarian wall; spaces be- tween lamellae common; only previtellogenic oocyte stages and possibly brown bodies and few atretic vitellogenic oocytes present. Most advanced oocytes are at yolk globule or migratory nucleus stage; atretic oocytes or brown bodies possibly present. Most advanced oocytes at yolk vesicle stage; atretic oocytes or brown bodies possibly present. Similar to stage IVa but large, irregularly shaped, clear to lightly coloured hydrated oocytes are present. Always even white color over entire surface; smooth surface texture; lobes quite small (typi- cally <2 cm long) and thin (mean GW=0.33 g; meanGSI=0.24^). Even white to cream or tan color over gonad sur- face; surface may be smooth or somewhat convo- luted; small white stage II ovaries are difficult to distinguish from stage I without histology I mean GW=1.01 g; mean GSI=0.43%). Color sometimes white but more often cream to tan; surface is commonly convoluted; difficult to distinguish from stage II without histology (mean GW=1.18 g; mean GSI=0.53% I. Color tan to brown or mustard with opaque speck- les that become larger and more dense as late stage oocytes become more numerous; convoluted surface sometimes with prominent vasculariza- tion (mean GW=4.04 g; mean GSI=1.399S \. External appearance identical to stage IVa and can only be differentiated histologically (no sam- ples found at Palm Island group). classes compared with the distribution of body sizes offish at stage II (Fig. 2). Stage-Ill (ripening) ovaries contain oocytes at the yolk vesicle vesicle stage, which some authors classify as vitel- logenic (e.g. Samoilys and Roelofs, 2000) and others classify as previtellogenic (e.g. West 1990). Like stage-II ovaries, stage-Ill ovaries can, but do not necessarily, contain brown bodies or atretic oocytes as evidence of probable prior spawning. Although the fish might not have spawned pre- viously, stage III is considered to be a mature stage in the present study because the appearance of yolk vesicles is associated with the initial development of the yolk globule and represents advanced development of the oocyte beyond perinucleolar stages (West, 1990). Therefore, the fish is pre- paring for spawning and will soon be part of the mature population if it is not already. Mean age and size of stage-II (4.4. years and 219 mm FL), stage-Ill (5.0 years and 222 mm FL), and stage-IV (6.5 years and 261 mm FL) females were much more similar to one another than they were to stage-I females (1.9 years and 119 mm FL). Moreover, size-frequency distributions of fish at stages II, III, and IV showed considerable overlap and similarity with one another and were all quite distinct from the size-frequency distribution for stage-I females (Fig. 2). This suggests a division between immature fish and those that are spawn- ing or are nearly ready to do so. The pronounced difference in GW and GSI between stage-I and stage-Ill ovaries and similarity in these metrics between stage-II and stage-Ill fish (Table 1) further support this division. Most immature ovaries and all ripe ovaries could be identified macroscopically. Because certain macroscopic fea- tures were common to multiple ovarian stages, additional histological features was required to separate the largest immature from the smallest resting ovaries and all ripening from resting ovaries among the samples remaining after the initial comparison betw-een histological and macroscopic features. Only one ovary with fully hydrated oocytes, col- lected at the Lizard Island group, was found among the samples prepared for histological analysis; therefore stages IVa and IVb were treated as a single stage. Stage IV suf- ficiently represents final development toward spawning on the broad seasonal time scale adopted in this study but encompasses a wide range of ovarian characteristics and would need to be divided into more detailed stages for finer temporal scale studies of lunar or diel spawning patterns. No samples exhibited features of truly "spent" ovaries. Sex-specific demography Differences were not apparent in early growth of L. car- ponotatus between the island groups (ANCOVA: df=l, 46; F=1.07; P=0.301); therefore the data were pooled to Kntzer: Sex-specific growth and mortality, spawning season, and female maturation of Lut/anus carponotatus 99 80 70 60 50 H 40 30 H 20 10 0 rzL estimate an early growth rate of 0.76 mm/d, assuming daily period- icity of micro-increments (Fig. 3). This rate of growth represents quite rapid growth, given that fish are adding 100 mm of length in around 4 months, increasing from approxi- mately 20 to 120 mm FL (Fig. 3). The x-intercept of the early growth curve (=-17.98 d) was divided by 365 d/yr to estimate a common t0 (=-0.049 yr) for all VBGF models. Although size at age for both sexes at both island groups was character- ized by substantial individual vari- ability, different growth trajectories were evident for males and females (Fig. 4, A and B). Estimates (Table 2) and 95% joint confidence regions (Fig. 4C) for the VBGF parameters indicated that the primary differ- ences in these trajectories at each island group lay in LM (which indi- cated that males grow larger than females). In contrast, the common range of K values spanned by the sexes within each island group indicated similar curvature (Table 2, Fig. 4C). However, use of a common t0 restricts the range of possible fitted lvalues (Kritzer et al., 2001). In addition to the differences between the sexes, the data revealed a general pattern of larger body sizes at the Lizard Island group (Table 2, Fig. 4). Mortality estimates at the Palm Island group were slightly higher when all age classes beyond 1 year were included compared with exclusion of age classes with n < 5 (Fig. 5). These higher mortality estimates contrast with Murphy's (1997) finding that truncation of the age structure results in higher least-squares estimates of Z. The differ- ences between mortality rates estimated with and without age classes with n < 5 were minor for both males (ANCOVA: df=l, 20; F=0.009; P=0.92) and females (ANCOVA: df=l, 23; F=1.35; P=0.26). Therefore, for comparisons between the sexes, the estimates that included all age classes greater than 1 yr were used. In contrast to the sex- specific growth differences, Z estimates of 0.26/yr and 0.29/yr (Fig. 5) corresponding to annual survivorship of 77% and 75% for females and males, respectively, at the Palm Island group were similar between the sexes (ANCO- VA: df=l, 27; F=0.505; P=0.483). Murphy's (1997) results also suggested that least-squares mortality estimates are likely to be around 30% less than the true mortality rate when n = 200 and the true Z = 0.2/yr. Correcting these mor- tality estimates based upon this potential bias results in Z estimates up to 0.37/yr and 0.41/yr for females and males, respectively, with corresponding annual survivorship of 69% and 66%-. However, the catch curve estimates (Fig. 5) corresponded well with estimates based upon Hoenig's (1983) empirically derived relationship between Z and n In I I □ Stage I ■ Stage II □ Stage III El Stage IV I ■ ■ Bfl 51 1 I i ' ' i ii, mi i m co,,3"ir>cor--ooa>o-<-OT- t-t-t-t-^t-t-CMJ(N(MCM and Lizard iBl island groups and estimated 959! joint confidence regions of the parameters A" and l. (C), Parameter estimates are presented in Table 2. Spawning season Mature female LSI values were highest in August through October with a maximum in September ( Fig. 7A). The peak in GSI lagged that of LSI by two months with the high- est values occurring from October through December and with a maximum in November (Fig. 7A>. The absence of a January sample unfortunately leaves some ambiguity as to whether GSI, and therefore presumably spawning activity, would still be high at this time or if it would have begun to decline. Male GSI values also exhibited a November maximum (Fig. 7B). Male LSI values, however, did not show any clear trend of increase and decline throughout the year and peaks in April, May, and August that did not correlate with future GSI values as clearly as seen in the female data (Fig. 7). Unlike LSI values for females, monthly mean male LSI values were always greater than the corresponding GSI values. The seasonal pattern of L. carponotatus spawning activity suggested by monthly trends in the proportions of mature ovarian stages can be interpreted as differ- ent from that suggested by GSI values. The lowest GSI values in the October-December peak period were close to twice as great as the next highest values in Septem- ber and February < Fig. 7A). However, the percentage of stage-IV ovaries in the September sample was greater than 50%. which is well over half the percentage of the October sample; whereas the February sample comprised approximately the same percentage of stage-rV ovaries as October (Fig. 8). Also, more than 50% of the March sample was stage-rV ovaries (Fig. 8). whereas its GSI value was close to that of the months with relatively few ripe ovaries (Fig. 7A). Furthermore, September and March had the highest proportions of ripening (stage-Ill I females and thus far fewer resting mature (stage-II) females than the April to August period of limited spawning activity I Fig. 8). Therefore, regardless of whether September, February, and March are defined as nonspawning months or months of limited spawning activity based upon GSI, analysis of ovarian stage frequencies suggests these to be periods of greater spawning activity than might be predicted with GSI. Clearly, the presence of advanced oocytes is a much better indication of imminent spawning than any measure of gonad size; therefore the reproductive stage- frequency data undoubtedly provide the more accurate picture of L. carponotatus spawning patterns. Of 59 ovaries staged from the October 1997 Lizard Island group sample, eight were at stage I, two were at stage II, and 49 (96% of mature females in the sample) were at stage PV. This finding suggests that the island groups share at least October as a common period of ac- tive spawning. Reproductive differences between locations and among size classes The variation in GW among females of like body sizes during peak spawning months increased to some degree with increasing TW, but there was a generally homoge- neous spread of data around the predicted regression Kntzer: Sex-specific growth and mortality, spawning season, and female maturation of Lut/anus carponotatus 101 male ages 2+: y = -0.289x + 4.319 r2 = 0.896 male ages with n 0.844 female ages 2+: y = - 0.261 x + 4.557 r2 = 0.872 4: female ages with n > 4: 0.272X + 4.282 y = - 0.203x + 4.289 0.879 6 8 10 12 Age class (years) 18 Figure 5 Age-based catch curves for female ■ higher elevation lines i and male (♦. lower elevation lines) Lutjanus carponotatus at the Palm Island group fitted to all age classes >1 (solid lines I and age classes >1 with n >4 (dashed lines I. Open symbols represent age class 1, which was not used in the analysis. Table 2 Sex-specific von Bertalanffy growth parameters for Lu tja ms carpom tatus at the Palm and Lizard Island groups , Great Barrier Reef, n is sample size; LF is the mean fork length ( mm ) K is the Brody growth :oefficient per yr) L. is the mean asymptotic fork length (mm); a common t -, of -0.049 yr was used n all growth models. Standarc errors are provided below parameter estimates. n LF A" L. r2 Palm Island group females 263 224.2 12.11) 0.77 (0.032) 246.3 (2.25) 0.515 males 202 224.7 (2.78) 0.69 (0.028) 264.3 (3.26) 0.629 sex ratio 1.3:1 Lizard Island group females 65 239.9 (4.76) 0.56 (0.043) 263.5 (4.24) 0.618 males 62 256.4 (4.77) 0.51 (0.032) 284.8 (4.03) 0.714 sex ratio 1.1:1 lines across body sizes (Fig. 9A). This suggests that on average GW at stage IV during peak spawning months is a linear function of TW. Lizard Island group fish generally had larger ovaries at a given size than did Palm Island group fish (Fig. 9A), a difference supported by ANCOVA (df=l, 125; F=34.7; P<0.001). In fact, regression slopes of 0.25 and 0.52 suggest relative ovary weights at the Lizard Island group were approximately twice as large as those at the Palm Island group. There were no differences in the GW-TW relationship among October, November, and December at the Palm Island group, and therefore the dif- ferences in this relationship between the island groups was consistent whether only the Palm Island group October data were used or whether the October through December data were used. Although GW is a linear function of TW, the nonzero regression constants (Fig. 9A) mean that GW is not a con- stant proportion of TW. Consequently, GSI increases with increasing TW ( Fig. 9B ). The relationship between TW and GSI is not strong, with regression slopes close to zero and low r2 values at both island groups (Fig. 9B). Despite this, the relationship is statistically strong at both the Palm ( ANOVA: df=l,82; F=12.70; P=0.006) and Lizard (ANOVA: df= 1,42; F=22.95; P<0.0001) Island groups. Also, there is 102 Fishery Bulletin 102(1) some suggestion that, like the GW-TW relationship, the GSI-TW relationship varies between the island groups, although to a much lesser extent (ANCOVA: df= 1,125; F=7.44;P=0.007). o o A 40 34 22 19 12 16 18 12 4 5 2 1 1 4 1 1.0 - 8 33 6 29 5741 2 1 56 /o E'"B 0.8 - 5/ .' 0.6 - a ■* 0.4 - 15 /"-' 0.2 - 3/ 00 - There is some indication that larger fish spawn over a longer period at the Palm Island group. During the September-February spawning season, mean GSI values were always higher for mature Palm Island group females >230 mm FL compared with mature fe- males <230 mm FL at the same location (Fig. 10). This pattern is likely due in part to the higher relative gonad weights of larger fish (Fig. 9B) but also seems to be driven by greater proportions of stage-IV ovaries among larger mature females in September, October, and February com- pared with fish <230 mm FL (Fig. 10). During these months, 13%, 13% and 25% more large fish were at stage IV, respec- tively, than were small fish. 0 1 2 3 4 5 6 7 B 9 10 11 12 13 14 15 16 17 M Age class (years) 53 44 25 11 3 2 1.0 0.8 0.6 0.4 0.2 0.0 10 50 90 130 170 210 250 290 330 Size class midpoint (mm fork length) Figure 6 Proportion of mature female Lutjanus carponotatus and estimated age-spe- cific (A) and size-specific (B) logistic maturation schedules at the Palm ■ solid lines) and Lizard (□, broken lines) island groups. Sample sizes for the Palm (top value) and Lizard (lower value) Island groups are presented above the data for each age or size class. Parameters of the maturity functions are provided in Table 3. Discussion Demography and reproduction of L. carponotatus Growth of L. carponotatus is rapid for the first two years of life, slows over the next two years, and nearly ceases by age 4. The slowing and cessation of growth coincide with the ages at 50% and 100% maturity, respectively, and support the argument of Day and Taylor (1997) that maturation represents a pivotal physiological trans- formation and consequently a fundamen- tal shift in the growth trajectory. Further supporting the idea that reproductive development occurs at the expense of somatic growth is the apparently longer average spawning season among larger fish that have ceased most somatic growth. The limited growth over much of the lifes- Table 3 Parameters of age- and size- specific logistic maturation schedules anc estimated ages and fork 1 engths at 50', maturity of female Lu tja n 11 s ca rpon otatus at the Palm and Lizar d Island gi oups, Great Barrier Reef. a adjusts th e posit on of the logi stic function along the abscissa; r determ ines the steepness of the logistic function. f i;n is the age at 50% maturity; Lr 0 is the fork length at 509S maturity. Standard errors are provided below parameter estimates. a r r2 *S0 OI" £50 Age-specific Palm Island group 6.40 (1.42) 3.42 (0.12) 0.985 1.9 years Lizard Island group 4.16 (0.48) 1.73 (0.19) 0.990 2.4 years Size-specific Palm Island group 14.72 (1.49) 0.081 (0.008) 0.994 182 mm Lizard Island group 11.61 (3.84) 0.061 (0.020) 0.908 189 mm Kritzer: Sex-specific growth and mortality, spawning season, and female maturation of Lut/anus carponotatus 103 pan of L. carponotatus can explain the apparently constant mortality rate over many age classes (evidenced by high catch curve r2 values) given that mortality is often largely a function of body size (Roff, 1992). The development and regression of visceral fat stores preceding increases in ovary weight is a pattern that has been observed in other reef fishes, in- cluding tropical surgeonfishes (Acan- thuridae: Fishelson et al., 1985) and groupers (Serranidae: Ferreira, 1995) and temperate rockfishes (Scorpaeni- dae: Guillemot et al., 1985). These pat- terns suggest that the stored lipid is fuelling the energetic costs of spawning. The lack of a similar pattern for males supports the idea that energetic costs associated with production of sperm are low in relation to eggs (Wootton, 1985) thus enabling male L. carponotatus to attain larger sizes, as also reported by Newman et al. (2000). Alternatively, males might spawn more frequently throughout the year than females and the lack of seasonal patterns in lipid storage among males might reflect a more regular energetic demand that precludes energy storage. In any case, these sex-specific growth patterns, coupled with similar mortality rates between the sexes and sex ratios that are at unity or that are at most only slightly female-biased (see below), sug- gest that females are limiting reproduction of this species. Therefore stock dynamics should be modeled in terms of female biology (Hilborn and Walters, 1992). The apparently female-biased sex ratio at the Palm Island group starkly contrasts with the heavily male-biased sex ratio reported for mid-shelf reefs of the central GBR by Newman et al. (2000). However, neither a male- nor fe- male-biased sex ratio would be expected from a nonhermaphrodite that is not known to possess a complex mating system such as defense of females or territories. It is possible that the spawning sex ratio (i.e. excluding juveniles) is closer to unity if males mature earlier than females, but this ratio is not possible to assess because male maturation has not yet been examined for this species. The difference between the sex ratio reported in this study and that by Newman et al. (2000) might be due to variation in mating systems across a cross- shelf density gradient (Newman and Williams, 1996). Alternatively, the sampling by traps and line fishing conducted by Newman et al. (2000) could be more heavily biased toward males than the sampling by spear fishing used in the present study because of larger % 25 to 8 2.0 1.5 1.0 0.5 0.0 Figure 7 Monthly mean gonadosomatic index IGSI ±SE; ■) and lipidsomatic index (LSI ±SE; □) values for mature female (A) and all male (Bl Lutjanus carponotatus at the Palm Island group. B -1.0 P CO - - 0.8 ■ - 0.6 v -H" - 0.4 - 0.2 April June Aug Oct i i Dec Feb Month (1997-98) 100% 80% I" 60% - CD f 40°= 20% 0% li i i I D Stage II ■ Stage III D Stage IV April June Aug Oct Dec Feb Month (1997-98) Figure 8 Monthly frequencies of ovarian stages of mature Lutjanus carpono- tatus at the Palm Island group. Stage descriptions are provided in Table 1. size, wider gape, or more aggressive behavior toward bait among males (Cappo and Brown, 1996). Furthermore, it is likely that a female-biased sex ratio as observed at the 104 Fishery Bulletin 102(1) 30 -i Lizard group: 25 - y = 0.052x -6.33 Ol £ 20 J 1 15 - .-'•" " r2 = 0.711 5 10 - o 5 - ° -■" . °^^ 0 200 400 600 800 1000 6.0 - (J 5.0 - B Lizard group: _»--' y = 0.0071X + 0.64 « 4.0- ■ ° aS°a!-'' f2 = 0353 c g 3.0 - to o 2.0 - c/i o 13 1.0- c <§ 0.0- □ D* u m a £ . ' ° a ° ' ° •*!..■■'' - ^—~—~' ■ n ^4 " ■ D □ ■ m ' • °~ " — m • ■ ' <£J-zi~*~^° ' Palm group: _*^*~""^° ■ ■ ' . y = 0.0029x + 1.02 ."^1 "■ " r2 = 0.134 0 200 400 600 800 1000 Whole body weight (g) Figure 9 Fixed ovary weight (A) and gonadosomatic index iBi at fresh whole body weight for mature female Lutjanus carponotatus at ovarian stage IV (see Table 1) collected during peak spawning months (Oct-Dec) at the Palm ■ solid lines) and Lizard (□, dashed lines) island groups. Sep Oct Nov Dec Jan Month (1997-98) Feb Mar Figure 10 Mean gonadosomatic index (GSI ±SE) for mature female Lutjanus carponotatus at the Palm Island group during the September through March spawning season for small (<230 mm fork length; ■> and large (<230 mm fork length; □) size classes. The percentage of fish at stage IV (see Table 1) is indicated above each data point. Palm Island group is not a prevalent feature of L. carponotatus populations. Rather, the strong statistical suggestion of a sex ratio quite different from unity might be due to the fact that sex ratios often show temporal variability (e.g. Stergiou et al., 1996) coupled with the propensity to achieve statistically significant differences when using large sample sizes (Johnson, 1999). Maturation schedules and sex-specific growth differences were consistent between the island groups, but overall growth pat- terns differed, with Lizard Island group fish reaching larger asymptotic body sizes. Given the vast distance between the island groups, these differences might be due to inherent genetic differences between the populations. Or, effects of temperature (the Palm Island group sits at a higher latitude), turbidity, freshwater run-off (the Palm Island group sits closer to a river mouth and has more developed mangrove systems), or other environmental factors could be driving the differences. Of course, these possibilities are not mutually exclusive. The larger ovaries observed among Liz- ard Island females might be due to further spatial differences or might be an effect of timing of sampling. The temporal resolution of sampling aimed to identify the extent of the spawning season but was too coarse to account for intramonth differences in ovar- ian development. Large changes in ovary size might occur within stage IV, and the final progression to immediate prespawning stages can be rapid (e.g. Davis and West, 1993). The Lizard Island group sample was collected from 17 to 23 October 1998, whereas the corresponding Palm Island group sample was collected from 11 to 12 October 1998. The October 1998 new moon was on the 20th, and P. leopardus, the only GBR species for which lunar spawning patterns have been reported, spawns primarily around the new moon (Samoilys, 1997). If L. carponotatus spawning is also centered around the new moon, the spatial differences in ovary weight at body weight might be due to more advanced develop- ment toward full hydration within the Lizard Island group sample. In fact, the higher proportion of stage-FV ovaries within the October Lizard Island group sample (96%) compared with the October Palm Island group sample (78'i ), coupled with the higher relative ovary weights at the Lizard Island group in October, can be taken as preliminary evidence that L. carponotatus spawns at the new moon. Comparison with other reef fishes The growth differences between male and female L. carponotatus contrast with a general trend of larger body sizes among female lutjanids observed in Atlan- Kritzer: Sex-specific growth and mortality, spawning season, and female maturation of Lut/anus carponotatus 105 tic, Caribbean, and Hawaiian species (Grimes, 1987). However, the pattern observed in the present study seems common in the Indo-Pacific where males frequently ( Davis and West, 1992; McPherson and Squire, 1992; Newman et al., 1996, 2000). but not universally (Hilomen, 1997), are the larger sex. As noted above, these differences are consis- tent with predictions based on energetic costs of producing sperm and eggs. Lutjanus carponotatus spawning patterns identified by using both GSI and ovarian stage frequencies show pro- nounced seasonal differences: there are at least five months of very limited or no spawning activity from April through August. This finding supports Grimes's ( 1987) observation that continental lutjanid populations tend to have more restricted spawning seasons than populations associated with oceanic islands, which spawn more or less continu- ously throughout the year. Although seasonal patterns ex- ist, the prominence of ripe gonads over seven months from September through March suggests an extended spawning season and supports the general observation that tropical reef fishes spawn over longer periods within the year than do cooler water species (Lowe-McConnell, 1979). However, a study with finer temporal resolution is needed to verify that spawning actually occurs in months with a high pro- portion of stage-IV ovaries. Female L. carponotatus mature on average at approxi- mately 75% of their mean asymptotic size, 54% of their maximum observed size, and 119c of their maximum longevity. The relative size at maturity contrasts with Grimes's ( 1987) observations that shallow-water continen- tal lutjanid populations like those of L. carponotatus on the GBR typically mature at smaller relative sizes (=42% maxi- mum size) compared to deep-water populations associated with oceanic islands (=50% maximum size). Two sympatric shallow -water species, L. russelli (Sheaves, 1995) and L. fulviflamma (Hilomen, 1997), likewise contrast with the general familial trend and mature at approximately 50% and 75% of their maximum size, respectively. Hence, a general pattern of relative size at maturity might exist among shallow-water lutjanids in the GBR region that is different from those regions covered by Grimes's ( 1987 ) review. Lutjanids on the GBR are generally lightly fished (Mapstone et al.1); therefore the geographic difference in sizes at maturity might be due to fishing pressure selecting for smaller sizes at maturity in other regions. The relative age at maturity of L. carponotatus cannot be as readily placed in a broader familial context given that ages at maturity were not widely estimated for lutjanids at the time of Grimes's (1987) review. However, an array of published studies suggests that many tropical and sub- tropical demersal fishes share the absolute, but not relative, ages of L. carponotatus at 50% and 100% maturity at 2 and 4 years, respectively. These include other small gonochores on the GBR (Sheaves, 1995; Hart and Russ, 1996; Hilomen, 1997), as well as a range of gonochores in other regions (Grimes and Huntsman, 1980; Davis and West, 199.3; Ross et al., 1995 ) and hermaphrodites on the GBR and elsewhere (Ferreira, 1993, 1995; Bullock and Murphy, 1994). The ubiquity of this maturity schedule, despite a wide array of maximum body sizes (160-1200 mm) and longevities (6-56 years) among these species, perhaps suggests a common physiological threshold toward which many species gravi- tate in order to maximize lifetime reproductive success. More comprehensive analysis of life history trade-offs (e.g. Roff, 1992) is needed to test this hypothesis. Fisheries management Harvest of L. carponotatus is currently restricted to fish greater than 250 mm total length ( approximately 233 mm FL) with the aim of allowing 50% offish to spawn at least once, and this regulation is proposed to remain after revi- sion by the GBR fishery management plan (Queensland Fisheries Management Authority3). The estimated size at 50% maturity of 190 mm FL suggests that the regula- tion is meeting its objective. However, the objective itself might not adequately protect the reproductive potential of L. carponotatus and similar species if individuals require multiple spawning years to ensure sufficient replenish- ment of the stock. The extensive longevities of many reef fishes have been hypothesized to be a mechanism for coping with low and irregular recruitment rates through a process dubbed the "storage effect" (Warner and Chesson, 1985). The rationale behind the storage effect hypothesis is that fish must reproduce during many breeding seasons in order to endure poor recruitment years and realize high repro- ductive success during the unpredictable and intermittent good recruitment years. If this process is important for population dynamics of L. carponotatus and other species, management will need to protect an intact natural popula- tion structure in some areas within the fishery. Protecting older age classes cannot be achieved by using maximum size limits for species like L. carponotatus that have a pro- nounced asymptote in the growth trajectory because body sizes are similar over a broad range of age classes and size is therefore poorly correlated with age. Protecting natural age structure could be accomplished through a system of strategically designed marine protected areas that allow some populations to experience natural survival free of fishing mortality. Proposed closures of the GBR line fishery during nine- day periods around the new moon in October, November, and December are aimed at protecting spawning activity and particularly spawning aggregations of P. leopardus and other harvested species (Queensland Fisheries Man- agement Authority3). Lutjanus carponotatus shares a peak spawning period during these months with P. leopardus (Ferreira, 1995; Samoilys 1997) and several other sym- patric exploited species (McPherson et al., 1992; Sheaves, 1995; Hilomen, 1997; Brown et al.5). In addition, the larger ovaries of the Lizard Island group fish, which were collected closer to the new moon, may indicate that, like P. leopardus (Samoilys, 1997), L. carponotatus spawns at 5 Brown, I. W., P. J. Doherty, B. Ferreira, C. Keenan, G. McPher- son, G. Russ, M. Samoilys, and W. Sumpton. 1994. Growth, reproduction and recruitment of Great Barrier Reef food fish stocks. Final report to the Fisheries Research and Development Corporation, FRDC Project 90/18, Queensland Department of Primary Industries, 154 p. Southern Fisheries Centre, GPO Box 76. Deception Bay, Queensland 4508, Australia. 106 Fishery Bulletin 102(1 the new moon. Therefore, the timing of the proposed spawn- ing closures seems appropriate. However, it is not known whether L. carponotatus aggregate to spawn; therefore the goal of protecting spawning aggregations might not be rel- evant for this species. In fact, the prevalence and ecological importance of spawning aggregations for any species on the GBR is largely unknown; therefore the efficacy of the proposed closures is difficult to predict. Beyond the implications for management regulations, these data have implications for modeling L. carponotatus stock dynamics. In particular, the results suggest that reproductive output by a unit of L. carponotatus biomass cannot be predicted on the basis of that biomass alone. Relative ovary weight increases slightly with increasing body size and there is evidence that larger fish spawn more frequently. The greatest difference in the proportion of ripe ovaries between size classes occurred in February 1998 af- ter severe flooding in January. It is possible that the lower proportion of ripe ovaries among small fish in February was due to stresses caused by changes in salinity or increased run-off and is not a regular trait. However, increased resil- ience to environmental stresses that allows more frequent spawning would also increase the relative reproductive success of large fish. Therefore, a population comprising fewer larger fish is likely to show greater annual egg pro- duction than a population with equivalent biomass that comprises more numerous but smaller fish. Additionally, the sex-specific patterns reported in this study further suggest gross biomass might be an inadequate index of replenishment potential and that female biomass needs to be considered. Therefore, stock structure, in terms of sex ratio and the frequency of size classes, and not simply overall biomass needs to be considered when predicting reproductive potential. Acknowledgments I thank the numerous assistants who participated in fieldwork, as well as Sam Adams and Sue Reilly for assis- tance with histological examinations. The manuscript was greatly improved by comments from Howard Choat, Carl Walters, Tony Fowler, Campbell Davies, Sam Adams, Bruce Mapstone, an anonymous thesis examiner, and two anony- mous reviewers. This work was conducted while the author was supported by an international postgraduate research scholarship from the Commonwealth of Australia and a postgraduate stipend from the CRC Reef Research Centre. Final preparation of the manuscript took place while the author was supported by a postdoctoral fellowship funded jointly by the University of Windsor and the Canadian National Science and Engineering Research Council (col- laborative research opportunity grant no. 227965-00) to Peter Sale and others). Literature cited Adams, S., B. D. Mapstone, G. R. Russ, and C. R. Davies. 2000. 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Croom Helm, London. 108 Abstract— The increase in harbor seal (Phoca vitulina richardsi) abundance, concurrent with the decrease in sal- monid [Oncorhynehus spp.) and other fish stocks, raises concerns about the potential negative impact of seals on fish populations. Although harbor seals are found in rivers and estuaries, their presence is not necessarily indicative of exclusive or predominant feeding in these systems. We examined the diet of harbor seals in the Umpqua River, Oregon, during 1997 and 1998 to indi- rectly assess whether or not they were feeding in the river. Fish otoliths and other skeletal structures were recov- ered from 651 scats and used to identify seal prey. The use of all diagnostic prey structures, rather than just otoliths, increased our estimates of the number of taxa, the minimum number of indi- viduals and percent frequency of occur- rence C^FO) of prey consumed. The *7fFO indicated that the most common prey were pleuronectids, Pacific hake (Merluccius produetus), Pacific stag- horn sculpin [Leptocottus armatus), osmerids. and shiner surfperch (Cyma- togaster aggregata ). The majority ( 76%) of prey were fish that inhabit marine waters exclusively and fish found in marine and estuarine areas (e.g. anad- romous spp. ) which would indicate that seals forage predominantly at sea and use the estuary for resting and opportu- nistic feeding. Salmonid remains were encountered in 39 samples (6%); two samples contained identifiable otoliths, which were determined to be from Chi- nook salmon (O. tshawytscha). Because of the complex salmonid composition in the Umpqua River, we used molecular genetic techniques on salmonid bones retrieved from scat to discern species that were rare from those that were abundant. Of the 37 scats with salmo- nid bones but no otoliths, bones were identified genetically as chinook or coho (O. kisutch) salmon, or steelhead trout (O. mykiss) in 90'? of the samples. Examination of the foraging habits of Pacific harbor seal (Phoca vitulina richardsi) to describe their use of the Umpqua River, Oregon, and their predation on salmonids Anthony J. Orr Adria S. Banks Steve Mellman Harriet R. Huber Robert L. DeLong National Marine Mammal Laboratory Alaska Fisheries Science Center, NMFS, NOAA 7600 Sand Point Way NE Seattle, Washington 98115 E-mail address (for A. J. Orr, contact author) tony.orr gnoaa.gov Robin F. Brown Oregon Department of Fish and Wildlife 2040 S E. Marine Science Drive Newport, Oregon 97365 Manuscript approved for publication 9 October 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull. 102:108-117 (2004). The Pacific harbor seal (Phoca vitulina richardsi) is found along the west coast of North America from the Aleutian Islands, Alaska, to the San Roque Islands. Baja California (King, 1983; Reeves et al., 1992). Before the pas- sage of the Marine Mammal Protection Act (MMPA) of 1972, harbor seals in Oregon were kept at relatively low numbers (fewer than 500 animals in 1968) because of bounties offered by the state and harassment from commercial and sport fishermen (Pearson and Verts, 1970). Since passage of protective leg- islation, harbor seals in Oregon have increased an average of 6^ to 7% annu- ally between 1978 and 1998, although, in recent years, numbers appear to be leveling at about 8000 individuals (Brown and Kohlmann. 1998). The rapid increase in harbor seal numbers has revived fishery-manag- ers' interest in seal diet because of the potential for increased consumption of commercial fish species. In addition, there has been a heightened concern about greater harbor seal abundance in rivers and estuaries during migra- tions of depressed salmonid popula- tions because of the potential negative impact on the recovery of these fishes (NMFS, 1997). Because of the tenuous status of many salmonid (Oncorhyn- ehus spp. I species along the west coast, the National Marine Fisheries Service ( NMFS ) recommended that the United States Congress modify the MMPA to allow lethal removal of seals from river mouths where they may prey on de- pressed salmonid populations (NMFS. 1997 ). Predation of salmonids by harbor seals in Oregon has been documented (Brown, 1980; Harvey. 1987; Brown et al., 1995; Riemer and Brown, 1997; Beach et al.1). The proportion of salmo- nids in the diet of harbor seals varied from 1% to 30'r depending on area, season, and sampling method (NMFS, 1997). Pinniped prey consumption can be determined from direct observations in some systems, if prey is consumed at 1 Beach, R.. A. Geiger. S. Jefferies. S. Treacy, and B. Troutman. 1985. Marine mam- mals and their interactions with fisheries of the Columbia River and adjacent waters, 1980-1982. NWAFC (Northwest Alaska Fisheries Science Center) processed rep. NWAFC 85-04, 316 p. NWAFC, National Marine Fisheries Service, Seattle, WA, 98115. Orr et al.: Foraging habits of Phoca vitulma richardsi in the Umpqua River, Oregon 109 Pacific Ocean A N the surface (Bigg et al., 1990); however, consumption is typically determined by examining scat (fecal) samples. In the past, species-specific sagittal otoliths found in scats were used exclusively to determine the identification of prey taxa. However, because otoliths can be partially or completely digested, or are not present in scats (because the head of the prey was not consumed ), they are not always an adequate representation of di- et. Recently, investigators have begun to use additional structures (e.g. cranial el- ements, vertebrae) recovered from scats to identify prey (e.g. Olesiuk et al., 1990; Cottrell et al., 1996; Riemer and Brown, 1997; Browne et al., 2002; Lance et al.2). These structures usually are more com- mon than otoliths and frequently can be identified to species; however, bones of some species can be identified to family only (e.g. salmonids). Consequently, the National Marine Mammal Laboratory (NMML) collaborated with the Conser- vation Biology Molecular Genetics Laboratory (CBMGL; Northwest Fish- eries Science Center, Seattle, WA) to develop molecular genetic identification of salmonid species (Purcell et al., 2004). Because of the complex salmonid species composition in the Umpqua River, genetic identification was vital to distinguish species that were rare from those that were abundant. The original impetus of this study was to assess the impact of harbor seal predation on the recovery of the Umpqua River sea-run cutthroat trout (O. clarkii) that were listed as endangered under the Endangered Species Act (ESA) during 1996 (Johnson et al., 1999). Umpqua River cutthroat trout were removed from the ESA in 2000 because they were identified to be part of the larger Oregon Coast evolutionary significant unit (U.S. Fish and Wildlife Service, 2000). The present study was continued despite the "delisting" of cutthroat trout because the Umpqua is inhabited year-round by harbor seals that haul out sev- eral kilometers upriver and is, thus, ideal for determining whether the presence of a pinniped species within a sys- tem is indicative of substantial feeding on fish species of concern within that environment. In addition, the Umpqua River contains several other salmonid species whose status is precarious (NMFS, 1997). Therefore, the development of genetic identification techniques was considered valuable for this system, as well as for future foraging studies in which species-specific identification may be desirable but impossible by way of conventional identification methods. Oregon L mpquu River hauiouts 2 Lance, M., A. Orr, S. Riemer, M. Weise, and J. Laake. 2001. Pinniped food habits and prev identification techniques pro- tocol. AFSC Proc. Rep. 2001-04, 36 p. AFSC, NMFS, NOAA. 7600 Sand Point Way NE, Seattle. WA 98115. Figure 1 Map of the lower section of the Umpqua River, Oregon, where scat samples were collected at two haulout sites during 1997 and 1998. The objectives of this study were 1 ) to determine by an examination of diet if harbor seals that haul out in the Umpqua River feed primarily in the river or elsewhere, and 2) to apply genetic techniques to identify salmonid prey species. Materials and methods Study area The Umpqua River, located in southern Oregon ( Fig. 1 ). is a natal river for sea-run cutthroat trout, as well as chinook (O. tshawytscha), coho (O. kisutch) salmon, and steelhead trout (O. mykiss). The Umpqua estuary is also inhabited year-round by approximately 600-1000 harbor seals and has been designated as an area where pinnipeds and sal- monids significantly co-occur (NMFS, 1997). Scat samples for this study were collected from two hauiouts located within 4.8 km of the river's mouth and within 1.6 km of each other (Fig. 1). Scat collection and analysis Samples were collected during two seasons: "spring" (March through June) and "fall" (August to December). "Spring" corresponded to the migration of anadromous cutthroat trout adults and some juveniles to the ocean and "fall" coincided approximately with the freshwater return of spawning anadromous adults. The migratory and spawn- 110 Fishery Bulletin 102(1) Table 1 Collection dates of harbor seal scats and numbers of scats wi th identifiable prey remains, without identifiable remains and without remains from the Umpqua River, Oregon, during 1997 and 1998 Fall and spring periods correspond to timing of cutthi oat trout runs on the Umpqua River. Collection dates With identifiable remains Without dentifiable remains Without remains Total Fall, 1997 16-23 Sep 26 1 2 29 27 Sep-6 Oct 5 0 3 8 12-24 Oct 31 0 7 38 31 Oct-lONov 21 0 6 27 12-25 Nov 36 0 10 46 Total 119 1 28 148 Spring 1998 24-25 Mar 27 5 2 34 13-15 Apr 59 5 7 71 26-27 Apr 45 4 4 53 13-14 May 41 0 4 45 27-28 May 12 0 1 13 11-12 Jun 35 2 1 38 Total 219 16 19 254 Fall 1998 5-6 Aug 142 1 1 144 19-20 Aug 111 1 3 115 6-9 Sep 28 3 3 34 19-21 Sep 13 0 0 13 7-8 Oct 19 0 1 20 Total 313 5 8 326 ing periods of chinook and coho salmon, and steelhead trout also occur during these times. During fall 1997, all harbor seal scats present at the haulouts were collected every other day during the day- time low tide, weather permitting (Table 1). In 1998. bi- weekly attempts were made to pick a minimum of 50 scats during low tides at the haulout sites (Table 1). Scats were collected, placed in individual plastic bags, and frozen for later processing. At the laboratory samples were thawed and rinsed in nested sieves (1.0 mm, 0.71 mm, and 0.5 mm in 1997; 1.4 mm, 1.0 mm, and 0.5 mm in 1998). Fish struc- tures were dried and stored in glass vials and cephalopod remains were stored in vials with 70"* isopropyl or ethyl alcohol. Prey were identified to the lowest possible taxon by using sagittal otoliths, skeletal, and cartilaginous remains from fish and beaks and statoliths from cephalopods. Other in- vertebrate remains were discarded from analysis because of the uncertainty of identifying them as primary or sec- ondary prey. Unknown prey were categorized as "unidenti- fied" and "unidentifiable" (Browne et al., 2002). Items that were categorized as "unidentifiable" were excluded from analyses because they could not be distinguished from prey already identified in the sample. Otoliths, beaks, and diagnostic bones were identified by using an extensive ref- erence collection at the NMML and voucher samples veri- fied by Pacific Identifications (Victoria, British Columbia). After identification, otoliths were separated by side (left, right, or unknown ) and enumerated to determine minimum number of specific prey. Unique diagnostic structures (e.g. quadrates, angulars, basioccipitals, vomers) were used for identification and enumeration offish. Non-unique skeletal structures such as gillrakers and teeth were used to iden- tify but not enumerate taxa (i.e. their presence indicated only a single individual) unless the structures were from different size classes. Vertebrae were treated like other non-unique structures; however, for salmon, if the number of vertebrae reflected more than one individual, then they were used for enumeration. Cephalopod beaks were sepa- rated by side (upper, lower, or unknown) and enumerated to determine number of prey. To discern where harbor seals were feeding, identified prey were categorized as those exclusively found in rivers or estuaries (e.g. gobiids, cyprinids), those found exclu- sively in marine waters (e.g. gadids, mvxinids), and those that could potentially be found in either environment (e.g. anadromous species, osmerids, petromyzontids) by using Eschmeyer et al. (1983). A seal was considered to feed in the river-estuary system if all the prey taxa identified in the scat were definitely or could potentially be found in the system. For example, a sample containing remains of pea- mouth chub iMylocheilus caurinus), threespine stickleback ( Gasterosteus aculeatus ), river lamprey iLampetra ayresii ), and chinook salmon would be classified as a riverine- Orr et al.: Foraging habits of Phoca vitulina richardsi in the Umpqua River, Oregon 111 estuarine species because these prey items could feasibly be consumed in the river. It was assumed that the seal was feeding in the marine environment if a sample contained exclusively marine prey, such as Pacific hagfish (Eptatretus stoutti). Pacific hake (Merluceius productus), and rockfish (Sebastes spp. ). If a scat comprised prey taxa that poten- tially could be found in a riverine-estuarine system or marine waters (e.g. salmonids, osmerids), as well as those found exclusively in marine waters, then it was assumed that the feeding environment was marine or mixed. Salmonid skeletal remains were sent to the CBMGL for species identification. Remains to be analyzed genetically were selected by number or size (or both) to represent dif- ferent species or individuals present in each scat. For ex- ample, if a scat had 95 approximately equal-size vertebrae (a salmonid has approximately 65 vertebrae; Butler, 1990). then at least two vertebrae (potentially representing at least two individuals) were sent for genetic identification. Also, if a sample had a very large gillraker and three small vertebrae, then the gillraker and one vertebra were sent for genetic identification. The size of diagnostic structures was also used to categorize salmon remains as juvenile or adult, when possible. The CBMGL identified salmonid spe- cies by direct sequencing of mitochondrial DNA or analysis of restriction fragment length polymorphism (Purcell et al., 2004). The abundance of prey taxa in harbor seal diet for each period was described by using the minimum number of individuals (MNI) and percent frequency of occurrence (%FO). We compared the effect of including bone on the number of prey consumed by estimating MNI using the greater number of right or left otoliths and then again using all diagnostic skeletal remains. Cephalopod MNI was estimated from the greater number of upper or lower beaks. The % FO of prey taxon i was defined as I°" %FO, x 100, where Oll; = absence (0) or presence (1) of taxon i in scat k\ and s = the total number of scats that contained identifiable prey remains. The presence of taxon ;' in scat k was determined by using otoliths and then again using all structures. To account for variability in diet, point estimates of %FO for a prey taxon were determined during each sampling period and then averaged for each season. Results Scats Over 725 scats were collected during all periods. The number of scats collected with identifiable remains was 119 (99%; n=148) in fall 1997, 219 (93%; ?z=254) in spring 1998, and 313 (98%; n=326) in fall 1998 (Table 1). Of the 651 samples with identifiable prey remains, 605 (93%) con- tained fish bones, 347 (53%) had fish otoliths, 231 (36%) contained remains from cartilaginous fish, and 41 (6% ) had cephalopod beaks. A majority (65% fall 1997, 65% spring 1998, 63% fall 1998) of scats with identifiable remains had one to three prey taxa present and less than 4% contained more than ten taxa. Approximately 40 prey taxa, repre- senting at least 25 families, were identified throughout the study (Tables 2 and 3). For nearly all prey taxa, MNI was greater when all skel- etal remains were identified than when otoliths were used exclusively (Table 2). For several species, such as Pacific hake. Pacific herring (Clupea pallasii), and Pacific sardine {Sardinops sagax), MNI at least tripled when all structures were used for enumeration (Table 2). For most salmonids, cartilaginous fishes, three-spine stickleback, Irish lords (Hemilepidotus spp.), and Pacific mackerel {Scomber ja- ponicus), no otoliths were recovered; therefore other skel- etal elements had to be used for identification (Table 2). For a few prey, such as cyprinids, gobiids, and butter sole (Isopsetta isolepis), only otoliths were recovered (Table 2). Foraging habits The %FO for most prey taxa was greater when all struc- tures were used than when j ust otoliths were used ( Table 3 ). The %FO indicated that the prey most frequently con- sumed were pleuronectids. Pacific hake. Pacific staghorn sculpin {Leptocottus armatus), osmerids, and shiner surf- perch (Cymatogaster aggregata). Prey frequently found in scats included those that were exclusively marine (e.g. Pacific hake, rex sole (Glyptocephalus zachirus), English sole (Parophiys vetulus), and myxinids), and those that occur in both marine and estuarine waters (e.g. Pacific staghorn sculpin. and shiner surfperch (Table 3] ). Only 24% of scats were composed entirely of prey taxa that could be found in riverine-estuarine systems (Fig. 2). Consequently, a majority of the scats contained prey species that were exclusively marine (.v=25.3%) or were a mixture of marine and potentially marine species (x=50.8%\ Fig. 2). Salmonids Salmonid remains were found in only 6% (39/651) of the samples. Five chinook smolts were identified from otoliths in two samples collected during fall 1997; in the remaining 37 samples, salmonid bones were unidentifiable to species with conventional techniques. With the cooperation of CBMGL, we examined 116 salmonid bones using molecular genetic techniques. Species identification was successful for 67% (78/116) of the bones and teeth from 90% (35/39) of the scat samples that contained salmonid structures. In the four samples that remained unidentified, three con- tained only a single salmonid bone that failed to produce any DNA. Most of the other bones where DNA could not be extracted were small or fragmented and highly digested. Seventeen of the samples contained chinook salmon bones (including the two samples with chinook salmon otoliths); 11 contained coho salmon bones, four contained steelhead trout bones, and three contained bones from two salmonid 112 Fishery Bulletin 102(1) Table 2 Minimum number of individuals ( MNI ) offish prey derived from sagittal otoliths and all structures retrieved from harbor seal scats collected at the Umpqua River during 1997 and 1998. s represents the number of scats with identifiable remains, na indicates taxon did not have sagittal otoliths to be used for identification. Fall 1997(s=119i Spring 1998(s=219i Fall 1998(s=313) MNI MNI MNI MNI MNI MNI Family Species otoliths all structures otoliths all structures otoliths all structures Ammodytidae Pacific sand lance 205 208 317 321 3 7 Bothidae Pacific sanddab 12 13 9 9 1 2 Clupeidae American shad 1 2 4 11 1 15 Pacific herring 6 22 3 10 121 345 Pacific sardine 0 0 50 235 39 185 Cottidae Pacific staghorn sculpin 44 65 25 48 30 85 unidentified cottid 0 0 0 0 0 8 Cyprinidae peamouth chub 1 1 4 4 4 4 Embiotocidae shiner surfperch 104 109 209 274 23 104 Engraulididae northern anchovy 1 3 0 0 1 2 Gadidae Pacific hake 1 35 10 44 58 199 Pacific tomcod 9 21 19 52 8 26 Gasterosteidae threespine stickleback 0 1 0 0 0 0 Gobiidae unidentified gobiid 2 2 1 1 0 0 Hexagrammidae lingcod 0 1 0 0 1 1 Myxinidae Pacific hagfish 0 20 0 13 0 61 Ophidiidae spotted cusk-eel 0 0 4 4 2 2 Osmeridae unidentified osmerid 42 54 14 41 105 132 Petromyzontidae Pacific lamprey na 5 na 89 na 41 river lamprey na 2 na 1 na 0 Pholididae saddleback gunnel 3 7 1 3 0 1 Pleuronectidae English sole 38 41 37 39 75 84 Dover sole 1 4 5 6 27 51 slender sole 1 1 18 24 28 42 butter sole 1 1 15 15 2 2 rex sole 19 44 44 53 96 125 petrale sole 0 0 0 0 1 1 starry flounder 10 17 8 12 6 31 Rajidae unidentified rajid na 1 na 7 na 4 Scombridae Pacific mackerel 0 2 0 3 0 2 Scorpaenidae Sebastes spp. 0 15 6 19 2 3 Trichodontidae Pacific sandfish 0 0 0 1 2 3 Zoarcidae unidentified zoarcid 0 0 0 0 2 2 Salmonidae coho salmon unknown 0 4 0 0 0 0 juvenile 0 1 0 4 0 2 adult 0 0 0 1 0 3 Steelhead or rainbow trou t unknown 0 0 0 2 0 2 juvenile 0 0 0 0 0 1 chinook salmon unknown 5 6 0 0 0 3 juvenile 0 5 0 2 0 5 adult 0 1 0 0 0 0 unidentified salmonid unknown 0 2 0 1 0 2 juvenile 0 1 0 0 0 1 Orr et al.: Foraging habits of Phoca vitulina richardsi in the Umpqua River, Oregon 113 Table 3 Mean percent frequency of occurrence (%FO) of common prey recovered from harbor seal scat samples collected at haulout sites in the Umpqua River, Oregon, during 1997 and 1998. SD indicates standard deviation. Family Species Fall 1997 Spring 1997 Fall 1998 Mean(±SD) Mean(±SD) Mean(±SDl Ammodytidae Pacific sand lance 12.5 ±8.3 12.6 ±8.3 9.1 ±8.9 Bothidae Pacific sanddab 11.4 ±7.5 4.1 ±2.5 3.0 ±3.2 Clupeidae American shad 4.3 ±0.6 13.0 ±2.3 5.3 ±3.1 Pacific herring 16.9 ±13.7 7.3 ±6.9 35.9 ±21.8 Pacific sardine 0 16.1 ±12.2 17.9 ±9.1 Cottidae Pacific staghorn sculpin 23.9 ±8.5 21.0 ±19.0 11.8 ±4.5 unidentified cottid 16.5 ±20.4 3.2 ±0.7 0.8 ±0.1 Cyprinidae peamouth chub 3.8 2.3 ±0.6 2.8 Embiotocidae shiner surfperch 18.2 ±8.2 23.6 ±19.4 7.0 ±2.9 Engraulididae northern anchovy 5.5 ±3.2 0 2.1 ±2.0 Gadidae Pacific hake 27.9+9.7 17.0 ±5.7 41.6 ±25.5 Pacific tomcod 15.4 ±7.8 16.1 ±7.0 12.3 ±8.3 Gasterosteidae threespine stickleback 2.8 0 0 Gobiidae unidentified gobiid 7.7 1.7 0 Hexagrammidae lingcod 3.8 0 0.7 Loliginidae market squid 12.8 ±10.2 3.5 ±1.3 0 Myxinidae Pacific hagfish 17.5 ±7.9 6.7 ±3.5 16.5 ±9.4 Octopodidae Octopus rubescens 3.8 ±1.4 8.3 ±2.6 8.4 ±7.0 Ophidiidae spotted cusk-eel 0 0 0.9 Osmeridae unidentified osmerid 20.8 ±11.3 14.6 ±8.2 19.5 ±10.0 Petromyzontidae Pacific lamprey 7.7 ±8.2 20.5 ±10.1 8.2 ±2.9 river lamprey 5.6 3.7 0 Pholididae saddleback gunnel 14.7 ±16.9 2.6 ±0.3 5.3 Pleuronectidae English sole 21.9 ±1.7 8.7 ±5.2 17.5 ±12.0 Dover sole 7.4 ±5.9 4.6 ±0.7 13.5 ±13.6 slender sole 0 11.0 ±7.2 14.9 ±14.9 butter sole 3.8 7.2 ±3.7 1.4 rex sole 27.4 ±12.1 14.2 ±9.6 19.9 ±20.5 petrale sole 0 0 0.7 starry flounder 15.8 ±7.4 3.7 ±1.0 5.8 ±1.2 Rajidae unidentified rajid 2.8 5.0 ±1.6 2.8 Scombridae Pacific mackerel 3.8 ±1.4 4.6 ±4.0 0.8 ±0.1 Scorpaenidae Sebastes spp. 15.7 ±8.3 9.1 ±2.6 2.1 Trichodontidae Pacific sandfish 0 1.7 2.1 unidentifed bothid/ unidentified flatfish 38.5 ±15.9 20.2 ±10.3 14.8 ±2.5 pleui-onectid Zoarcidae unidentified zoarcid 0 0 1.4 Salmonidae coho salmon unknown 5.8 ±3.6 0 0 juvenile 4.8 3.3 ±2.3 0.7 adult 0 2.4 6.2 ±6.2 steelhead/rainbow trout unknown 0 2.7 ±1.4 0.7 juvenile 0 0 0.9 adult 0 0 0.9 chinook salmon unknown 7.6 ±3.5 0 0.8 ±0.1 juvenile 4.0 ±1.1 3.4 3.6 ±3.0 adult 4.8 0 0 unidentified salmonid(s) unknown 4.3 ±0.6 2.4 0.8 ±0.1 juvenile 4.8 0 7.7 114 Fishery Bulletin 102(1) species (two with coho and chinook salmon and one with coho salmon and steelhead trout, Table 2). No cutthroat trout were identified with conventional or molecular genetic techniques. Using otoliths and other diagnostic skeletal struc- tures, we enumerated at least 54 individual salmonids in 39 scats (Table 2). All individuals identified as adults I n =5 ) were coho salmon, except one chinook salmon from spring 1997. Individual juveniles identified as steelhead trout (n=l), coho salmon (re=7), chinook salmon («=12), or unidentified salmonids (/2=2) were present during all periods. Because of the difficulty of determining age from size-variable structures such as gillrakers and teeth, most individuals («=27) were designated as "unknown age." Discussion Investigating diet is essential to assessing the role of harbor seals in marine and freshwater ecosystems in order to quantify their interactions with fisheries and determine their impact on the recovery of endangered species. All methods used to investigate diet of seals and other pinnipeds have some limitations (Murie and Lavigne, 1985, 1986; Harvey, 1989). With scats, it is assumed that the relative frequency of prey identified from undigested remains reflects the frequency of prey eaten (Tollit et al., 1997). However, several investigators have determined that this assumption may be seriously biased in several ways (Hawes, 1983; da Silva and Neilson, 1985; Jobling, 1987; Dellinger and Trillmich, 1988; Harvey, 1989; Pierce and Boyle, 1991; Cottrell et al., 1996; Tollit et al., 1997; Bowen, 2000; Orr and Harvey, 2001). No diet study can estimate detrimental or lethal impacts to prey resulting from harassment by pinnipeds. In addition, once a prey is captured, a seal might consume only the soft tissue (especially of larger prey), which would not leave identifiable evidence in scats. Additionally, because skel- etal remains from different prey species pass through the alimentary canal and erode at different rates they may not reflect the true number or proportions of prey consumed (Hawes, 1983; Harvey, 1989; Pierce and Boyle, 1991; Cottrell et al., 1996; Tollit et al., 1997). Therefore, preda- tion estimates determined from scat samples should be regarded as a measure of minimum impact. Although there are complications inherent in the use of scats to describe the diet of seals, scat analysis remains useful because many scats can be collected quickly, with minimum effort and without harm to the animals (Harvey, 1989). Scats Recently, skeletal remains other than otoliths and beaks have begun to be used to identify and enumerate prey of pinnipeds (e.g. Olesiuk et al., 1990; Cottrell et al., 1996; Riemer and Brown, 1997; Browne et al., 2002). There are constraints, however, for using all skeletal elements to identify prey species, including the need for a reference col- lect ion and the extensive training of personnel to identify Fall I9M7 Q nverine-estuanne marine or mixed Scat categorization Figure 2 Mean percentage plus standard deviation (SD) of scats that were classified as "riverine-estuarine" (i.e. samples composed of prey taxa that are exclusively or potentially (e.g. anadromous species, osmerids) found in rivers or estuaries), "marine" (i.e. samples composed exclusively of prey that inhabit marine waters l, and "marine or mixed" (i.e. samples composed of prey taxa exclusively found in marine waters or those that might inhabit marine waters at some stage in their life). digested prey structures (Cottrell et al., 1996). Moreover, there is usually a bias in the recovery and recognition of prey structures from different taxa (Cottrell et al., 1996; Laake et al., 2002). This bias may be a significant problem in estimating relative abundance of prey or biomass con- sumption by harbor seals and is the reason these indices were not considered in this study. Despite these complications, the use of all available structures increased our estimates of prey diversity, MNI, and % FO for most prey taxa. Examination of all diagnostic structures also allowed us to consider a greater sample size because 93% of scats with identifiable remains contained bones, whereas only 53% of scats contained otoliths. Spe- cies not represented by otoliths, such as salmonids (during 1998) and cartilaginous fishes, were detected because all structures were used. In addition, the MNI of important prey such as Pacific hake. Pacific herring, and Pacific sar- dine would have been greatly underestimated had otoliths been used exclusively because the MNI derived by using all structures was at least threefold greater. Although there are complexities associated with estimating MNI from all structures, this method avoids the use of numerical correc- tion factors determined from recovery rates of otoliths fed to captive seals during laboratory experiments (Browne et al., 2002). Results from captive experiments are highly variable between repeated trials for the same individual and among different individuals (Harvey, 1989; Bowen et al., 2000; Orr and Harvey, 2001 1, Foraging habits Harbor seals in the lower Umpqua River consumed prey from over 35 taxa; however, only a few prey taxa were dominant in their diet, as reflected by %FO. Overall, the five most abundant families of prey were Clupeidae, Cot- Orr et al.: Foraging habits of Phoca vitulina nchardsi in the Umpqua River, Oregon 115 tidae, Embiotocidae, Gadidae, and Pleuronectidae. These are similar to those reported in other studies of harbor seal diet in Oregon (Riemer and Brown, 1997; Browne et al., 2002; Riemer et al.3-4). It was evident by the presence of prey like Pacific hake. Pacific sardine, hagfish, and various flatfishes that seals fed offshore in pelagic and demersal areas. Harbor seals also consumed prey (e.g. Pacific staghorn sculpin) com- monly found inshore or in estuarine waters. The NMFS recommendations to remove pinnipeds from systems where endangered prey also occur, rely on the assumption that pinnipeds are primarily feeding (on ESA-listed species) in that system. Our study indicated that this was not the case. Although the seals at the Umpqua hauled out several kilometers up river, they foraged primarily at sea. Because of the life histories of many of the prey taxa, our foraging habitat categories must be considered estimations of where the prey might have been consumed. For example, we estimated that 24% of scats contained prey attributable to the riverine-estuarine environment. However, this may actually be an overestimation because some of these spe- cies potentially inhabit the marine environment at some time in their life and may have been consumed there. Ad- ditionally, scats categorized as marine or mixed may reflect that the seal fed solely in the marine environment (because all the taxa can potentially be found in marine waters) or fed at sea and within the river. Nevertheless, these catego- ries are useful for a broad apportioning of foraging habitat. Even though we were able to determine that approximately 76% of the scats contained marine and potentially marine prey taxa, we were unable to assess whether this reflected a seal population with homogeneous or heterogeneous for- aging patterns. In other words, because the scats could not be attributed to a particular individual, we had no way of discerning: 1) whether the entire seal population foraged roughly three-fourths of the time at sea and one-fourth of the time in the river, or 2) whether 76% of the seals fed at sea whereas 24% foraged closer to shore and in the river. This distinction may be important if only a subgroup of seals is feeding in the river and preying on fish that are seasonally abundant in the estuary, such as salmonids. Studies that incorporate radio- or satellite-telemetry or genetic identification of individual prey items in scats may reveal these distinctions in the future. Because the seals haul out almost 5 km upriver and have been observed as far as 32 km upriver, it is clear that 3 Riemer, S. D., R. F. Brown, and M. I. Dhruv. 1999. Monitoring pinniped predation on salmonids in the Alsea and Rogue River estuaries: fall. 1997. //; Pinniped predation on salmonids: pre- liminary reports on field investigations in Washington, Oregon, and California, p. 104-152. Compiled by National Marine Fisheries Service, Northwest Region. [Available from ODFW, 7118 NE Vandenberg Avenue, Corvallis, OR 97330.] 4 Riemer, S. D., R. F. Brown, and M. I. Dhruv. 1999. Monitoring pinniped predation on salmonids in the Alsea and Rogue River estuaries: fall, 1998. In Pinniped predation on salmonids: pre- liminary reports on field investigations in Washington, Oregon, and California, p. 153-188. Compiled by National Marine Fisheries Service, Northwest Region. [Available from ODFW. 7118 NE Vandenberg Avenue, Corvallis, OR 97330.] seals use the river environment. However, the prevalence of marine fish remains in the scat samples indicates that the seals that haul out at the Umpqua River do not feed exclusively in the river. The predominance of marine prey may reflect a foraging strategy in which the effort required to find marine sources of food is offset by the energy gained by exploiting large aggregations of marine schooling fish (e.g. Pacific hake and Pacific sardine). In this scenario, the seals in the Umpqua estuarine-riverine system may depend on marine resources while taking advantage of protected estuarine waters that provide a sheltered place to rest and occasionally feed. Salmonids We used two methods to estimate the number of salmonids eaten by harbor seals: prey remains and genetic analyses of scat samples. Analysis of skeletal remains was of lim- ited value because the majority of salmonid structures recovered from scat samples were bones, which could be identified only to family. This study represents a novel application of genetic techniques to identify salmonid spe- cies from bones found in scats. These techniques allowed us to determine species for a majority of the salmonid samples that would have otherwise remained unidentified because they did not contain otoliths. Salmonid bones or otoliths were found in 6% of the har- bor seal scats collected during our study — a finding that is comparable to the 5% found by Laake et al. (2002) at the Columbia River. However, it is about one-half of what was found by Riemer and Brown ( 13% ; 1997 1 at selected sites in Oregon. Brown et al. (1995) found salmonids in 12% of gastrointestinal tracts of harbors seals taken incidentally by commercial salmon gillnet fishing operations, and Roffe and Mate (1984) observed that salmonids made up 30% of the prey for harbor seals surface feeding in the Rogue Riv- er. Regardless of sampling method, in these studies, most of the salmonids could be identified only to family because few otoliths were recovered and genetic techniques to identify bones to species had not yet been developed. Salmonids are present in the Umpqua River year-round although species and age composition change throughout the year. In this study, most salmonid prey of known age were juveniles; however, we could determine age of only one-half of the individuals. Juveniles are found in the Umpqua River system year-round and may be easier for seals to catch than adults. Alternatively, perhaps seals did not consume many adult skeletal elements because adult salmonids are large fish, which may be ripped apart rather than swallowed whole. Our sampling seasons encompassed at least some por- tion of the migrations of all salmonids, all of which (except cutthroat trout ) were prey of harbor seals. The fact that portions of all migrations were included in the sampling design was noteworthy because there were a large num- ber of seals in the river throughout the year and yet we found no evidence through genetic or otolith identification that seals consumed cutthroat trout in the Umpqua River. The genetic identification tools developed and applied in our collaboration with CBMGL were useful in discerning 116 Fishery Bulletin 102(1) scarce from abundant salmonids. These techniques may be useful in identifying other pinniped prey that lack spe- cies-specific structures and would allow managers to better assess the impact of pinniped predation on threatened or endangered species. Acknowledgments This study was proposed and initiated in collaboration with Joe Scordino. Scat collection and harbor seal counts were conducted by Lawrence Lehman, Kirt Hughes, Mer- rill Gosho, Sharon Melin, and Robert DeLong. The U.S. Coast Guard Umpqua River Station provided boat storage and a location for keeping a chest freezer during the 1997 field season. We would like to thank the Oregon Institute of Marine Biology, Charleston, OR, where the samples col- lected during 1997 were processed. We greatly appreciate the collaboration with Conservation Biology Molecular Genetics Laboratory, which resulted in the identification of our salmon remains based on genetic methods. We would also like to thank Susan Reimer who kindly helped us with difficult identifications, as well as Lawrence Lehman and Jason Griffith for their verification of bone and otolith identifications. We thank Patience Browne, Patrick Gearin, John Jansen, Mark Dhruv, and three anonymous review- ers for providing helpful comments on earlier drafts of this manuscript. Literature cited Bigg, M. A.. G. Ellis, P. Cottrell, and L. Milette. 1990. Predation by harbour seals and sea lions on adult salmon in Comox Harbour and Cowichan Bay, British Columbia. Can. Tech. Rep. Fish. Aquat. Sci. 1769, 31 p. Bowen, W. D. 2000. Reconstruction of pinnipeds diets: accounting for complete digestion of otoliths and cephalopod beaks. Can. J. Fish. Aquat. Sci. 57:898-905. Brown, R. F. 1980. Abundance, movements and feeding habits of the harbor seal, Phoea vitulina, at Netarts Bay, Oregon. M.S. thesis, 69 p. Oregon State Univ., Corvallis, OR. Brown, R. F., S. D. Riemer, and S. Jefferies. 1995. Food of pinnipeds collected during the Columbia River Area Commercial Salmon Gillnet Observation Program, 1991-1994. ODFW (Oregon Dep. Fish Wildlife), Wildlife Diverstiy Program Tech. Rep. 95-6-01, 16 p. Brown, R. F, and S. Kohlmann. 1998. Trends in abundance and current status of the Pacific harbor seal {Phoea vitulina richardsi) in Oregon: 1977-1998. ODFW, Wildlife Diverstiy Program Tech. Rep. 98-6-01, 16 p. Browne, P., J. L. Laake, and R. L. DeLong. 2002. Improving pinniped diet analyses through identifica- tion of multiple skeletal structures in fecal samples. Fish. Bull. 100:423-433. Butler. U. L. 1990. Distinguishing natural from cultural salmonid dep- osits in Pacific Northwest North America. Ph.D. diss., 218 p. Univ. of Washington, Seattle, WA. Cottrell, P. E., A. W. Trites, and E. H. Miller. 1996. Assessing the use of hard parts in faeces to identify harbour seal prey: results of captive-feeding trials. Can. J. Zool. 74:875-880. da Silva, J., and J. Neilson. 1985. Limitations of using otoliths recovered in scats to estimate prey consumption in seals. Can. J. Fish. Aquat. Sci. 42:1439-1442. Dellinger, T, and F. Trillmich. 1988. Estimating diet composition from scat analysis in otariid seals (Otariidae): is it reliable? Can. J. Zool. 66: 1865-1870. Eschmeyer. W. N, E. S. Herald, and H. Hammann. 1983. A field guide to Pacific Coast fishes of North America, 336 p. Houghton Mifflin Co., Boston, MA. Harvey, J. T. 1987. Population dynamics, annual food consumption, move- ments and dive behaviors of harbor seals, Phoca vitulina richardsi, in Oregon. Ph.D. diss., 177 p. Oregon State Univ., Corvallis, OR. 1989. Assessment of errors associated with harbor seal (Phoca vitulina) faecal sampling. J. Zool., Lond. 219: 101-111. Hawes, S. 1983. An evaluation of California sea lion scat samples as indicators of prey importance. M.S. thesis, 50 p. San Francisco State Univ. San Francisco, CA. Jobling, M. 1987. Marine mammal faeces samples as indicators of prey importance — a source of error in bioenergetics studies. Sarsia 72:255-260. Johnson, O, M. Ruckelshaus, W Grant. F. Waknitz, A. Garrett. G. Bryant, K. Neely, and J. Hard. 1999. Status review of coastal cutthroat trout from Washing- ton, Oregon, and California. NOAA Tech. Memo. NMFS- NWFSC-37, 292 p. King, J. 1983. Seals of the world, 240 p. Comstock Publishing Assoc, Cornell Univ. Press, New York, NY. Laake, J. L., P. Browne. R. L. DeLong, and H. R. Huber. 2002. Pinniped diet composition: a comparison of estimation models. Fish. Bull. 100:434-447. Murie, D., and D. Lavigne. 1985. A technique for the recovery of otoliths from stomach contents of piscivorous pinnipeds. J. Wildl. Manag. 49: 910-912. 1986. Interpretation of otoliths in stomach content analy- ses of phocid seals: Quantifying fish consumption. Can. J. Zool. 64:1152-1157. NMFS (National Marine Fisheries Service). 1997. Investigation of scientific information on the impacts of California sea lions and Pacific harbor seals on salmo- nids and on the coastal ecosystem of Washington, Oregon, and California. NOAA Tech. Memo. NMFS-NWFSC-28, 172 p. Olesiuk, P. E, M. A. Bigg, G. M. Ellis. S. J. Crockford, and R. J. Wigen. 1990. An assessment of the feeding habits of harbour seals (Phoca vitulina i in the Strait of Georgia. British Columbia, based on scat analysis. Can. Tech. Rep. Fish. Aquat. Sci. 1730. 135 p. Orr, A. J., and J. T. Harvey. 2001. Quantifying errors associated with using fecal sam- ples to determine the diet of the California sea lion {Zalo- phu* califbrnianus). Can. J. Zool. 79:1080-1087. Orr et al.: Foraging habits of Phoca vitultna richardsi in the Umpqua River, Oregon 117 Pearson, J., and B. Verts. 1970. Abundance and distribution of harbor seals and northern sea lions in Oregon. Murrelet 51:1-5. Pierce, G. J., and P. R. Boyle. 1991. A review of methods for diet analysis in piscivorous marine mammals. Ocean. Mar. Biol. Ann. Rev. 29:409-486. Purcell, M, G. Mackey, E. LaHood, H. Huber, and L. Park. 2004. Molecular methods for the genetic identification of salmonid prey from Pacific harbor seal ( Phoca vitulina richardsi) scat. Fish. Bull. 102:213-220. Reeves, R., B. Stewart, and S. Leatherwood. 1992. The Sierra Club handbook of seals and sirenians, 359 p. Sierra Club Books, San Francisco. CA. Riemer, S. D., and R. F. Brown. 1997. Prey of pinnipeds at selected sites in Oregon identi- fied by scat (fecal) analysis, 1983-1996. ODFW Wildlife Diversity Program Tech. Rep. 97-6-02. 34 p. Roffe, T, and B. Mate. 1984. Abundances and feeding habits of pinnipeds in the Rogue River, Oregon. J. Wildl. Manag. 48:1262-1274. Tollit, D. J., M.J. Steward, P. M. Thompson, G J. Pierce, M. B. Santos, and S. Hughes. 1997. Species and size differences in the digestion of oto- liths and beaks: implications for estimates of pinniped diet composition. Can. J. Fish. Aquat. Sci. 54:105-119. U. S. Fish and Wildlife Service. 2000. Endangered and threatened wildlife and plants: final rule to remove the Umpqua River cutthroat trout from the list of endangered wildlife. Federal Register: 26 April 2000, 65181:24420-24422. 118 Abstract— Larval development of the sidestriped shrimp ^Pandalopsis dis- par) is described from larvae reared in the laboratory. The species has five zoeal stages and one postlarval stage. Complete larval morphological charac- teristics of the species are described and compared with those of related species of the genus. The number of setae on the margin of the telson in the first and second stages is variable: 11+12, 12+12, or 11+11. Of these, 11+12 pairs are most common. The present study confirms that what was termed the fifth stage in the original study done by Berkeley in 1930 was the sixth stage and that the fifth stage in the Berkeley's study is comparable to the sixth stage that is described in the present study. The sixth stage has a segmented inner fla- gellum of the antennule and fully devel- oped pleopods with setae. The ability to distinguish larval stages of P. dispar from larval stages of other plankton can be important for studies of the effect of climate change on marine communities in the Northeast Pacific and for marine resource management strategies. Larval development of the sidestriped shrimp (.Pandalopsis dispar Rathbun) (Crustacea, Decapoda, Pandalidae) reared in the laboratory Wongyu Park School of Fisheries and Ocean Sciences University of Alaska Fairbanks Juneau, Alaska, 99801-8677 E-mail address: wparkig'uaf edu R. Ian Perry Pacific Biological Station, Fisheries and Oceans Nanaimo, British Columbia, V9R 5K6, Canada Sung Yun Hong Department of Marine Biology Pukyong National University Pusan, 608-737, Korea Manuscipt approved for publication 23 June 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull. 102:118-126 (2004). Sixteen species of the genus Pandalop- sis have been recognized in the South- western Atlantic and North Pacific Oceans (Komai, 1994; Jensen, 1998; Hanamura et al., 2000). Most members of the genus attain a large body size and are valuable as commercial fishery resources (Holthuis, 1980; Baba et al., 1986). In the North Pacific, P. dispar, P. ampla, P. aleutica, P. longirostris, P. lucidirimicola, and P. spinosior have been reported. Of these, Pandalopsis dispar is an important component of the commercial shrimp fisheries along with several species of the genus Pandalus. Commercial landings of shrimp during 1999 totaled approximately 19 million tonstPSMFC, 1999). Knowledge of the life histories of these species, including the duration and growth of their larvae, is important for stock assessment and management. However, remarkably little is known about their early life histories because most species of the genus live at con- siderable depths. Of the 16 Pandalopsis species, the larvae of only three species have been described partly or com- pletely from plankton samples or from larvae reared in the laboratory. The larvae of Pandalopsis japonica were described completely from specimens reared in the laboratory by Komai and Mizushima (1993). Kurata (1964) described the first stage of P. cocci nata from plankton samples and from larvae hatched in the laboratory. Thatje and Bacardit (2000) assumed that larvae of P. ampla occurring in Argentine waters were similar to those of P. dispar and Pandalopsis coccinata. Berkeley ( 1930) described four larval stages of P. dispar based on samples collected in British Columbia coastal waters. The first stage was obtained from ovigerous females, whereas the larvae of the other stages were separated from plankton samples. In addition, the stage described as the fifth stage was not clearly defined. In this study, we describe the complete series of larval stages of P. dispar using specimens reared in the laboratory. Materials and methods Ovigerous females of Pandalopsis dispar were collected on 25 March Park et al.: Larval development of Pandalopsis dispor 119 1999 by using a small shrimp trawl fished at depths of about 40 m near Gabriola Island in the vicinity of the Pacific Bio- logical Station, Nanaimo, Brit- ish Columbia (latitude 49°13', longitude 123°55'). Water tem- perature at the collection site was around 9°C, and salinity was 29.0: rostrum not strongly curved upwards; 5-6 prominent dorsal denticles and 3-4 weak ventral spines; rostrum shorter than carapace; supraorbital spine present. Eyes (Fig. 2A) Stalked; separated from carapace. Antenna (Fig. 2A) General shape unchanged; longer than that of 1st Antennule (Fig. 2B) Peduncle 3- Park et al.: Larval development of Pandalopsis dispar 121 segmented; inner flagellum with 2 distal setae; outer flagellum with 2, 3, 4, 2 aesthetascs on inner margin. Mandible (Fig. 2C) General shape unchanged; bigger than that of 1st stage. Maxillule (Fig. 2D) Coxal and basal endite with serially developed strong spines and multiple setae; endopodite with 2+3 spines; a strong subtermi- nal seta. Maxilla (Fig. 2E) Palp with 2, 2, 2, 3 setae; broad scaphogna- thite with narrow posterior lobe having a long naked seta; coxal endite with 6 distal setae; basal endite with 7 distal setae. First maxilliped (Fig. 2F) Epi- pod bilobed; endopod with 3+1, 2+1, 2+1, 3 setae; exopod unsegmented with 14 plumose natatory setae. Second maxilliped (Fig. 2G) One long and several intermedi- ate sized spines in basal endite; endopod 5-segmented; exopod with 24 plumose natatory setae. Third maxilliped (Fig. 2H) En- dopod 5-segmented, armed with many spines; exopod of 36 plu- mose natatory setae. Pereiopods (Fig. 2, I— M) Not chelate; 1st pereiopod of 4 spines in basal endite; 3 strong and two weak spines in dactylus of 1st pereiopod; general shape unchanged from 2nd pereiopod through 5th pereiopod. Pleopods (Fig. 2N) Bilobed buds, not functional; no seta and hair on buds; no further devel- opment from the 1st stage. Telson (Fig. 20) Unchanged. Third stage Measurement bars represent 1 mm. Figure 3 Carapace (Fig. 3A) CL, 2.7 mm (SD: 0.12, rc=67); longer rostrum than that of 2nd stage; almost 0.9 times as long as carapace; rostrum with 5-6 dorsal spines and 1-2 ventral spines. Antennule (Fig. 3B) Inner flagellum 2-segmented with 0, 2 setae; outer flagellum 2-segmented with 3+3+3, 3+3 aesthetascs. Antenna (Fig. 3A) General shape unchanged; longer than 2nd stage. Mandible (Fig. 3C) Molar and incisor processes present; incisor process with 6-9 teeth; molar process with heavy teeth on biting edge. Maxillule (Fig. 3D) Palp with 2+3 setae; a small subtermi- nal spine; basal and coxal endite with numerous spines. Maxilla (Fig. 3E) Protopodite unsegmented; palp with 1, 2, 2, 1+2 setae and with 4 lobes; broad scaphognathite with narrow posterior lobe bearing numerous setae. First maxilliped (Fig. 3F) Epipod bilobed; endopod 4-seg- mented with 4, 2, 2, 2 setae; exopod with 15-16 plumose natatory setae; Second maxilliped (Fig. 3G) Coxal endite with an epipod and a strong spine; endopod 5-segmented with 3, 2, 2, 4, 6 setae exopod with setae. Third maxilliped (Fig. 3H) Coxal endite with one long and 122 Fishery Bulletin 102(1) one short spine; basal endite with 2 long and 2 interme- diate sized spines; endopod 5-segmented with numerous setae; exopod with 25-26 plumose natatory setae. Pereiopods (Fig. 3, 1— M) General shape unchanged except addition of setae. Pleopods (Fig. 3N) Buds biramous; much longer than that of 2nd stage. Uropods (Fig. 30) Biramous; endopod with a fused spine at distal quarter of outer margin and numerous setae on inner distal margin; exopod with 4 spines on outer margin and numerous setae on inner distal margin. Telson (Fig. 30). With 12 pairs of posterolateral spines plus a median spine. ,F,I-M,Q, ,B,G,H,N Measurement bars represent 1 mm Figure 4 Fourth stage Carapace (Fig. 4A) CL. 3.1 mm (SD: 0.13. re=59); Rostrum slightly longer than carapace and directed forward; ros- trum with 15 dorsal spines and 6 ventral spines. Antenna (Fig. 4A) General shape unchanged; longer than that of 3rd stage. Antennule (Fig. 4B) Much longer inner flagellum than that of 3rd stage; inner flagellum about 0.9 times as long as outer flagellum, 2-segmented with 0, 2 setae; outer fla- gellum 6-segmented with 1, 2, 2, 3, 4 aesthetascs. Mandible (Fig. 4C) Similar to third stage. Maxillule (Fig 4D) General shape unchanged except addi- tion of setae on endites. Maxilla (Fig. 4E) Palp with 3, 2, 2, 3 setae; endites and scaphog- nathite added numerous setae. First maxilliped (Fig. 4F) Expod with 15 plumose natatory setae. Second maxilliped (Fig. 4G) Basal endite with an epipod and a long spine; exopod with 28-29 plumose natatory setae. Third maxilliped (Fig. 4H) Ex- popod with 36-37 plumose nata- tory setae. Pereiopods (Fig. 4, l-M) Num- ber of spines increased. Pleopods (Fig. 4N) Lobes much longer than those of third stage. Uropods (Fig. 40) Endopod and exopod with numerous setae on inner distal margin. Telson (Fig. 40) With 12 pairs of spines on posterolateral margin; a pair of lateral spines at distal third. Fifth stage Carapace (Fig. 5A) CL. 3.6 mm (SD: 0.15, re=51); Rostrum directed forward and upward, slightly longer than cara- pace; rostrum with 17-18 dorsal spines and 7-8 ventral spines. Antennule (Fig. 5B) Inner flagellum 3-segmented and about 0.9 times as long as outer flagellum: outer flagellum with 2+2+3+3+3+2+3+5 aesthetascs and distal third 6-segmented. Mandible (Fig. 5C) More ad- vanced development than that of 6th; not much change in biting surface. Maxillule (Fig. 5D) General shape unchanged except addi- tion of setae on endites. Park et al.: Larval development of Pandalopsis dispar 123 Maxilla (Fig. 5E) General shape unchanged. First maxilliped (Fig. 5F) Exo- pod with 16 plumose natatory setae. Second maxilliped (Fig. 5G) Exopod with 31-33 plumose natatory setae. Third maxilliped (Fig. 5H) Exo- pod with 46-48 plumose nata- tory setae. Pereiopods (Fig. 5, l-M) Is- chium slightly expanded in first pereipod. Pleopods (Fig. 5N) Much more developed than pleopods of 4th stage; exopod with 13, 1 setae; endopod with 6 setae and ves- tiges of appendix interna. Uropod (Fig. 50) Exopod with numerous minute spines on outer margin Telson (Fig. 50) Both lateral margins parallel; 19 termi- nal spines; 2 pairs of lateral spines. Sixth stage Carapace (Fig. 6A) CL, 4.0 mm (SD: 0.21, n=48); adult-like. Antennule (Fig. 6B) Inner flagellum as long as outer fla- gellum; inner flagellum with multisegments; outer flagellum with numerous segments. Mandible (Fig. 6C) Incisor part separated from molar process and extended anteriorly. Maxillule (Fig. 6D) 9 terminal spines on basal endite. Maxilla (Fig. 6E) Palp with 2, 2, 2, 1+2 spines; broad scaphogna- thite with narrow posterior lobe bearing 3 long setae. First maxilliped (Fig. 6F) Exop- odite with 4+2, 2, 2 3 long and 1 short spines. Second maxilliped (Fig. 6G) spines; vestigial dactylus. Third maxilliped (Fig. 6H) Propodus armed with many spines; dactylus with 2 spines. Pereiopods (Fig. 6, l-M) 1st pereipods with subchelated terminal segment; 1st pereiopod with slightly expanded ischium. Pleopods (Fig. 6N) Endopod and exopod with numer- ous plumose natatory setae; endopod with epipod almost adult-like. Uropods (Fig. 60) Biramous; larger than those of fifth stage; adult-like. Measurement bars represent 1 mm. Figure 5 Basal endite with 2 long Telson (Fig. 60) Telson with 20 terminal spines and 4 pairs of lateral spines. Discussion The first stage larva of Pandalopsis dispar described by Berkeley (1930) is identical to the larva described in the present study. However, we found that she overlooked some important characteristics. She described the first stage larva as having 24 setae on the margin of the telson. We found, however, that the number of setae is variable, and that the larvae have 11+12, 12+12, or 11+11 marginal 124 Fishery Bulletin 102(1) setae. Of these, 11+12 pairs are more common than the others. Berkeley ( 1930) described the fifth stage based on plank- ton materials. In the present study, what was described by Berkeley ( 1930) as the fifth stage larva turned out to be the sixth stage because the larvae of this stage have fully devel- oped pleopods. Although the larvae of the fifth stage have somewhat natatory setose on their pleopods, they appear not to be completely functional. Compared to the larvae of P. japonica, P. dispar has one more stage than that of P. japonica. The pleopod development of P. japonica from the fourth stage to the fifth stage is very obvious, whereas that off! dispar has another stage and the changes in its fea- tures between the fourth and sixth stages are easily seen. Measurement bars represent 1 mm. Figure 6 The major characteristics of the six larval stages of P. dispar are summarized in Table 2. This table can be used for the identification of the larval stages of this species. Komai ( 1994) reviewed the morphological characters of the first larval stage of three Pandalopsis spp.: P. dispar. P. coc- cinata, and P.japonica.The larvae of P. dispar at this stage are quite different from those of the other two species. The larvae of P dispar have a triangular telson, whereas those of P coccinata and P. japonica have a semicircular telson. The adults of the genus Pandalopsis differ from those of other pandalid shrimps by having a laminated expansion on the first pereiopod (Schmit, 1921; Butler, 1980). This character is also present in larvae of P coccinata and P. japonica, whereas it is not present in larvae of P. dispar. From the third stage the is- chium does indicate expansion, however, it is not distinctive. It is assumed that in P dispar. the expansion should be distinctive after the larval stages. In P. coccinata and P. japonica f ) 'vi_— the ischium of the first pereio- pod has a laminated expansion; however, in P. dispar it has no lamination. The structure of the ischium of the first pereiopod can be a diagnostic feature of P. dispar in addition to the shape of the telson. Interspecific variation in the larval stages of pandalid shrimp is large, ranging from three to thirteen stages (Rothlisberg, 1980; Komai and Mizushima, 1993). Haynes (1980, 1985) assumed that P. dispar might have seven pelagic stages, or at least more than four. The pres- ent study has determined that P. dispar has five zoeal stages prior to the juvenile stage. Pandalopsis dispar is one of the four principal target species of shrimp trawl fisheries in both offshore and inshore areas of the NE Pacific Ocean (PICES, 2001) but has undergone very large fluctuations in abundance, particularly in Alaska where it was reduced to extremely low levels during the late 1980s and through the 1990s. These fluc- tuations appear to have been associated first with climate fluctuations (Anderson, 2000), and second with intense har- vesting (Oresanz et al., 1998). Anderson (2000) has suggested that pandalid shrimp population changes are one of the early in- i i< Park et al.: Larval development of Pandalopsis dispor 125 Table 2 Major characters of Pandalopsis dispar larvae. Characters Larval stage' 1 2 3 4 5 6 (postlarva) Antenna Inner flagellum One strong spine One peduncle with a few small spines 2 segments 2 segments 3 segments Multisegmented over 14 Outer Not segmented Slightly developed 2 segments 6 segments 7 segments 12 segments Telson 12+11, 12+12, or 11+11 10 pairs of terminal spines, 2 pairs of uropods One spine on each midlateral margin 2 spines on each lateral margin 4 spines on each lateral margin Pleopod development Wide as much as long Longer than wide Almost separated lobes Longer lobes than those of stage 3 Lobes separated completely with natatory setae Adult-like, with many natatory setae on both lobes ' Eyes of the first stags are sessile on carapace, whereas those of the second and later stages are stalked. dicators of shifts in marine communities in this region. Orensanz et al. (1998) have suggested it is important to recognize that crustacean stocks can have multiscale spatial structures; species have possibly both widely dis- tributed populations (such as in the oceanic offshore) and populations with discrete and localized distributions (as may occur in the nearshore inlets). The ability to distinguish the larval stages of Pandalopsis dispar from routine plankton samples is therefore of use in studying both these problems of population fluctuations and population distributions. Early identification of trends in strong versus weak year classes can provide rapid indica- tions of possible changes in large-scale climate conditions. Unambiguous identification of planktonic stages of P. dis- par is also essential for studies of the spatial structure of its populations, for studies of transport pathways and potential mixing rates among populations, and ultimately for under- standing the metapopulation structure of these populations. This latter point is critical for the development of improved management approaches, which may include identification of reproductive refugia (Orensanz et al., 1998). Acknowledgments We wish to thank Jim Boutillier and Steve Head for their support with this study. This study was supported by the Korea Research Foundation Grant (KRF-2002-013- H00005). Literature cited Anderson, P. J. 2000. Pandalid shrimp as indicators of ecosystem regime shift. J. Northw. Atl. Fish. Sci. 27:1-10. Baba, K., K. Hayashi, and M. Toriyama. 1986. Decapod crustaceans from continental shelf and slope around Japan, 336 p. Japan Fisheries Resource Conserva- tion Association, Tosho Printing Co. Ltd., Tokyo. Berkeley, A. 1930. The post-embryonic development of the common pan- dalids of British Columbia. Cont. Can. Bio. Fish., New Series 6:79-163. Butler, T H. 1980. Shrimps of the Pacific coast of Canada. Can. Bull. Fish. Aquat. Sci. 202, 280 p. Hanamura. Y., H. Khono. and H. Sakai. 2000. A new species of the deepwater pandalid shrimp of the genus Pandalopsis (Crustacea: Decapoda: Pandalidae) from the Kuril Islands, North Pacific. Crust. Res. 29:27-34. Haynes, E. 1980. Larval morphology of Pandalus tridens and summary of the principal morphological characteristics of North Pacific pandalid shrimp larvae. Fish. Bull. 77:625-640. Haynes, E. 1985. Morphological development, identification, and biol- ogy of larvae of Pandalidae, Hippolytidae and Crangoni- dae (Crustacea: Decapoda) of the northern North Pacific Ocean. Fish. Bull. 83:253-288. Holthuis, L. B. 1980. Shrimps and prawns of the world, an annotated catalogue of species of interest to fisheries. FAO species Catalogue. FAO Fish. Syn. 125, FIR/S 125 Vol.1, 271 p. Jensen, G. C. 1998. A new species of the genus Pandalopsis (Decapoda: Caridea: Pandalidae) from the Eastern Pacific, with notes on its natural history. Species Diversity 3:81-88. Komai. T. 1994. Deep-sea shrimps of the genus Pandalopsis (Decap- oda: Caridea: Pandalidae) from the Pacific Coast of eastern Hokkaido, Japan with the description of two new species. J. Crust. Biol. 14:538-559. Komai, T, and T. Mizushima. 1993. Advanced larval development of Pandalopsis japonica 126 Fishery Bulletin 102(1) Balss, 1914 (Decapoda: Caridea: Pandalidae) reared in the Laboratory. Crustaceana 64:24-39. Kurata, H. 1964. Larvae of deacapod Crustacea of Hokkaido. 3. Pandalidae. Bull. Hokkaido Reg. Fish. Res. Lab. 28:23- 34. Orensanz, J. M., J. Armstrong, D. Armstrong, and R. Hilborn. 1998. Crustacean resources are vulnerable to serial deple- tion— the multifaceted decline of crab and shrimp fisheries in the greater Gulf of Alaska. Rev. Fish Biol. Fisheries 8: 117-176. Pike, R. B„ and D. I. Williamson. 1964. The larvae of some species of Pandalidae (Decapoda). Crustaceana 6:265-284. PICES. 2001. Commercially important crabs, shrimps and lobsters of the North Pacific Ocean. PICES Scientific Report 19. 79 p. North Pacific Marine Science Organization, Sidney, B.C., Canada PSMFC (Pacific States Marine Fisheries Commission). 1999. 52nd annual report of the Pacific States Marine Fish- eries Commission (A. J. Didier. ed.l, 37 p. Pacific States Marine Fisheries Commission, Gladston, OR. Rothlisberg, P. C. 1980. A complete larval description of Pandalus jordani Rathbun (Decapoda, Pandalidae i and its relation to other members of the genus Pandalus. Crustaceana 38:19-48. Schmitt, W. L. 1921. The marine decapod Crustacea of California with special reference to the decapod Crustacea collected by the United States Bureau of Fisheries Steamer "Albatross" in connection with the biological survey of San Francisco bay during the years 1912-1913. LTniv. California, Publ. Zool., vol. 23, 470 p. Thatje, S., and R. Bacardit. 2000. Larval development of Austropandalus grayi (Cun- ningham, 1871) (Decapoda. Caridea. Pandalidae) from the southwestern Atlantic Ocean. Crustaceana 73:609-628. 127 Abstract— This study was undertaken to resolve problems in age determina- tion of sablefish (Anoplopoma fimbria). Aging of this species has been ham- pered by poor agreement (averaging less than 45%) among age readers and by differences in assigned ages of as much as 15 years. Otoliths from fish that had been injected with oxytetracycline (OTC) and that had been at liberty for known durations were used to determine why age determinations were so difficult and to help determine the correct aging procedure. All fish were sampled from Oregon southwards, which represents the southern part of their range. The otoliths were examined with the aid of image processing. Some fish showed little or no growth on the otolith after eight months at liberty, whereas otoliths from other fish grew substantially. Some fish lay down two prominent hyaline zones within a single year, one in the summer and one in the winter. We classified the otoliths by morphological type and found that certain types are more likely to lay down multiple hyaline zones and other types are likely to lay down little or no zones. This finding suggests that some improvement could be achieved by detailed knowledge of the growth char- acteristics of the different types. This study suggests that it may not be possible to obtain reliable ages from sablefish otoliths. At the very least, more studies will be required to under- stand the growth of sablefish otoliths. Sources of age determination errors for sablefish (Anoplopoma fimbria)* Donald E. Pearson Santa Cruz Laboratory National Marine Fisheries Service 1 10 Shaffer Road Santa Cruz, California 95060 E-mail address Don Pearsom&Noaa Gov Franklin R. Shaw Alaska Fisheries Science Center National Marine Fisheries Service 7600 Sand Point Way NE Seattle, Washington 98118 Manuscript approved for publication 14 July 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish Bull. 102:127-141 (2004). Sablefish (Anoplopoma fimbria) are a valuable groundfish resource off the west coast of North America. The fish- ery in California, Oregon, and Wash- ington is tightly regulated according to periodic stock assessments. Between 1990 and 1998 landings averaged more than 8000 metric tons per year and an average exvessel (retail) value of 12.5 million dollars per year (PFMC, 1999). Sablefish are distributed in the northeastern Pacific Ocean from Baja California to the Bering Sea and southeast to northern Japan (Miller and Lea, 1972). Males and females are sexually mature between 55 and 67 cm, although there is considerable variation (Fujiwara and Hankin 1988a; Hunter et al, 1989). Off Washington, Oregon, and California, sablefish spawn from Octo- ber through April and spawning peaks in January and February. Sablefish are oviparous, releasing eggs that float near the surface (Hunter et al., 1989). After hatching, larvae and juveniles in- habit surface waters offshore for several years after which they migrate inshore and settle to the bottom. Sablefish are found on the continen- tal slope and are commercially fished at depths from 200 to 1400 meters (Leet et al., 1992). Adult sablefish feed on fish, cephalopods, and crustaceans (Laidig et al., 1998). They reach a maximum length of 102 cm (Miller and Lea, 1972) and are believed to be a very long-lived species (possibly 100 years or more). Many physical features have been used to age this species, including scales, finrays, thin-sectioned otoliths, and broken and burned otoliths, but all methods have resulted in less than 45% agreement among readers (Lai, 1985; Fujiwara and Hankin 1988b; Kimura and Lyons, 1991; Heifetz et al. 1999). The broken and burned otolith method (Chilton and Beamish. 1982) is the principal method used in aging of the species in both the United States and Canada. Typically, age readers agree on ages less than 50% of the time, and for fish older than 7 years, agreement drops to less than 15% (Kimura and Lyons. 1991). There have been repeated efforts at validating sablefish ages and develop- ing aging criteria. Beamish et al. ( 1983) successfully used oxytetracycline (OTC ) marking to validate ages and repeated his experiment in 1995 when additional marked fish were recovered (MacFar- lane and Beamish, 1995). Lai (1985) validated the use of otoliths for aging sablefish. Fujiwara and Hankin ( 1988b) examined otolith growth characteristics to help refine aging criteria. Heifetz et al. (1999) validated the currently ac- cepted aging practices and examined sources of error in the aging of sablefish. Kastelle et al. (1994) used radiometric methods to generally validate the aging criteria currently used. Even with all of these studies that have validated age * Contribution 119 from the Santa Cruz La- boratory, National Marine Fisheries Ser- vice, Santa Cruz, CA 95060. 128 Fishery Bulletin 102(1) ?8W 126"W 124°W 122 W 120W 118"W 116°W Figure 1 Map of California and southern Oregon showing the locations ( black dots ) of sablefish sampling and tagging in September and October of 1991. determinations, independent age readings seldom are in agreement. This suggests that the methods used to validate the ages were insufficient to allow development of precise aging criteria. The lack of reliable age data has made stock assessments difficult and controversial (Crone et al., 1997 ). and in addition, accurate aging is needed to support eco- logical and habitat studies. In September and October of 1991, a tagging and oxytet- racycline (OTC) injection study was included as part of a fish trap survey of the abundance of sablefish in southern Oregon and California. The purpose of this study was to attempt, once more, to improve our ability to reliably age sablefish, thereby improving our ability to manage the species. Methods trarily selected fish at each station, and the rest of the fish were tagged with blue spaghetti tags. Three of every four tagged fish were injected intraperitoneally with 30 mg of OTC per kilogram offish (Beamish et al., 1983) and the fourth fish was used as a control. A complete description of the survey can be found in Parks and Shaw ( 1994 ). A scientist visited the major commercial fishing ports in California and southern Oregon to make port samplers, commercial dealers, and fishermen aware of the impor- tance of the study and to explain handling procedures in the study. A $50.00 reward was offered for the return of whole tagged fish. When a tagged fish was returned, the port sampler measured it (fork length in mm), determined the sex, and removed the otoliths. The otoliths were cleaned and stored in painted glass vials (because the OTC mark was light labile) with a 5095 ethanol solution. Capture, tagging, injection, and recovery In September 1991, the fisheries research vessel Alaska was chartered by the National Marine Fisheries Service to conduct a trap survey from Coos Bay, Oregon, to Cortez Bank, California (Fig. 1). A total of nine sites were visited. At each site seven strings of ten traps were deployed in various depths between 250 and 1900 meters. The traps were retrieved after 24 hours, the catch was removed, and the traps reset for an additional 24 hours. All the sablefish were counted, otoliths were removed from the first 20 arbi- Processing of the otoliths Two pairs of otoliths were initially selected to develop the procedures to be used in the study. It was found that the OTC mark was very faint and upon heating (as required by conventional age determination methods), the mark disappeared. Accordingly, we developed a method to obtain images of the otoliths before and after heating, and to superimpose the two images of the same otolith; the first viewed under UV light and, the second, after heating, under white light. Pearson and Shaw: Age determination errors for Anoplopomo fimbria 129 OTC Mark Alignment point Alignment point Pasted UV Image Figure 2 Composite image of a sablefish otolith. The otolith was first viewed under UV light and an image was captured. It was then baked and a second image was captured by using white light. Then a small rectangle from the UV image was electronically cut and pasted on the image of the baked otolith. The fiourescent mark produced by the OTC appears as a dark line on the UV section. Points on the otolith used for correct positioning of the pasted section are shown. The otoliths were embedded in epoxy casting resin. After the resin hardened, the blocks containing the otoliths were sliced in half across the dorsoventral axis with a diamond saw. Images were captured in a two-stage process. The first stage used ultraviolet light to reveal the OTC mark, and the second stage used white light to reveal the growth marks used for age determination. In the first stage, the room was completely darkened and an image of the otolith, including the OTC mark, was captured by using a video camera capa- ble of capturing images under low light conditions. We used an ultraviolet lamp which produced a strong beam of light at 365 angstroms. The otolith was viewed on a compound microscope using reflected light. The camera and image pro- cessing system were connected to a PC computer equipped with a frame grabber card. A version of NIH Image, a pub- lic domain image processing software (Scion Corporation, Frederick, MD), was used to process the images. The embedded otolith was placed on the microscope and a drop of mineral oil was placed on the surface of the oto- lith. The limited amount of UV light available to the cam- era required the use of frame averaging. Usually 30 frames were sufficient to produce a sharp view of the otolith and the fluorescing mark. In some cases, the mark was too faint to allow an image to be captured. When there was sufficient fluorescence, two composite images were captured, one at 4x and one at 40x. In the second stage, the same embedded otolith was placed in a small toaster oven at 270°C and heated for 20 to 25 minutes until it had turned dark brown. This baking process enhanced the growth rings for visual analysis and approximated what age readers see using the break and burn method; however, the latter process results in darker hyaline zones than those obtained with this method. After cooling, the otolith was viewed under white light. A second set of images was then captured. A section of each UV im- age was then electronically cut and pasted onto the image captured under visible light. With some experimentation it was found that the pasted sections could be aligned exactly over the visible light images, creating a final composite im- age as shown in Figure 2. Initial examination of the otoliths Initially, all OTC-marked otoliths were examined with knowledge of the year and season of release, but without any other information about the fish. Composite UV and white light images were obtained as previously described. The age reader determined the following: whether or not the OTC mark was visible; whether the OTC mark was in a hyaline or opaque zone; the number of annual hyaline zones visible beyond the OTC mark (and whether or not the edge was included in the count); edge type (hyaline, narrow opaque, wide opaque, or unidentifiable), and the shape of the otolith. In some cases the OTC mark could not be identified or the mark was too faint to be captured as a composite image; these specimens were excluded from subsequent analyses. 130 Fishery Bulletin 102(1) : v.; - ■*■-■ count from here Figure 3 Example of an image of a baked sablefish otolith which has been annotated with a mark. The image is an example of one of the images provided to three researchers in order to obtain cross-reading comparisons. Following standard age determination procedures (Chil- ton and Beamish. 1982), if a hyaline zone was not visible on the edge between January and March, then the edge was counted. If a mark was not visible on the edge between April and May and there was a wide opaque zone, then the edge was counted as a mark. If a mark was visible on the edge and the month was after May, the edge was not count- ed. This procedure is used to properly assign the fish to an annual cohort. Because the reader was not given the month of recapture, the ages were adjusted based on the count of hyaline zones, the month of recapture, and whether the edge had been counted. This adjustment provided a cor- rected reader count of annual marks. The corrected count was compared to the number of annual marks that would have been present if marks were laid down annually. Previous experience suggested that there are differ- ent patterns of sablefish otolith growth. We attempted to classify and characterize these different types of growth patterns based on morphology of the otoliths as seen in cross section. After the otoliths had been examined, we developed a standard classification scheme of morphologi- cal classes and types which could be used to classify the most commonly observed morphological types. The otoliths were re-examined and reclassified to see if difficulties and discrepancies in aging were associated with morphological type. It was hoped that this process could be used to refine the aging criteria and improve precision. Because sample size was small, we used a Fisher exact test (Agresti, 1990) to test for independence of morphological type versus tendency to over-estimate, correctly estimate, or under-estimate the number of annual marks. The columns in the test indicated whether the fish had been over-aged. correctly aged, or under-aged. The rows in the test were the four morphological types identified in this study. Examination of the otoliths by the age readers To determine how age readers would count the marks on the otoliths, we selected a subsample of 25 otoliths to be aged at four West Coast fisheries laboratories. The otolith selection was based on having good quality images and otoliths. The images of the baked otoliths (not the compos- ite images ) were annotated with a mark ( Fig. 3 ). The mark was placed in a location which could be readily located on the actual otolith by the readers — on the zone just inside of the OTC mark. Readers were given the following: a set of printed images, an electronic file of the images for viewing on a computer screen, the embedded otolith, the month of capture, the size and sex of the fish from which the otolith came, and a set of instructions for examining the otoliths. Readers were not told where the mark on the image was placed in relation to where the OTC mark was in order to reduce bias from readers who may have known when the fish were injected and recaptured. Readers were asked to provide the following: the number of annual marks vis- ible outside the mark on the image, whether the edge was counted, how confident they were of their readings, and any comments they might have. Three readers participated in this analysis, two of whom had extensive, long-term experience in aging sablefish. The readings and age determination criteria (including edge count criteria) were compared to each other and to the time known to have passed between OTC marking and recapture. Pearson and Shaw: Age determination errors for Anop/opoma fimbria 131 Figure 4 Images of four otolith morphological types. (A) Otolith is a wide type, (B ) otolith is a wide, wedge subtype. (C) otolith is a thick type, and (D) otolith is a thick, wedge subtype. To determine if age determination difficulties were relat- ed to sex, size, area of capture, depth of capture, or otolith morphological type; Fisher exact tests were performed. In each test, the variables were compared to whether the fish had been correctly aged, over aged, or under aged. Results Recoveries A total of 2575 fish were tagged at the nine sites, and 368 tagged fish were recaptured. Of the recaptured fish, 284 had been injected with OTC. Of the 284 injected fish, usable otoliths were recovered from 191 fish; for the remaining fish, otoliths either were not recovered or were too badly damaged during removal to be used. Otolith morphological types After examination of all the otoliths, "wide" and "thick" morphological types were identified, and each type had a "wedge" subtype ( Fig. 4 ). Each otolith in the study was then classified according to this scheme. The wide type (Fig. 4A) is characterized by new growth that steadily increases cross sectional width along the dorsal and ventral surfaces. In the wedge subtype (Fig. 4B), initial growth increases the width, but the most 132 Fishery Bulletin 102(1) recent growth is concentrated on the medial or lateral surface at the sulcus, decreasing towards the dorsal and ventral surfaces, resulting in a wedgelike appearance. The thick type (Fig. 4C) is characterized by new growth that increases the thickness of the otolith without increas- ing the cross sectional width, causing the annulii to appear closely spaced on the lateral surfaces. In the wedge subtype (Fig. 4D), the most recent growth is concentrated at the sul- cus and narrows towards the dorsal and ventral surfaces, forming a wedge shape. It should be noted that these types and subtypes are not always clearly defined. It should also be noted that clas- sification to the subtype is based on the most recent one or more hyaline zones. A wedge subtype is formed when a single hyaline zone widens near the sulcus and comes to a point at the outer edge. Of the 191 otoliths examined, 63 (33.0%) were classified as "wide" types, 76 (39.7% ) were classified as "wide, wedge subtypes," 32 (16.8%) were classified as "thick" types, 5 (2.6%) were classified as "thick, wedge subtypes,' and 15 i 7.99? ), could not be classified by this scheme. Position of the OTC mark There was no detectable OTC mark in 22 of 191 otoliths. The absence of marks appeared to be a random event, occurring in otoliths from several different recovery years and equally likely to be found among different sexes, otolith types, different depths, and locations. Of the 169 otoliths with detectable marks, the OTC mark was found in a hyaline zone in 129 otoliths (76.3%), in an opaque zone in 36 otoliths (21.3%), and could not be reli- Pearson and Shaw: Age determination errors for Anoplopoma fimbria 133 Table 1 Frequency of otoliths with an OTC mark appearing on the edge versus those with the marks inside the edge. All fish were injected between September and October of 1991. Mark Mark Year Month on edge not on edge 1991 Oct 2 1 Nov 1 3 Dec 4 2 1992 Jan 2 4 Feb 1 6 Mar 7 4 Apr 3 1 May 7 26 Jun 2 Jul 1 7 Aug 1 4 Sep 1 2 Oct 5 Nov 3 Dec ably determined in four otoliths (2Ac/c) because the marks were between a hyaline and opaque zone. Of the .36 otoliths with the mark in an opaque zone, the mark occurred just after a hyaline zone in four otoliths. In 24 of the 36 otoliths with the mark in an opaque zone, the mark was on the edge where it can be difficult to determine whether it is opaque or hyaline. In no case did the reader indicate that the mark was in a hyaline zone at the edge and thus the edge appeared to be opaque in most cases. The OTC mark occurred on the otolith edge in 30 of the otoliths recaptured prior to 1993 (up to 16 months after injection). Examination of the monthly distribution of oto- liths with marks on the edge ( Table 1 ) indicated that some fish exhibited little or no otolith growth for substantial lengths of time. Otoliths from fish recaptured in 1992 with marks on the edge (i.e. showing little growth) were examined and classi- fied by morphological type (Table 2). This examination indi- cated that the thick type is more likely to have little growth Table 3 Number of visible hyaline zones occurring after an OTC mark on otoliths from fish recaptured in 1992. This is shown by three-month interval to show the progression of development of the hyaline zones. All fish were injected in September and October of 1991. Interval No. of hyaline zones 0 1 2 Jan-Mar 12 8 1 Apr-Jun Jul-Sep Oct-Dec 5 14 4 6 2 3 1 because 32"* of the otoliths with marks on the edge were the thick type, yet they made up only Y1CA of the otoliths in the study. Conversely, only 18% of the otoliths with the mark on the edge were of the wide type; however, they made up 33^ of the otoliths in the study. This trend was not statistically significant, however, because the P-value was 0.106. Number of visible hyaline zones The number of prominent hyaline zones after the OTC mark for fish recaptured in 1992 at three-month intervals is shown in Table 3. This distribution shows the otoliths that had no detectable growth but also shows that a hya- line zone forms in many fish during the winter. It also shows that in some fish, a summer hyaline zone is formed; however, the sample size for October-December was small and this is a period when a summer hyaline zone would be expected to be fully visible. The number of visible and prominent hyaline zones after the OTC mark for fish recaptured after 1992 (Table 4), com- pared with the number of zones which should have been counted, showed that if a reader had counted each of the prominent hyaline zones as an annulus, the count would have overestimated the age of the fish. An example of an otolith with a larger number of prominent hyaline zones than expected is shown in Figure 5. It should be noted that a reader would not necessarily have counted each of the Table 2 Number of otoliths in 1992 with OTC marks on the edge by otolith morphological type. Also shown is morphological types in the present study. All fish were injected in September and October 1991. the overall percentage of the Otolith type Wide Wide, wedge Thick Thick, wedge No. Percent No. Percent No. Percent No. Percent 1992 otoliths 4 Otoliths in this study 63 18 10 45 7 33 76 40 32 32 17 1 5 5 3 134 Fishery Bulletin 102(1) Figure 5 Image of a sablefish otolith having more prominent hyaline zones than should have been present. The fish was caught after eight months at liberty. A single hyaline zone should have formed; however, there is a zone on the edge and one midway between the dark OTC mark. Table 4 Counts of the number of prominent hyaline zones versus the number of annual hyaline zones that should have been present after an OTC mark. These counts are for fish recaptured more than 15 months after initial capture. Agreement between counts and number of expected annual hyaline zones is shown in bold. Year Expected number 1993 1994 1995 1996 1997 No. of prominent hyaline zones 10 3 1 3 1 5 1 2 1 1 1 2 1 1 2 Table 5 Percent and number (in parentheses I of sablefish otoliths with more hyaline zones than were expected, with the expected number of hyaline zones (correct count), and with fewer hyaline zones than were expected for each otolith type. Otolith type More zones Expected number of zones Fewer zones Thick 10.3% (3) 41.4%(12) 48.3% ( 14 1 Thick, wedge 0 (0) 40.09! (2) 60.09! (3) Wide 39.39! (22) 48.29! (27) 12.5% (7) Wide, wedge 35.29! (25) 45.19! (32) 19.79! (14) prominent hyaline zones as an annulus (they might have considered them to be checks). In many of these otoliths, there were less prominent zones that were not counted and which were interpreted as checks. Thick type otoliths and thick, wedge subtype otoliths tend to have fewer visible hyaline zones than expected (Table 5). In contrast, wide type and the wide, wedge sub- type otoliths are more likely to have more hyaline zones than expected. The Fisher exact test yielded a significant P-value of 0.001. Blind comparisons of reader counts A comparison of the counts of annual hyaline zones for each reader to the expected number of annual hyaline zones Pearson and Shaw: Age determination errors for Anoplopoma fimbria 135 Table 6 Comparison of number of annual hyaline zones by reader 1 versus the expected number of annua zones that should have been counted. Agreement expected counts are shown in bold. counted hyaline with the Expected count Reade • 1 count 1 2 3 4 5 6 7 1 2 7 2 1 2 2 4 1 1 3 1 1 4 2 5 1 Table 8 Comparison of number of annual hyaline zones counted by reader 3 versus the expected number of annual hyaline zones which should have been counted. Agreement with the expected counts are shown in bold. Reader 3 count Expected count 1 2 3 4 5 6 7 1 10 2 2 2 1 3 1 1 4 2 5 1 3 1 1 Table 7 Comparison of number of annual hyaline zones counted by reader 2 versus the expected number of annual hyaline zones that should have been counted. Agreement with the expected counts are shown in bold. Reader 2 count Expected count 12 3 4 5 6 7 1 5 2 3 1 2 3 2 2 3 1 1 4 5 1 1 1 1 1 after the OTC mark are shown in Tables 6, 7, and 8. In these tables, it is assumed that the readers should not have counted the zone in which the OTC mark occurred because that mark is presumed to have formed in the summer of 1991. Readers 1 and 2 tended to overestimate, whereas reader 3 (the least experienced age reader) had generally good agreement. Reader 1 agreed with the expected count 24% of the time, reader 2 agreed with the expected count 4% of the time, and reader 3 agreed with the expected count 44% of the time. The result for reader 3 is deceptive, how- ever, because that reader did not follow accepted methods of when to count the edge. Reader 1 and reader 2 agreed on whether to count the edge of the otolith in 24 of 25 otoliths (Table 9). Reader 3 agreed with reader 1 on whether to count the edge in 16 of 25 otoliths and 17 of 25 otoliths with reader 2. Had reader 3 followed accepted practice, agreement with the expected count would have been much less. Efforts to determine what factors (depth of capture, loca- tion of capture, sex, size of the fish, and otolith morphologi- cal type) resulted in a miscount of the true number of an- nual marks were inconclusive. We first corrected the count for the fact that all readers counted the mark in which the OTC mark had occurred by subtracting one from their counts, and we then eliminated the readings from reader 3 because of his lack of experience and anomalous age de- termination criteria. Then we examined the relationship of how many otoliths had been over-aged, correctly aged, and under-aged to the above factors. Depth of capture was divided into two groups: less than 600 m and 600 or more m. Location was divided into two groups: north and south of latitude 39 north. Sizes were divided into two groups: <55 cm FL and ;>55 cm FL. And finally, we tested each of the four otolith morphological types. We used Fisher exact tests to determine the probability that differences were due to chance alone. There were no detectable differences from the null hypothesis for depth, sex, or location of capture (Table 10); however, there was some evidence that fish length and otolith morphological type might be related to miscounting. Small fish showed a slightly greater tendency to be over counted (more rings than should have been present) than larger fish (P=0.150). Otolith morphological type showed some departure from randomness: thick types appeared to be more likely to be undercounted (fewer rings than should have been pres- ent) and wide types were more likely to be over counted (P=0.066). Discussion Position of mark There was no visible mark on 22 of the 191 otoliths ( 11.5%). Beamish et al. ( 1983 ) reported that 14 of 129 OTC-injected fish ( 10.9%) had no detectable mark. They attributed this to improper handling of the fish after recapture. The similar- ity in the number of otoliths failing to show the OTC mark between their study and our study suggests that some portion of the population may not absorb sufficient OTC to produce a visible mark. The finding that most of the OTC marks were in a hyaline zone is important. This indicates that many of the sablefish in our study laid down a prominent hyaline zone in the summer. Age readers who conventionally as- sume that an annual mark is laid down only in the winter 136 Fishery Bulletin 102(1) Table 9 Blind reading results of 25 s ablefish otoliths by 3 readers. All fish had been captured and injected with OTC in September and Octo- ber of 1991. The counts thev providec are the number of annual marks outside of the OTC mark "Expected count" indicate s how many winter hya ine zones should have been present. The columns labeled "Edge' refer to whether or not the edge was included in the age reader' s counts. Fish ID no. Recapture date Expected count Reader 1 Reader 2 Reader 3 Count Edge Count Edge Count Edge 10375 4 May 92 1 2 Y 2 Y N 10030 14 May 92 1 1 Y 2 Y N 10267 17 May 92 1 2 Y 3 Y N 10408 17 May 92 1 2 Y 2 Y Y 10417 18 May 92 1 2 Y 3 Y N 10630 25 May 92 1 4 Y 4 Y N 12148 26 May 92 1 3 Y 4 Y N 12176 26 May 92 1 2 Y 5 Y N 12431 26 May 92 1 2 Y 2 Y Y 10568 29 Jul 92 1 1 N 2 N N 11121 lOct 92 1 2 N 6 N N 11117 16 Oct 92 1 3 N 4 N N 10400 12 Jan 93 2 3 Y 5 Y 3 Y 10370 14 Jan 93 2 4 Y 4 Y 3 Y 10870 15 Feb 93 2 2 Y 7 Y 5 Y 10246 15 Apr 93 2 3 N 3 Y 3 Y 11735 16 May 93 2 3 Y 3 Y 2 Y 11586 18 May 93 2 2 Y 4 Y 4 Y 11106 3 Aug 93 2 3 N 3 N 1 N 10617 2 Dec 93 2 5 N 5 N 1 N 10580 23 Mav 94 3 2 Y 4 Y 2 Y 10714 9 Dec 94 3 5 N 3 N 1 N 11516 3 Aug 95 4 4 N 7 N 1 N 11524 16 Dec 95 4 4 N 6 N 1 N 11761 25 Apr 96 5 3 Y 5 Y 3 N Table 10 Comparison of the lumber offish under counted. correctly counted, and over counted by two experienced age readers versus depth of capture, location ( north or south of 39 degrees latitude), sex, fork length and otolith moi •phological type. The P-value from the Fisher exact test is shown indicating the level of significance. LInder counted Correctly counted Over counted P Depth <600 meters 7 14 15 0.987 >600 meters 3 6 5 Location South 4 8 10 0.606 North 3 12 7 Sex Male 3 2 5 0.381 Female 7 18 15 Length <55 cm 4 1 1 15 0.150 255 cm 6 6 5 Otolith type Thick 4 2 2 0.066 Thick, wedge 1 1 0 Wide 2 5 11 Wide, wedge 3 12 7 Pearson and Shaw: Age determination errors for Anop/opoma fimbria 137 Figure 6 Image of a baked sablefish otolith with an electronically pasted section taken from an image captured under UV light. The dark OTC mark is clearly located within a hyaline zone, and the hyaline zone persists through the entire otolith. The fish was injected with OTC on 5 October 1991. would probably mis-age these fish. Because the age read- ers who examined the otoliths without knowledge of the recapture information were not informed that the point they were counting from was just inside the summer mark, it was interesting to note that all three of them counted the hyaline zone in which the OTC mark had occurred as an annual hyaline zone in all cases. In other words, the summer hyaline zone did not appear to be a check to the readers. The readers indicated that the manner of prepa- ration of the otoliths (embedded and baked) was not the manner in which they were accustomed to view otoliths and may have influenced their results. The fact that the hyaline zones were not as dark with the baking method as opposed to the burning method may have influenced the readers age estimates; however, some otolith burns can be quite light and experienced readers recognize the various levels of burning, particularly when cross reading otoliths from other age readers. Readers sometimes use multiple sections and are free to manipulate the otolith to improve viewing, which was not possible in the present study. Beamish et al. (1983) indicated that when readers knew how many marks to look for, they were able to iden- tify false annual marks (checks). According to their study, a check is not persistent throughout the otolith. In Figure 6, the hyaline zone in which the OTC mark appeared clearly persists throughout the otolith. If the hyaline zone which contained the OTC mark began to be laid down in the win- ter, then there would be very little time for the formation of a wide opaque zone to form after injection in the fall. Because the age readers counted the hyaline zone in which the OTC mark occurred, they clearly assumed that it was not a check. If the age readers had known that the hyaline zone (in which the OTC mark occurred) had formed in the summer, then they presumably would not have counted it. It is therefore of interest to see the effect on agreement between reader counts minus the hyaline zone where the OTC mark occurred and the actual number of hyaline zones that should have been present. When we adjusted the reader counts by subtracting one year from their original counts and compared their adjusted counts to the expected number of annual marks (Table 11 ), agreement for readers 1 and 2 improved, whereas it decreased for reader 3 (the least experienced reader). Also of importance is the fact that on some otoliths, even after eight months at liberty, no growth had occurred, as evidenced by the fact that the OTC mark was on the edge. For example, otoliths from two fish, recaptured after eight months at liberty showed marked differences in otolith growth ( Fig. 7 ). On otolith A there was no detectable growth with the OTC mark on the edge, whereas on otolith B there was substantial growth. The OTC marks on both otoliths were very prominent. These otoliths came from similar fish; that is, otolith A came from a 597-mm female fish caught in 680 meters of water at 40°52' latitude, and otolith B came from a 610-mm female fish caught in 480 meters of water at 41°54' latitude. This provides strong evidence that otolith growth, and presumably fish growth, varies greatly among individual sablefish. Beamish et al. (1983) reported that the OTC mark was on or near the edge in 28 otoliths (18.1%) of 154 fish which had been at liberty for two to three years. In a similar time interval, we found that 34 of 126 (27.0%) had the OTC marks on or near the edge. Both the finding of a summer hyaline zone and the differences in growth of the otolith among individual fish 138 Fishery Bulletin 102(1) Figure 7 Images of otoliths from two sablefish showing differences in otolith growth rate. Both fish were injected with OTC in early October of 1991 and were recaptured in May of 1992. (Al Otolith was from a 597-mm female caught in 680 meters of water at 40°52' latitude. iBl Otolith was from a 610-mm female fish caught in 480 meters of water at 41°52' latitude. The OTC mark in A was on the edge, whereas the position of the OTC mark in B is shown on the insert. are important factors in developing reliable and consistent age determination criteria. The importance of using the same age determination criteria among readers cannot be overestimated. In the blind comparison, the readers were asked whether they had included the edge in their count of annual zones. With standard age determination methods, if no hyaline mate- rial is visible on the edge up to about May, then the edge is counted. This procedure is based on the assumption that a zone is in the process of forming but is not yet clearly vis- ible. On the other hand, if hyaline material is observed on the edge after May, it is not counted because it is assumed to be either a check or the beginning of the next winter's hyaline zone. Reader 1 and reader 2 (the two most expe- rienced age readers) agreed on whether to count the edge 96% of the time, indicating that they were using the same criteria. Reader 3, however, agreed with reader 1 only 64% of the time and with reader 2 only 68% of the time which suggests that reader 3 was using different edge-interpreta- tion criteria. Pearson and Shaw: Age determination errors for Anoplopoma fimbria 139 Table 11 Percent agreement between number of hyaline zones counted by three age readers and the number which should have been pres- ent. Also shown is the effect of removing the count of a hyaline zone which formed in the summer and which should not have been counted as an annual mark. Reader 1 Reader 2 Reader 3 Original Corrected Original Corrected Original Corrected 24% 44% 36% 44% 20% Effect of ages on stock assessments Crone et al. (1997) noted that one of the problems with stock assessments of sablefish is that the size at 50% sexual maturity is between 55 and 67 cm ( age 5-7 ) and that there is considerable variability in the these estimates. Further, they noted that there has been difficulty in determining age-specific selectivity because of problems with the ages used in previous assessments. Crone et al. (1997) further noted that there is a considerable discrepancy in ages among the age determination laboratories on the west coast. Finally, the model used to perform stock assessments has estimated that in order to obtain a good fit with the data, the actual level of aging error should be higher than has been reported. The lack of reliable age data has been used to criticize stock assessments. Age and length at sexual maturity has been found to vary substantially by depth (Fujiwara and Hankin, 1988a). Fujiwara and Hankin found that both males and females had a length of 550 mm for the length at 50% sexual matu- rity in shallow water (<600 meters ). In depths greater than 600 m, the size at 50% sexual maturity was 450 mm for males and 500 mm for females. To determine age, they used sectioned otoliths and methods that may not have been directly comparable to the methods used in other studies or the methods used in the present study; nonetheless, they found that both males and females matured at a younger age in deeper water. Saunders et al. (1997) also reported differences in length at maturity related to depth and loca- tion of capture. Methot1 found that ontogenetic movement into deeper water for spawning was more closely related to age than size. If sexual maturity is more closely related to age than length as suggested by Methot, then unreliable ages may explain the variable maturity schedule for sable- fish. In our study, fish were captured over a 900 nmi range at depths from 200 to more than 1000 m. If depth is related to growth of sablefish, then it is possible that the different morphometric types of otoliths observed in our study may also be a function of depth. If depth is responsible for the morphological types, it also suggests that reliability of ages may be a function of the depth at which the sablefish are found. Further, if depth influences growth, a fish which 1 Methot. R. D. 1995. Geographic patterns in growth and maturity of female sablefish off the U.S. west coast. Unpubl. manuscript, 39 p. NOAA, NMFS, Northwest Fisheries Science Center, Seattle, WA. changes its depth over time, may exhibit different patterns of growth throughout its life which would further compli- cate the problem of determining reliable ages. Potential sources of error in this study This study used sablefish caught in the southern part of the sablefish range. Many species show latitudinal varia- tion in growth (June and Reintjes, 1959; White and Chit- tenden, 1977; Leggett and Carscadden, 1978; Shepherd and Grimes, 1983; Pearson and Hightower, 1991). It is possible that the results of this study do not apply to the northern portion of their range. Another potential source of error in our study is the effect of tagging on the growth of the sablefish. MacFarlane and Beamish ( 1990 ) found that tagged sablefish grew slower than untagged fish. If this is true, then the results of this study are much more difficult to interpret. MacFarlane and Beamish did not use OTC and as a result they based their ages on conventional aging methods. If they had injected the fish, it would have been interesting to note whether the ages for the fish in their study would have been inter- preted differently. If fish do grow differently after tagging, many age, growth, and validation studies will need to be re-evaluated. Conclusion Obtaining accurate ages, with reasonable precision, for sablefish is very difficult. Previous aging studies of sable- fish have obtained results similar to ours, even when the readers knew how many annual marks should have been present (Beamish et al. 1983; MacFarlane and Beamish, 1995). We found that some fish lay down two marks a year and others may not lay down any. We also found that certain morphological types of otoliths may be indicative of slow growing fish and others may be indicative of rapidly grow- ing fish (assuming otolith growth relates to fish growth). The fact that agreement among readers or with the cor- rect age consistently ranges between 30% and 45% sug- gests that this imprecision may be inherent in sablefish aging. A substantial fraction of the population may not be able to be reliably aged: some otoliths do not appear to grow and others grow very rapidly, laying down prominent summer hyaline zones that even experienced age readers cannot differentiate from winter hyaline zones. 140 Fishery Bulletin 102(1) We believe the wide type and wide, wedge subtypes are often over-aged, and the thick type and thick, wedge sub- types are occasionally under-aged and further propose that readers be made aware that a hyaline zone typically forms in the winter, but that it is not uncommon for a second mark to form in the summer. Another, less desirable approach, would be for age read- ers to record the morphological type of otolith as a routine part of aging. Users of the data could then incorporate this information into their studies by using a correction factor for fish likely to be under-aged and for fish likely to be over-aged. This factor could be in the form of an aging error matrix as suggested by Heifetz et al. ( 1999 ). This approach may not be practical until more data are available on the true effect on ages for the morphological types described in this study, including how many years would need to be added or subtracted for each type. Finally, a more complete description of the morphological types would be needed to assist the age readers. Acknowledgments We would like to express our gratitude to Delsa Anderl (Alaska Fisheries Science Center), Kristin Munk (Alaska Department of Fish and Game), Shayne MacLellan (Pacific Biological Station, Canadian Department of Fisheries and Oceans), and Bruce Pederson (Oregon Department of Fish and Wildlife) for participating in the otolith blind reading component of this paper. We would also like to thank Dan Kimura and Craig Kastelle of the Alaska Fisheries Science Center for their assistance in developing the design of this study. We would like to thank Michael Mohr (Southwest Fisheries Science Center, Santa Cruz, CA) for his valuable contribution to the statistical analyses used in this study This study could not have been completed without the sup- port of Gary Stauffer (Alaska Fisheries Science Center) who provided funding for the recovery of the sablefish. Additionally, this study would never have been completed without the assistance of numerous commercial market samplers, port biologists, commercial fishermen, and deal- ers who were responsible for collecting and processing the sablefish when they were caught. And finally, we would like to thank William Lenarz (Southwest Fisheries Science Center, retired) for his support of this study. Literature cited Agresti, A. 1990. Categorical data analysis, 558 p. Wiley. New York. NY. Beamish. R. J.. G. A. MacFarlane, and D. E. Chilton. 1983. Use of oxytetracycline and other methods to validate a method of age determination for sablefish. In Proceedings of the second international sablefish symposium, p. 95-116. Alaska Sea Grant Rep. 83-8, Univ. Alaska, Fairbanks, AK. Chilton, D. E„ and R. ■). Beamish. 1982. Age determination methods for fishes studied by groundfish program at the Pacific Biological Station. Can Spec Publ. Fish. Aquat. Sci. 60:19-22. Crone, P. R., R. D. Methot, R. J. Conser, R. R. Lauth. and M. E. Wilkins. 1997. Status of the sablefish resource off the U.S. Pacific Coast in 1997. Appendix G in Status of the Pacific Coast groundfish fishery through 1997 and recommended accept- able biological catches for 1998, 135 p. Pacific Fisheries Management Council 1998. Fujiwara, S., and D. G. Hankin. 1988a. Sex ratio, spawning period, and size and age at matu- rity of sablefish Anoplopoma fimbria off northern California. Nippon Suisan Gakkaisha 54(8):1333-1338. 1988b. Aging discrepancy related to asymmetrical otolith growth for sablefish Anoplopoma fimbria in northern California. Nippon Suisan Gakkaishi 54(1):27-31. Heifetz, J., D. Anderl, N. E. Maloney, and T. L. Rutecki. 1999. Age validation and analysis of aging error from marked and recaptured sablefish. Anoplopoma fimbria. Fish. Bull. 97:256-263. Hunter, J. R., B. J. Macewicz, and C. A. Kimbrell. 1989. Fecundity and other aspects of the reproduction of sablefish, Anoplopoma fimbria, in central California waters. CalCOFI Rep. 30, 1989, p. 61-72. June, F. C, and J. W. Reintjes. 1959. Age and size composition of the menhaden catch along the Atlantic coast of the United States, 1952-55: with a brief review of the commercial fishery. U.S. Fish and Wildl. Serv., Spec. Sci. Rep. Fish. 317, 65 p. Kastelle, C. R, D. K. Kimura, A. E. Nevissi, and D. R. Gunderson. 1994. Using Pb-210/Ra-226 disequilibria for sablefish. Ano- plopoma fimbria, age validation. Fish. Bull. 92:292-301. Kimura, D. K., and J. J. Lyons. 1991. Between-reader bias and variability in the age deter- mination process. Fish. Bull. 89:53-60. Lai, H. L. 1985. Evaluation and validation of age determination for sablefish. pollock and yellowfin sole; optimum sampling design using age-length key; and implications of aging variability in pollock. Ph.D. diss., 426 p. Univ. Washing- ton, Seattle, WA Laidig, T. E., P. B. Adams, and W M. Samiere. 1998. Feeding habits of sablefish, Anoplopoma fimbria, off the coast of Oregon and California. NOAA Tech. Rep NMFS 130:65-79. Leet, W. S., C. M. Dewees, and C. W. Haugen. 1992. California's living marine resources and their utiliza- tion. Calif. Seagrant Publication UCSGEP-92-12, 257 p. Univ. California Davis, Davis, CA. Leggett, W. C, and J. E. Carscadden. 1978. Latitudinal variation in reproductive characteristics of American shad (Alosa sapidissima): evidence for popu- lation specific life history strategies in fish. J. Fish. Res. Board Can. 35:1469-1478. Macfarlane, G. A., and R. J. Beamish. 1990. Effect of an external tag on growth of sablefish ffice. Fish. Bull. 102:142-155 (2004). Growth, mortality, and hatchdate distributions of larval and juvenile spotted seatrout {Cynoscion nebulosus) in Florida Bay, Everglades National Park Allyn B. Powell Robin T. Cheshire Elisabeth H. Laban National Ocean Service National Oceanic and Atmospheric Administration Center for Coastal Fisheries and Habitat Research 101 Pivers Island Road Beaufort, North Carolina 28516 E-mail address (for A. B Powell): allyn powellgnoaa gov James Colvocoresses Patrick O'Donnell Florida Fish and Wildlife Commission Florida Marine Research Institute 2796 Overseas Highway, Suite 119 Marathon, Florida 33050 Marie Davidian Room 209, Patterson Hall 2501 Founder's Drive North Carolina State University Raleigh, North Carolina 27695 The spotted seatrout (Cynoscion nebu- losus) is an important recreational fish in Florida Bay and spends its entire life history within Florida Bay I Rutherford et al.,1989). The biology of adult spotted seatrout in Florida Bay is well known (Rutherford et al., 1982, 1989), as are the distribution and abundance of juveniles in the bay, including a description of the juvenile habitats and their diets (Het- tler, 1989; Chester and Thayer, 1990; Thayer et al., 1999; Florida Department of Environmental Protection1). The temporal and spatial distribution and abundance of larval spotted seatrout in Florida Bay and adjacent waters, and the spatial and temporal spawning habits of these larvae also have been determined (Powell et al., 1989; Rutherford et al.. 1989; Powell, 2003). The early life history of spotted seatrout in other south Florida estu- aries also has been well documented. Peebles and Tolley ( 1988) described the distribution, growth, and mortality of larval spotted seatrout in Naples and Fakahatchee Bays, and McMichael and Peters (1989) described the size distri- bution, growth, spawning, and diet of spotted seatrout in Tampa Bay. Information on growth and mortality of larval and juvenile spotted seatrout in Florida Bay is lacking. Research on these topics would enhance our under- standing of the entire life history of this valuable species, and in particular aid in eventually developing a spatially ex- plicit model for spotted seatrout that is highly desired by the Program Manage- ment Committee for the South Florida Ecosystem Restoration Prediction and Modeling Program. In addition, these life history studies could help clarify ju- venile growth and survival and provide needed information for the restoration Florida Department of Environmental Protection. 1996. Fisheries-independent- monitoring program. 1995 annual report, 58 p. Florida Department of Environmen- tal Protection, Florida Marine Research Institute, 100 8th Avenue SE, St. Peters- burg, FL 33701. Powell et al.: Growth, mortality, and hatchdate distributions for Cynoscion nebulosus 143 25°20' 25° 10' 2.V00' — 24"50' 81 "00' 80"45' so Mr Figure 1 Location of sampling sites for spotted seatrout (Cynoscion nebulosus) in Florida Bay, Everglades National Park, Florida, including Florida Bay Subdivisions. of the Everglades, including a return of historic freshwater flows into Florida Bay. Two conceptual frameworks have been advanced to couple the role of growth and mortality in influencing cohort dy- namics. Anderson ( 1988), in a review of hypotheses relating survival of prerecruits to recruitment, advocated a growth- mortality hypothesis as a rational framework for early life history studies that address recruitment variability. This concept predicts that survival of a cohort is directly related to growth rates during the early life stages. The growth- mortality framework, which includes several important in- tegrated components and is based on bioenergetic principles of growth and ecological theory that predict growth rate, is directly related to survival. If it can be demonstrated that survival is a function of growth during the early life stage, then a valuable tool becomes available for examining mecha- nisms influencing recruitment of marine fishes. Another framework suggests that the mortality rate does not operate alone in determining stage-specific survival, but it is the mortality:growth (M:G) ratio (mortality per unit of growth) that determines stage-specific survival (see citations in Houde, 1997 ). Houde ( 1997 ) advanced the idea of using the M:G ratio as an estimator of production and potential survivorship especially in early life stages when both mortality and growth are high and variable. This con- cept was partly based on the strong coupling of growth and mortality demonstrated by Ware ( 1975 ) who argued that when growth rate is poorer than average, larvae would be exposed to sources of mortality over a longer period and hence their mortality rate would increase. Growth and mortality values for successive cohorts would tend to form a cluster of points around a regression of mortality on growth based on average values for a particular species. Our intent is not to test the growth-mortality hypothesis (sensu Hare and Cowen, 1997) as outlined by Anderson (1988), nor fully to develop the M:G ratio concept (Houde, 1997), but rather to use these concepts as a framework for our study. The major goal is to provide information on growth and survival of larval and, mainly, juvenile spotted seatrout that can ultimately be used to develop a spatially explicit model that can be linked to Everglades restoration activities. Therefore, the major objectives of this paper are 1 ) to determine overall growth rates of larval and juvenile spotted seatrout in Florida Bay; 2) to determine and com- pare juvenile growth rates geographically; 3) to estimate natural mortality rates of juveniles; 4) to estimate hatch- date distributions; 5 ) to compare cohort growth and mortal- ity rates and G:M ratios for juveniles; and 6) to evaluate the effects of salinity and temperature on otolith growth — a surrogate for somatic growth. Methods and materials Field collections Larval fish used for otolith microstructure analysis were collected from September 1994 through July 1997, mainly in the Gulf transition, western, and central subdivisions (Table 1, Fig. 1). These subdivisions designated by the 144 Fishery Bulletin 102(1) Table 1 Florida Bay sampling stations where otoliths from spotted seatrout were collected Included are numbers in > of larvae and juveniles used in the otolith microstructure ar alysis. and subdivisions as defined by the South Florida Ecosystem Restoration Prediction and Modeling Program, Program Management Committee. Station Latitude Longitude Florida Bay Juveniles Larvae numbei (degrees and minutes) (degr ees and minutes) subdivisions Location (n) in) 1 25 06.81 81 05.27 Gulf transition Cape Sable 4 2 25 06.37 81 01.42 Gulf transition Middle Ground 1 10 3 25 06.40 80 58.58 Gulf transition Conchie Channel — 4 4 25 07.70 80 56.90 Gulf transition Bradley Key 119 — 5 25 07.12 80 56.07 western Murray Key 4 8 6 25 08.11 80 50.95 central Snake Bight 3 — 7 25 09.45 80 53.42 central Snake Bight 4 — 8 25 07.50 80 48.51 central Rankin Lake 12 — 9 25 05.06 80 47.30 central Roscoe Key 20 — 10 25 02.30 81 .1.12 Gulf transition Sandy Key 49 — 11 25 02.90 80 55.00 western Johnson Key Basin 125 — 12 25 06.00 80 52.50 western Palm Key Basin 110 — 13 25 04.50 80 45.15 central Whipray Basin 2 62 14 25 08.00 80 43.20 central Crocodile Point 9 — 15 24 56.70 80 57.20 Gulf transition Schooner Bank 2 — 16 24 54.70 80 56.31 Gulf Transition Sprigger Bank — 8 17 25 00.40 80 47.68 central Sid Key Bank 6 — 18 24 57.03 80 47.52 central Twin Key Basin 6 — 19 25 07.98 80 40.48 eastern Madeira Point 1 — 20 25 11.85 80 37.15 northern Little Madeira Bay 8 — 21 25 13.00 80 27.80 eastern Shell Key 5 — South Florida Ecosystem Restoration Prediction and Modeling Program, Program Management Committee, were based on modifications of the benthic mollusc com- munity (Turney and Perkins, 1972). In 1994 and 1995, we used 60-cm bongo nets fitted with 0.333-mm mesh fished from the port side of a 5.4-m boat. Beginning in 1996, we used a paired 60-cm bow-mounted push net with 0.333- mm mesh nets similar to that described by Hettler and Chester (1990). Juvenile spotted seatrout were obtained from monitor- ing programs established by the NOAA Center for Coastal Fisheries and Habitat Research (NOAA) and Florida Ma- rine Research Institute (FMRI). NOAA collections were made from May 1995 through September 1997. Juveniles were collected with an otter trawl towed between two 5-m boats. The otter trawl measured 3.4 m (headrope) and was fitted with a 3.2-mm mesh tailbag with 6-mm mesh. FMRI collections were made in 1995 with a seine and a trawl. The 21.4-m center-bag drag seine was fitted with a 1.8 m x 1.8 m x 1.8 m bag of 3.2-mm mesh. The 6.1-m (headrope) otter trawl was fitted with a body of 38.1 -mm stretch mesh and a 3.2-mm mesh tailbag. The majority of juveniles (86ri ) from NOAA and FMRI collections were collected in 1995. Otolith microstructure analysis Otolith processing Otolith removal and preparation gen- erally followed the methods of Secor et al. (1991). All oto- liths, except for the right sagitta, were mounted on a slide with mounting media and archived. The right sagittal oto- lith was embedded for transverse sectioning or polishing (or both). The left sagitta was embedded for transverse sec- tioning if the right was damaged. Sagittae were read with a light microscope at lOOOx magnification under oil immer- sion. The first increment was determined as that following the core increment; which was defined as a well-defined dark increment surrounding the core (Powell et al., 2000). Two blind counts of increments were made by one reader and if the counts differed by more than 5, then the otolith was read again. If the counts were within the acceptable range, the two counts were averaged. Based on a previous validation study (Powell et al., 2000), 2.5 days were added to the increment counts to obtain the daily age. A total of 582 sagittal otoliths were aged. This total included 96 from larval collections from September 1994 through July 1997, 139 juveniles from NOAA collections from June 1995 through September 1997, and 347 from FMRI collections from June 1995 through December 1995. Increment widths were measured on 347 otoliths from FMRI collections (1995) by using image analysis. The measuring path consisted of two segments: a ventral path from the core to the 21st increment and a ventral-medial path along the sulcus, from the 21st increment to the edge (Fig. 2). The 21st increment was selected as the transition point in these measuring paths by test reading 30 otolith sections representing the entire range of sample fish Powell et al.: Growth, mortality, and hatchdate distributions for Cynoscion nebulosus 145 Distal Edae Ventral Counting Path 2 (21 days to capture) Figure 2 Transverse polished section of a spotted seatrout ( Cynoscion nebulosus) ( 18 mm SL; age 48 days) otolith showing the counting paths. lengths. In all samples, the 21st increment could easily be traced in both measuring paths and in all samples the first 21 increments could be measured within the same image. Increment widths were averaged over a 7-day period. Age estimates were also obtained and we eliminated any oto- lith used to measure increment widths if the difference in total increment count between the two methods ( counts ob- tained directly from the microscope versus those attained by image analysis) was greater than 7 days or 10%. On this basis, 117 otoliths were removed from the increment width analysis. We believed counts obtained directly from the microscope were more accurate than those obtained by summing the number of increments measured on the computer moni- tor with the image analysis system. Counting increments directly through the microscope lens allows the reader to optically section the otolith (by varying the focus), which helps in detecting daily increments. "Frozen" multiple im- ages are a result of using the image analysis; hence optical sectioning is not possible. Data analysis Data from all years and sources were used for 1) overall growth (i.e. larval and juvenile); 2) juvenile growth; and 3) estimates of juvenile mortality. Data from NOAA larval and juvenile collections were used to estimate a body-length-otolith-radius relationship. Data from 1995 FMRI and NOAA collections, which was the most com- plete data set, were used for growth comparisons between cohorts, and hatchdate distributions. Data from 1995 FMRI collections were used for 1) growth comparisons between geographical subdivisions; 2) estimating a wet-weight-age relationship to compute the ratio of wet-weight specific- growth to mortality (G:M ratios), which assesses the rela- tive recruitment potential of individual cohorts (Houde, 1996; Rilling and Houde, 1999; Rooker et al., 1999); and 3) determining the influence of temperature on otolith incre- ment width. We used the FMRI data set exclusively for the above analyzes because collections were spatially more localized and wet weights were available. Natural mortality (M) estimates were derived by regress- ing log. unadjusted numbers on age classes (5-day bins); the resulting slope provided an estimate of total mortality (Ricker, 1975). However, on the basis of the age-frequency distributions (Fig. 3), we considered juveniles a40 days old fully recruited to our gear and juveniles >90 days old ap- peared to avoid our gear. Hence, only juveniles between 40 and 90 days old were used to calculate mortality. Hatchdate distributions were computed on a weekly ba- sis and adjustments for mortality were made on individual juveniles by the equation N =N,/e-z<, where N0 = estimated number at hatching; Nt = number at time t (Nt=l because N0 was calcu- lated for each individual fish); Z = instantaneous daily mortality coefficient; and t = age in days. Spotted seatrout cohorts were divided into weekly units, but comparisons between cohort growth was done on a monthly basis because of inadequate numbers for weekly comparisons. A test of heterogeneity of slopes was imple- 146 Fishery Bulletin 102(1) CJ ^f •* -a- Tt n 't m to n CO ■5f o CM o o o o o co -^- in co r*~ Age class (days) o CO o CD Figure 3 Frequency distribution of spotted seatrout {Cynoscion nebulosus) age classes used in deter- mining minimum age at full recruitment to the sampling gear, and mortality. 32 30 I 28 CO ra 26 a> Q. E CO merited by using a generalized linear model (SAS/STAT software, version 6.12, SAS Insti- tute, Cary, NO to test if growth differed among cohorts. A general linear test ( Neter et al., 1983 ) was used to compare growth between three geo- graphical subdivisions (Gulf transition, western, and central). This test is a function of the error sum of squares of the reduced model minus the error sum of squares of the full model. Adequate numbers of juveniles were not available to com- pare growth in eastern and northern subdivi- sions (Table 1). Circular statistics (Batschelet, 1981) were used to determine if spawning, as determined from hatchdate distributions, was uniform over the lunar month. The phase of the moon for 1995 was identified by the fraction il- luminated (U. S. Naval Observatory Applications Department, 1997). A 3-point moving average was used to test if spawning was cyclical. Cohorts (1995) were categorized according to the following hatchdates: cohort A, 29 March-2 May ("April"); cohort B, 3 May-6 June ("May"); cohort C, 7 June-4 July ("June"); cohort D, 5 July-1 August ("July"); cohort E, 2 August-5 September ("August"); cohort F, 6 September— 3 October ("September"). Comparisons of the relative recruitment potential of individual cohorts (G:M ratios) between all cohorts were unresolved. Although cohort mortality estimates could be generated, they were appropriate (by analyzing r2 and P-values from regression analysis) for only three cohorts (cohorts B, D, and F). A random coefficient model was used to investigate the relationship between growth rate of otoliths with age and 24 22 20 - (I O II 4U - 35 - 30 - < , * \ i \ I 25 - 20 - 15 - 10 - < t 5 - « » Station Figure 4 Mean and ranges of temperature and salinity data by station used in the otolith microstructure longitudinal analysis (relationship between increment width and temperature and salinity). For station locations relative to subdivisions, see Table 1 and Figure 1. temperature from juveniles collected in 1995. Most fish were exposed to salinities in a narrow range between 28 and 34 ppt; only 9 fish were exposed to salinities in the 5-13 ppt range (Fig. 4). Consequently, there was insufficient in- formation to obtain reliable inferences on the relationship of growth rate to salinity or the relationship to salinity and temperature for growth information obtained by using either otolith measuring path. This was a disappointment because growth responses to salinity were considered an important objective in relation to proposed Everglades water management activities. Thus, investigation was restricted to the relationship of growth with temperature. A separate model was fitted for the first ( 1-21 increments) Powell et al.: Growth, mortality, and hatchdate distributions for Cynoscion nebulosus 147 and second (22-60 increments) measuring paths because otolith increment width changed at a constant (age-inde- pendent) rate for each path. We did not include fish with >60 increments because the relationship past this number was determined for only 10% of the fish and included obvi- ous outliers. Letting Y be the otolith width measurement for fish ; at age a , where y indexes time, the model for each path was Y,j = «o, + «i where a0l and «1; are the fish-specific intercept and slope describing the relationship between increment width and age for fish i, and e is a normally distributed error term; thus, ah is the growth rate for fish i over the measuring path. Temperature exhibited only negligible change for any given fish over the measuring path; thus, temperature for fish i was summarized as tr the average temperature over the path for that fish. To determine an appropriate model for the relationship between intercept and growth rate and temperature, a preliminary analysis was performed in which ordinary least squares estimates of «0; and ah were obtained separately for each fish i and plotted against tem- perature. For the first measuring path (1-21 increments), the appropriate model was «o, = A)0 + 0oi'i + bor «i, = Pw + Put, + P\4? + bii> where b0l and bh are normally distributed random effects, allowing growth rates for fish at the same temperature to vary across fish. For the second measuring path (22-60 increments), the appropriate model was, «o, = Poo + /V, + Po-i'r + V «ii = Pw + 0ii'i + Put? + bu- By substitution, these considerations yielded models 1 and 2 for the first and second paths, respectively; Y„ = {Poo + fVP + Cfto + 011*1 + 012*^ a„ + bo blpii+eii (•Pw + Pnl, + 012^ aa + bo, + bi,a„ + e,j (1) (2) thus representing otolith increment width in each case as having a straight line relationship with age, where the slope (age-independent growth rate) depends on average temperature according to a quadratic relationship. The random effects allow observations on the same fish to be correlated, whereas observations across fish are inde- pendent. Models 1 and 2 were implemented in SAS Proc Mixed (SAS/STAT software, version 6.12, SAS Institute, Cary,NC). Daily temperature records were obtained from the Unit- ed States Department of Interiors National Park Service, Florida Bay monitoring stations and averaged over a 7-day period. In 1995, temperature records were available only for Johnson Key Basin ( JKB), Whipray Basin (WB), Little Blackwater Sound (LBS), and Little Madeira Bay (LMB), but spotted seatrout were also collected at other sites (Table 1). Daily temperatures were estimated for Sandy Key (SK) and Roscoe Keys (RK) from values recorded dur- ing sampling trips because both these stations are not in close proximity to National Park Service monitoring sites. Sandy Key values were regressed on JKB values (same dates). Sandy Key temperatures were collected from Janu- ary 1994 through August 1996. The regression model for temperature was SK = 0.76 + 0.9536 JKB [r2=0.89; w=25], Roscoe Key values were regressed on WB values (same dates). Roscoe Key temperatures were collected from Janu- ary 1994 through August 1996. The regression model for temperature was RK = 5.60 + 0.7976 WB [r2=0.87; n=31). Temperature values were available at Murray Key (MK) in 1997. To attain values for our 1995 analysis we regressed MK on JKB (same dates). The temperature regression model was MK = 0.77 + 0.9680 JKB [r2=0.99; re=342]. We reported measurements in standard length (SL). For preflexion and flexion larvae, standard length was mea- sured from the tip of the snout to the tip of the notochord. For postflexion larvae and juveniles, standard length was measured from the tip of the snout to the base of the hy- pural plate. Results Overall growth of larvae and juveniles (<80 mm SL) was best described by the equation log, standar-d length = -1.31 + 1.2162 (loge age) [«=582; r2=0.97]. Growth in body length of juveniles (12-80 mm SL) was best described by the linear equation standard length = -7.50 + 0.8417 {age) [n=486; /-2=0.84]; hence, juveniles between approximately age 20-100 days grew on average 0.84 mni/d. There were no significant differences in juvenile growth in body length among three geographical subdivisions [F*327=0.756; n=333] (Table 2), but there was a significant growth differ- ence in length for one of six 1995 cohorts (Table 3, Fig. 5). Growth in wet weight of juveniles ( 15-69 mm SL) was best described by the equation log(, wet weight = -AAA + 0.0748 (age) [n=347, r2=0.84]. There was a significant growth dif- ference in wet weight for one cohort (Table 4, Fig. 6). Weekly 1995 hatchdate distributions, determined by us- ing daily instantaneous mortality ( 0.0585. Fig. 7 ). indicated juveniles in collections (i.e. survivors) were from spawning that was cyclical and protracted (Fig. 8). The most intense successful spawning occurred during 21-27 June (9.2% of total). Using a 3-point moving average, we observed three similar cycles (Fig. 8). From data on survivors, -25% of ju- veniles were spawned by late May, 50% by early July, and 75% by late August and from data on cohorts, three cohorts (cohorts C, D, and E; early June-late August) comprised 55% of the total estimated spawn of spotted seatrout. There was no correlation between spawning and moon phase (pe- riodic regression r2=0.019, P=0.754) (Fig. 8). The relative recruitment potential (G:M ratio) of the 1995 year class estimated from the wet-weight specific growth coefficient (0.0748) and the instantaneous daily mortal- ity rate (0.0585, Fig. 7) was 1.28. The G:M ratio for three cohorts (B, May; D, July; and F, September) was greater than the ratio for the total 1995 year class because mortal- 148 Fishery Bulletin 102(11 Table 2 Summary of growth data used to compare best described by the linear equation: stan growth ir dard leng length of spotted th = a + b ( age in seatrout among days). three Florida Bay subc ivisions. Growth was Subdivision Intercept Slope n r- Size range (mm SD Gulf transition -11.07 0.8914 139 0.86 16-69 Central -12.23 0.9298 49 0.80 15-63 Western -10.56 0.8834 145 0.85 17-69 Table 3 Summary of statistics for a test for heterogeneity of slopes for cohort somatic growth rates rized according to month of hatchdate (see text). The base parameter is cohort F and all the base cohort. For growth equations, see Figure 5. of spotted seatrout. Cohorts were catego- parameter estimates are deviations from Parameter Estimate Standard error /-value P-value Intercept -7.97270 3.02829 -2.63 0.0088 Cohort A -0.86811 4.31970 -0.20 0.8408 Cohort B -11.85849 3.91387 -3.03 0.0026 Cohort C 1.65094 3.86470 0.43 0.6695 Cohort D -6.70820 4.74936 -1.41 0.1586 Cohort E 1.02077 3.50931 0.29 0.7713 Slope 0.82088 0.05613 14.62 <0.001 Cohort A 0.04054 0.08604 0.47 0.6378 Cohort B 0.24578 0.07106 3.46 0.0006 Cohort C -0.00113 0.07091 -0.02 0.9873 Cohort D 0.15741 0.08730 1.80 0.0721 Cohort E 0.04544 0.06821 0.67 0.5058 Table 4 Summary of statistics for a test for heterogeneity of slopes for cohort wet-weight growth rate of spotted seatrout. Cohorts were categorized according ,o month of hatch date (see text ). The base parameter is cohort F and all parameter estimates are deviations from the base cohort. For growth equations, see Figure 6. Parameter Estimate Standard error /-value P-value Intercept -4.27384 0.23763 -17.99 <0.0001 Cohort A 0.10201 0.37014 0.28 0.7830 Cohort B -0.46348 0.34092 -1.36 0.1749 Cohort C -0.27866 0.32116 -0.87 0.3862 Cohort D -0.19540 0.38352 -0.51 0.6108 Cohort E -0.38260 0.29853 -1.28 0.2009 Slope 0.06974 0.00439 15.88 <0.0001 Cohort A 0.00195 0.00729 0.27 0.7889 Cohort B 0.00679 0.00622 1.09 0.2754 Cohort C 0.00502 0.00575 0.87 0.3835 Cohort 1) 0.00759 0.00702 1.08 0.2808 Cohort E 0.01188 0.00564 2. 11 0.0359 Powell et al.: Growth, mortality, and hatchdate distributions for Cynoscion nebulosus 149 Cohort A Length = -8.84+0 8614 (Age) 60 40 20 0 20 60 Cohort B Length = -19.83 + 1.0667 (Age) F B0 ^ = 0.80 F n = 57 <9 en 60 o 'O a> 40 % o •n nftri'™ 20 --?-: c m rn 40 80 100 Cohort C i Length = -6.32 + 0.8198 (Age) ? = 0.90 B0 | n = 55 40 20 0 80 60 40 20 0 80 60 40 20 0 80 60 40 20 0 Cohort D Length = -14 68 + 0.9783 (Age) r2 = 0.80 n = 69 Cohort E Length = -6.95 + 0.8663 (Age) ^ = 0.89 n =99 20 40 60 Cohort F Length = -7.97 + 0.8209 (Age) r = 0.86 n = 50 Age (days) Figure 5 Comparison of growth in standard length among six spotted seatrout (Cynoscion nebulosus) cohorts collected in 1995. See text for cohort hatchdates. Table 5 Daily gr owth (wet weight in grams) rates and daily mort ality rates for three cohorts in Florida Bay in 1995. Cohorts were cate- gorized according to month of hatchdate (see text). The G:M ratio derived from the growth and mortality rates is also presented. For growth equations and associated r- values, see Figure 6. Cohort Hatchdate month Growth rate Mortality rate r2 G:M ratio Size range (mm SL) B May 0.0765 0.0445 0.54 1.72 28- -62 D July 0.0773 0.0565 0.82 1.37 37- -68 F September 0.0697 0.0354 0.67 1.97 37- -66 ity rates appeared relatively low compared to the overall mortality rate (0.0585) for juveniles (Table 5). However, differences in mortality rates among these three cohorts were not significant (F4.;i=1.414). There were no significant differences in weight-specific coefficients among the three cohorts (B, D, and F) (Table 4), but a significant difference in length-specific coefficients among the three cohorts was found (Table 3). Cohort B (May) had a significantly higher growth rate than the other two cohorts. There was a close relationship between otolith radius and body length (Fig. 9). A linear equation with the sagittal ven- tral radius, had a similar r2 as a curvilinear equation with the sagittal dorsal radius. However, we were unable to mea- sure increment widths along this plane and instead used a combination of a ventral path and a ventral medial path. As an initial demonstration that otolith increment width increased with age along the 1-21 increment measuring path and decreased along the 22-60 increment path, simpli- 150 Fishery Bulletin 102(1) 2 ! 1 o -1 -2 -3 Cohort A Loge weight = -4.17 + 0-0717 (age) 20 40 Cohort B Loge weight z2 = 0.88 n = 47 60 80 100 : -4.74 + 0.0765 (age) 20 40 60 80 100 Cohort C Loge weight = -4.55 + 0.0748 (age) ^ = 0.94 n = 47 JM o^°^ o 20 40 60 80 100 Cohort D Log B weight = -4.47 + 0.0773 (age) ^ = 0.81 n = 61 20 40 60 80 100 3 2 1 0 -1 •2 -3 -4 20 Cohort E Loge weight = ^1.66 + 0.0816 (age) ? = 0.85 n = 66 40 60 80 100 Cohort F Log e weight = -4.27 + 0.0697 (age) f2 = 0.86 n = 47 20 40 60 80 100 Age (days) Figure 6 Comparison of growth in wet-weight (grams) among six spotted seatrout \Cynoscion nebulosus) cohorts collected in 1995. See text for cohort hatchdates. fieri versions of Equations 1 and 2 ( see above ) were fitted, in which all coefficients of temperature were set equal to zero, so that Equations 1 and 2 represent simple linear relation- ships with age. For the first path, the estimate of slope was 0.153 fjm/d (P<0.0001); that for the second path was -0.065 fim/d (P<0.0001>. Addition of quadratic terms to each model was not supported (P=0.81 and 0.12, respec- tively). For the first path, whether intercept or growth rate were associated with temperature was determined by test- ing whether the parameters j301, /3n, and j312 were equal to zero. There was no evidence that any of these parameters were different from zero (P=0.45, 0.35, and 0.42, respec- tively); the latter two may indicate that the data do not support the contention that growth rate depends on tem- perature in this range (1-21 d). For the second path, tests "I /'„ ,=0 and /i12=0 offered strong evidence that these pa- rameters are different from zero (P<0.001 in each case). In particular, these results suggested for the age range 22-60 d, otolith growth rates decrease. The extent of the decrease is strongly associated with average temperature according to a quadratic relationship such that growth rates were more steeply decreasing with age for lower temperatures and then became shallower at higher temperatures. In summary, for temperatures at the lower and higher end of the observed temperature range, otolith growth rates for the age range 22-60 d were higher than they were in the middle of the observed temperature range. Discussion Growth in body length of juvenile spotted seatrout in Flor- ida Bay was faster than growth of juveniles from Tampa Bay (Table 6, McMichael and Peters, 1989). Florida Bay is generally considered an oligotrophic system (Fourqurean and Robblee, 1999). Nevertheless, seagrass beds in west- ern Florida Bay, where juvenile spotted seatrout are most common (Chester and Thayer, 1990), are significantly more dense than beds in northwestern Florida waters, slightly north of Tampa Bay (Iverson and Bittaker, 1986). Increased growth of juveniles in Florida Bay could be attributed to the dense seagrass beds that provide habitat for epifaunal crustaceans (Holmquist et al., 1989; Mathe- son et al., 1999), which are important in the diet of juve- Powell et al.: Growth, mortality, and hatchdate distributions for Cynosaon nebulosus 151 5.0 - Log, abundance = 6.83 -0.0585 (age) ^ = 094 4.5 - n n= 10 4.0 - ^^^\ 8 3.5- c CO T3 § 3.0 - .a CO Cu g1 2.5 - _j ^^\. 0 2.0 - \ 1.5 - o ^f ^f -^ Tf r}- ■3- in cd h~ co o o o o o ^f in cd r^. co Age class (days) Figure 7 Catch curve of juvenile spotted seatrout (Cynoscion nebulosus) used to estimate daily instantaneous mortality (Z). Z = slope = -0.0585. Spotted seatrout were fully recruited to the gear at age 40-44 days. Comparison of spotted seatrout growth (size Florida Bay, Florida (this study). Table 6 in mm SL at age) betw ;en Tampa Bay. Florida (McMichaels and Peters, 1989) and Age (days) Area 10 20 30 40 50 60 70 80 90 Tampa Bay 5.1 10.2 Florida Bay 4.4 10.3 15.3 20.3 16.9 23.3 25.4 31.4 30.5 35.6 39.2 47.2 40.7 55.6 45.8 64.1 nile spotted seatrout (Hettler, 1989; McMichael and Peters, 1989). Additionally, warmer water temperatures have been observed in Florida Bay (Boyer et al., 1999) compared to Tampa Bay (McMichael and Peters, 1989); these warmer temperatures could enhance growth if adequate food is available (Warren, 1971). However, our study and that of McMichael and Peters ( 1989) were quite a few years apart; hence differences that we observed could also be accounted for by interannual variability. In addition, differences in growth could also be attributed to differences in sampling gear between the two studies. Florida Bay is a heterogenous ecosystem and consists of ecologically distinct regions (Phlips and Badylak, 1996; Fourqurean and Robblee, 1999); however, we did not de- tect any differences in growth of juvenile spotted seatrout among our three subdivisions. In general, juvenile collec- tions from the central subdivision were from stations that were spatially dispersed; whereas, juvenile collections in the western and Gulf transition subdivisions were from relatively few stations (Table 1 ). Normally, the central sub- division is characterized by the highest salinities in the bay and the western and the Gulf transition are characterized by high salinities (Orlando et al., 1997). However, in our study, salinities in the three subdivisions were moderate and similar (Fig 4). and growth rates estimated for the three subdivisions could be useful as baseline rates, par- ticularly in the central subdivision where salinities are commonly hyperhaline (Orlando et al., 1997). The spawning habits of spotted seatrout throughout their entire range are generally similar. They have a protracted spawning season, are multiple spawners, and reach sexual maturity at an early age. Initiation of spawn- ing might be temperature dependent, with water tempera- tures between 20° and 23°C necessary to initiate repro- ductive development (Brown-Peterson and Warren, 2001). Hatchdate distributions calculated for spotted seatrout in 152 Fishery Bulletin 102(1) Birthweek Figure 8 ( Al Spotted seatrout {Cynoscion nebulosus) («=417) weekly hatchdate distributions adjusted for mortality, including moon phases (#=new moon; 0=full moon), and 3-point moving average (solid line) of hatchdate distributions. (B) Cumulative frequency of spotted seatrout (n=417) hatch- date distributions. Florida Bay in this study along with early stage larval collections (Powell, 2003) indicate that spotted seatrout spawn between March and October (based on hatchdate distributions) and that the majority of spawning occurs between 27° and 35°C , with very little spawning between 20° and 26°C (based on early stage larval collections). Spawning peaks, based on larval collections in 1994-96, occurred in June, August, and September (Powell, 2003), and early May, late June, and late August through early September based on 1995 hatchdate distributions (this study). However, Stewart (19611 reported that spotted seatrout in Florida Bay spawned throughout the year and that spawning peaked in spring and fall. Another larval fish study in Florida Bay indicated that some spawning occurs as early as February and continues into December (Rutherford et al., 1989). Loge Body length = -1 .64 + 0.7821 (dorsal radius) ? = 0.99 n = 232 80 -i 60 40 20 - E 1 ' 1 ' 1 ' 1 ' ' 1 B> 0 200 400 600 800 1000 1200 Sagittal dorsal radius (microns) £ 80 CO (7) au -i Body length = = 0.75 + 0.0503 (ventral radius) ? = 0.99 60 - n = 232 rffO 40 - CM§^> QqAO 20 - n- kd I ' I ' I ' I ' I 0 200 400 600 800 1000 1200 Sagittal ventral radius (microns) Figure 9 The relationship between sagittal otolith radius and standard length (top), and sagittal ventral radius and standard length (bottom) for spotted seatrout (.Cynoscion nebulosus). Peak spawning activity of spotted seatrout is highly variable (McMichael and Peters, 1989; Brown-Peterson and Warren, 2001). McMichael and Peters (1989) observed two spawning peaks; spring and summer. Older fish participate in two peak spawning periods ( Tucker and Faulkner, 1987 ), and a portion of the larger spring-spawned fish (age- 1+) en- ter the spawning population during their second summer, augmenting the number of summer spawning fish. We found that spawning activity and moon phase were uncorrelated, which is not in concordance with observations of McMichael and Peters (1989). They found that distinct peaks in spawning (based on hatchdate distributions of lar- val spotted seatrout) occurred at monthly intervals, and this periodicity might coincide with moon phase. However, this monthly periodicity was not observed when their data for juvenile spotted seatrout were examined. Moreover, statisti- cal tests were not performed on the data in their study. Powell et al.: Growth, mortality, and hatchdate distributions for Cynoscion nebulosus 153 Our inferences, from this study, in relation to spotted seatrout peak spawning are based on hatchdate distribu- tions and should be viewed with caution because hatch- dates are based on survivors. Differential survival for early life history stages can bias results. Hatchdate distributions are valuable when compared to egg or recently hatched lar- val densities and might suggest processes responsible for differential cohort survivorship. Because spotted seatrout undergo a protracted spawning period and because there is high variation associated with icthyoplankton samples ( Cyr et al., 1992), intensive and extensive sampling of recently hatched larvae would be required over a long duration to answer these process-oriented mortality questions. The daily instantaneous mortality rate of juvenile spot- ted seatrout was higher in Florida Bay than those reported from northwestern Florida systems (Nelson and Leffler, 2001). Mortality rates of juvenile spotted seatrout from Florida Bay were 5.7%/d; whereas, for the other systems, rates approximated 3%/d. In general, mortality rates might increase with increasing estuarine temperatures (Houde and Zastrow, 1993). Although we were unable to estimate instantaneous daily mortality rates for larval spotted seat- rout, these data have been estimated for larvae (3.5-6.5 mm) in two southwestern Florida estuaries (Peebles and Tolly, 1988). Highly variable rates were reported between the two Florida estuaries (Naples Bay: 0.70 or 50%/d; and Fakahatchee area: 0.37 or 31%/d). Houde ( 1996) reported a generalized instantaneous daily mortality rate for marine fish larvae of 0.239 (21%/d). Estimating mortality rates for larval spotted seatrout in Florida Bay will be critical for calculating G:M ratios in order to evaluate stage-specific survival and to develop credible spatially explicit models. Mortality rates of spotted seatrout cohorts could be cal- culated for only three of six cohorts (B, May; D, July; and F, September) because slopes were significantly different from zero for only these cohorts. Furthermore, mortality rates of two of the three cohorts (B and F) were associated with low r2 values (Table 5); hence the G:M ratios along with the mortality rates for these three cohorts should be considered "rough" estimates. Attaining more accurate mortality estimates for spotted seatrout would be valuable in linking cohort variability with potential recruitment and stage-specific survival. For example, larval cohorts of bay anchovy [Anchoa mitchilli) from Chesapeake Bay, a tem- perate estuary, exhibit growth rates that are temporally variable and mortality rates that are spatially and tem- porally variable (Rilling and Houde, 1999). Temperature, zooplankton prey and gelatinous predators are believed to influence growth and mortality rates of the bay anchovy. For striped bass iMorone saxatilis ) in a subestuary of Ches- apeake Bay, cohorts exhibited highly variable seasonal G:M ratios that were strongly influenced by temperature (Houde, 1997). In a subtropical estuary, cohort-specific mortality rates for juvenile red drum varied temporally; early and late season cohorts exhibited the highest mortal- ity rates, which coincided with highest growth rates and G:M ratios for midseason cohorts (Rooker et al., 1999). We agree with Houde ( 1997) that future research should focus on the variability and causes of variability in growth and mortality, both of which interact to determine stage-spe- cific survival. The developmental stage or age where G:M variability is greatest, along with the relationship of this variability to recruitment, need to be determined for spot- ted seatrout in Florida Bay. No doubt a relationship exists between G:M ratios and recruitment. Future research should also determine if cohort G:M ratios and somatic growth rates are seasonally or spatially variable. If they are, then a limited spatial and temporal sampling program could be designed to annually evaluate G:M ratios at highly variable stages or ages as an index of year-class strength of spotted seatrout in Florida Bay. Such an index could be verified by examining year-class catch rates on an annual basis or by virtual population analysis. In our study there was little temporal difference in growth of juvenile spotted seatrout cohorts. Larval growth and mortality, which was not treated adequately in our study, could be influenced by copepod prey — an important dietary component of larval spotted seatrout (McMichael and Peters, 1989). The copepod Acartia tonsa is dominant in Florida Bay, but egg production rates for this species are low in the bay compared to those in other systems (Kleppel et al., 1998). We suspect the "bottleneck" to recruitment of spotted seatrout could occur during the larval stage. Hence, future research should examine mortality and growth of larval and recently settled spotted seatrout; in particular the patterns of larval production potential (G:M ratios). Research in these areas should increase our understand- ing of the degree of variability in stage-specific survival and recruitment of spotted seatrout in Florida Bay (Houde, 1996). For most species, especially those with protracted spawn- ing habits, it is most informative to analyze cohort growth and mortality. For example, striped bass and bay anchovy cohorts in Chesapeake Bay exhibit highly variable growth rates, mortality rates, and stage durations (Rutherford and Houde, 1995; Rilling and Houde, 1999). This variabil- ity could cause differential survival for cohorts and result in frequency distributions of survivor hatchdates that do not resemble recently hatched larvae or egg-production frequency distributions (e.g. Crecco and Savoy, 1985; Rice etal., 1987). We are unable to interpret the significance of the abso- lute value of the G:M ratio for juvenile spotted seatrout, because interannual comparisons were not made, but we presented the ratio for future comparisons. Generally, the G:M ratio is <1.0 during the early larval stage, indicating a decline in biomass. However, the G:M ratio of a cohort will eventually exceed 1.0 as a result of a relative decline in mortality as larvae grow (Houde and Zastrow, 1993). Clearly, stage specific analysis of the spotted seatrout from egg through juvenile stage would have been more informa- tive in determining when the maximum G:M ratio occurs (when cohort biomass increases at a maximum rate) and in providing insight into stage-specific dynamics of spotted seatrout (Houde, 1997). A constraint of our study was our inability to estimate larval mortality rates; hence early life history stage dynamics could not be examined. Size-selective mortality in the juvenile life history stages can have important consequences for recruitment. Sogard ( 1997 ) argued that "within-cohort size-selective mortality" 154 Fishery Bulletin 102(1) is more evident in the juvenile stage than during the egg and larval stages when random mortality independent of fish size is more likely to occur (e.g. dispersal of eggs and larvae away from suitable nursery areas). In addition, vari- ation in size, which provides a "template" for size-selective processes, increases during the juvenile stage as larval size is constrained by egg size. Sogard ( 1997) cited a number of recent studies that suggest the early juvenile period plays a greater role in determining year-class strength than previously thought. We were unable to determine if salinity influenced incre- ment width (a surrogate for somatic growth) at early life stages. Understanding the relationship between salinity and growth is critical because Everglades restoration will most likely result in increased freshwater flows to Florida Bay, and during low rainfall periods, salinities in the north central portion of the bay can exceed 45 ppt (Orlando et al., 1997; Boyer et al., 1999). But, salinities were moderate and similar at most stations where juvenile trout were col- lected in the bay during 1995 I Fig. 4). Very few fish were collected at low salinities; in fact, juvenile spotted seatrout are not commonly collected at low-salinity stations (Table 1; Florida Department of Environmental Protection1), and hyperhaline conditions were not observed in 1995. There- fore, we were only able to determine if temperature could influence increment widths. The curvilinear relationship between otolith growth rate and temperature, although a statistically strong relationship, is difficult to explain biologically. Temperature could mask other factors, e.g. temporal variability in prey and predator availability, and optimal temperatures for growth (Rooker et al., 1999). We were able to demonstrate that one cohort grew faster than five other cohorts, possibly indicating differential prey availability in 1995. An individual-based bioenergetics model for spotted seatrout now in preparation (Wuenschel et al.2) should add to our understanding of the effects of salinity and temperature on larval and juvenile spotted seatrout Acknowledgments We are especially grateful to Al Crosby, Mike Greene, Mike LaCroix, and other Beaufort staff that participated in the field work. We thank James Waters of the NMFS Southeast Fisheries Science Center for computer programing assis- tance and Jon Hare of our laboratory for performing the circular statistics. We are grateful to Dean Ahrenholz, Jon Hare, Patti Marraro, Joseph Smith, and three anonymous reviewers for their valuable reviews of the manuscript. We also thank Steve Bobko at Old Dominion University for the image analysis macro used to obtain otolith increment widths. 2 Wuenschel, M. J., R. G. Werner, D. E. Hoss, and A. B. Powell. 2001. Bioenergetics of larval spotted seatrout (Cynoscion nebulosus) in Florida Bav. Florida Bay Science Conference, April 23-26, 2001, p. 215-216. Westen Beach Resort, Key Largo, Florida. Abstract. Center for Coastal Fisheries and Habitat Research, Beaufort Laboratory, 101 Pivers Island Road, Beaufort, NC 28516. Literature cited Anderson, J. T. 1988. A review of size dependent survival during pre-recruit stages of fishes in relation to recruitment. J. Northw. Atl. Fish. Sci. 8:55-66. Batschelet, E. 1981. Circular statistics in biology, 371 p. Academic Press, New York, NY. Boyer, J. N, J. W. Fourqurean, and R. D. Jones. 1999. Seasonal and long-term trends in the water quality of Florida Bay ( 1989-1997). Estuaries 22:417-430. Brown-Peterson. N. J., and J. W Warren. 2001. 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Philadelphia, PA. 156 Abstract— Age and growth of the night shark (Carcharhinus signatus) from areas off northeastern Brazil were determined from 317 unstained ver- tebral sections of 182 males (113-215 cm total length [TLI>, 132 females (111.5-234.9 cm! and three individuals of unknown sex ( 169-242 cm ). Although marginal increment (MI) analysis sug- gests that band formation occurs in the third and fourth trimesters in juve- niles, it was inconclusive for adults. Thus, it was assumed that one band is formed annually. Births that occur over a protracted period may be the most important source of bias in MI analysis. An estimated average percent error of 2.4'S was found in readings for individuals between two and seventeen years. The von Bertalanffy growth function (VBGF) showed no significant differences between sexes, and the model derived from back-calculated mean length at age best represented growth for the species (1^=270 cm, K= 0.11/yr, t0=-2.71 yr) when compared to the observed mean lengths at age and the Fabens' method. Length-frequency analysis on 1055 specimens (93-260 cm) was used to verify age determina- tion. Back-calculated size at birth was 66.8 cm and maturity was reached at 180-190 cm (age 8) for males and 200-205 cm (age ten) for females. Age composition, estimated from an age- length key, indicated that juveniles predominate in commercial catches, representing 74.3% of the catch. A growth rate of 25.4 cm/yr was esti- mated from birth to the first band (i.e. juveniles grow 38?< of their birth length during the first year), and a growth rate of 8.55 cm/yr was estimated for eight- to ten-year-old adults. Age determination and growth of the night shark (Carcharhinus signatus) off the northeastern Brazilian coast Francisco M. Santana Rosangela Lessa Universidade Federal Rural de Pernambuco (UFRPE) Departamento de Pesca, Laboratory de Dinamica de Populacoes Mannhas - DIMAR Dois Irmaos, Recile-PE, Brazil, CEP 52171-900 E-mail address (for R. Lessa. contact author) rplessaigig.com br Manuscipt approved for publication 26 June 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull. 102:156-167 (2004). The night shark (Carcharhinus sig- natus) is a deepwater coastal or semi- oceanic carcharhinid that is found in the western Atlantic Ocean along the outer continental or insular tropical and warm temperate shelves, at depths exceeding 100 meters (Bigelow and Schroeder, 1948). The species has been recorded from Delaware to Florida, the Caribbean sea (Cuba), and northern South America (Guayana) (Compagno, 1984). It has also been recorded in southern Brazil, Uruguay, and Argen- tina (Krefft, 1968; Compagno, 1984; Marin et al., 1998), and on the sea- mounts off northeastern Brazil (02°16' to 04°05'S and 033°43' to 037°30'W. Menni et al., 1995) where it is called "toninha." Since 1991, tuna longline vessels have targeted the night shark in northeast- ern Brazil (Hazin et al., 1998) because of its highly prized fins, the increasing value of shark meat in the local market, and their relatively large abundance and accessability on seamounts (Menni et al., 1995). This species is most im- portant in the area, making up 909; of catches over shallow banks (CPUE, in number, is 2.94/100 hook), and only 15% of catches on the surrounding deep area, yielding 0.04/100 hook (Amorim etal., 1998). Information on this species is re- stricted to taxonomic descriptions (Bigelow and Schroeder 1948; Cadenat and Blache, 1981; Compagno, 1984, 1988), and some biological aspects (Guitart Manday, 1975; Hazin et al., 2000). Night sharks reach >270-280 cm maximum total length (TL) (Compagno, 1984; Branstetter, 1990). Off northeast- ern Brazil, females mature at 200-205 cm TL, males at 185-190 cm. Litter sizes range from 10 to 15 pups and the gesta- tion period may last one year ( Hazin et al., 2000). The assumed size-at-birth off the United States is 60-65 cm TL (Com- pagno, 1984; Branstetter, 1990). Age and growth have not been estimated. The aim of this study is to present the first growth curve for Carcharhinus signatus from vertebral and length-fre- quency analyses. This information will permit the use of age-based stock as- sessment methods for the management of the species in the Exclusive Economic Zone (EEZ) off Brazil. Materials and methods Sampling data and vertebrae were col- lected from November 1995 to Novem- ber 1999 from commercial landings (Natal, Brazil) caught in deep (Aracati, Dois Irmaos, Fundo, Sirius) and shallow (Pequeno, Leste, and Sueste) seamounts with depths between 38 to 370 m at the summits (Fig. 1 ). Commercial vessels were equipped with -30 km Japanese-style multifila- ment longline gear (Suzuki et al., 1977). On average, each vessel used 970-980 hook per day; mainline sets began at -02:00 h and ended at -06:00 h. The retrieval of gear began at noon and fin- ished by dusk. The Brazilian sardinella (Sardinella brasiliensis), margined fly- ingfish (Cypselurus cyanopterus), and squid [Loligo sp. ) were used as bait (Hazin etal, 1998). A total of 1055 individuals, landed whole, eviscerated, or as carcasses (headless and finless). were sampled. The interdorsal space (posterior dorsal Santana and Lessa: Age and growth of Carcharhinus signatus off the northeastern Brazilian coast 157 0° 02°S - 04°S - 06°S ATLANTIC OCEAN Archipelago of Fernando de Noronha 40:W 38=W 36°W 34 =W 32W Figure 1 Location of the sampling area for the night shark iC. signatus) collected off northeastern Brazil. fin base to origin of the second dorsal fin [IDS, cm] ), total length (snout to a perpendicular line from the tip of the up- per caudal fin [TL, cm] ) and fork length (snout to fork of tail [FL, cm]) were measured. In carcasses, only IDS was mea- sured, and IDS, FL, and TL were recorded for eviscerated or whole individuals. A set of five or six vertebrae were removed from below the first dorsal fin in 317 specimens. Total length was measured as the "natural length" (without depressing the tail) according to Garrick ( 1982). To estimate TL for carcasses, relationships from sub- samples of IDS versus TL and FL versus TL were estab- lished for males and females separately. Linear regressions derived for each sex were tested for homogeneity and ana- lyzed for covariances (ANCOVA), resulting in TL=1.2049 FL + 1.7972 (r2=0.944.n=668,P=0.41) and TL = 3.3467 IDS + 30.879 (r2=0.824; rc=764, P=0.161). Whenever length is mentioned hereafter, we always refer to TL. Vertebrae were processed by removing excess tissue, fixed in 49c formaldehyde for 24 hours, and preserved in 70% alcohol. Each vertebra was embedded in polyester resin and the resulting block was cut to about a 1-mm thick sec- tion containing the nucleus by using a Buehler® low speed saw. Initially, alizarin-red-s stained sections (Gruber and Stout, 1983) were compared to unstained sections from the same individuals to define the best contrast for narrow and broad zones. In the first procedure, sections were immersed overnight in an aqueous solution of alizarin red s and 0.1% NaOH at a ratio of 1:9 and then rinsed in running tap water. In stained sections, narrow zones were visible as dark red and broad zones as light red, whereas in unstained sections translucent (narrow) and opaque (broad) zones were visible under transmitted light. Unstained sections produced com- parable results to alizarin stained sections and were used for band observation in the study. Bands counted in each section and distances from the focus to the margin of each narrow zone were recorded. Vertebral radius (VR) was measured by using a binocular dissecting microscope equipped with an ocular micrometer. Measure- ments were made at lOx magnification ( 1 micrometer unit=l mm) with both reflected and transmitted light. The same reader read sections from the same specimen twice at dif- ferent times without knowledge of the individual size or previous count. Whenever the counts differed between the two readings, a third reading was used for back-calculation of size-at-age. The index of average percentage error (IAPE) (Beamish and Fournier, 1981) to compare reproducibility of age de- termination between readings was calculated. IAPE = 1 / Ar]T ( 1 / R^ ( | Xtj - Xj \Xj)x 100, where N = the number of fish aged; R = the number of readings; Xt - the mean age off1' fish at the i'h reading; and Xj = the mean age calculated for the/,! fish. Marginal increment ( MI ) analysis to determine the time of band formation was used. The analysis was restricted to 1995-97, when samples were collected every month. The dis- tance from the final band to the vertebral's edge (MI) was expressed as a percentage of the distance between the last two bands formed on vertebrae (Crabtree and Bullock, 1998). The distance between the last and the penultimate band was divided by the distance between the nucleus and the 158 Fishery Bulletin 102(1) last band for each vertebra that was measured, and we then calculated the mean of this number for the entire sample: IK*. ,)-i?„)//2=0.13(SE = 0.0009). The expected distance between the last (Rn ) and the pen- ultimate (i?n_! ) bands was estimated as a function of the distance between the vertebral nucleus and the last band (MI). The percent marginal increment (PMI) was calcu- lated as PMI = [MI I (0. 13 x Rn )] x 100. Analysis of variance to test for differences in PMI by month was used. Post-hoc tests (Tukey honest significant differences ( [HSD] ) were performed to indicate which months were different. Characterization of the vertebral edge was used to de- termine the time period of band formation (Carlson et al., 1999). Under reflected light, a narrow dark zone (MI 0), a narrow light zone ( MI 0. 1 to 0.5 ), and a broad light zone ( MI 0.6 to 1 ) were observed. Absolute marginal increments ( MI ) were also analyzed by trimester for juveniles aged four and five years, and for adults ( more than eight years ) to confirm the time of translucent zone formation. The relationship between VR and TL was calculated by sex, tested for normality, and compared by ANCOVA (Zar, 1996). The final regression in both sexes did not pass through the origin, thus suggesting that the Fraser-Lee method was the most appropriate for back-calculation (Ricker, 1969). [TL]„ = (RJVR)({TL\-a) + a, where [TL] R = the back-calculated length at age n; - vertebral radius at the time of the ring n\ VR = the vertebral radius at capture; TL = the length at capture; and a = the intercept on the length axis. A von Bertalanffy growth function (VBGF) (von Berta- lanffy, 1938) was fitted to back-calculated and observed length-at-age data with the following equation. L . 1- kit („)i where Lt = predicted length at age t; Lr = mean asymptotic total length; K = growth rate constant; and t0 = the age when length is theoretically zero. To obtain parameters of VBGF, data were analyzed by using FISHPARM (Prager et al., 1987) for nonlinear least- squares parameter estimation. The Kappenman's method (1981), based on the sum of squares of the differences between observed and predicted lengths from a growth model, was used for comparing male and female growth curves. In addition, likelihood-ratio tests were used to com- pare parameter estimates of the von Bertalanffy equation between sexes (Cerrato, 1990). Von Bertalanffy parameters (Lx, K) were also estimated by the method of Fabens ( 1965 ) usually employed for recap- ture data and which takes into account the size at birth (L(l) instead of t0. This method reconfigures VBGF and forces the regression through a known size at birth: L, =Ljl-be- where b = (L., -L0)/Lx We used Fabens routine for growth increment data analysis of the FAO-ICLARM stock assessment tools (FI- SAT) program (Gayanilo et al., 1996), assuming that the time intervals (=At) for each size-at-age class were equal and had a periodicity identical to that obtained from the vertebral analysis. The lengths of 1055 individuals were divided into 5-cm intervals and analyzed by the Shepherd method ( 1987 ) with the length-frequency data analysis program ( LFDA ). Initial values of Lv were based on results from maximal lengths in the sample and from literature (Compagno. 1984). K values ranging from 0.05 to 1.8 were used as input into the program, which was run repeatedly until the highest score function was obtained. The Lx and /f values were then used to calculate t0 (Sparre et al., 1989): tQ = t + {l/K)(\nlL. -lt])/LJ. Using an age-length key, based on 317 individuals for which vertebrae were read, we evaluated the age composi- tion of the sample (Bartoo and Parker, 1983). Maximal ages in the sample were calculated by employing the inverted VBGF (Sparre et al.. 1989). Further, the formula by Fa- bens (1965) [5(ln2)/AT for longevity estimation was used. All statistical inferences were made at a significance level of 0.05. Results The total sample size consisted of 1055 individuals: (551 males [93-248 cm], 499 females [110-252 cm], and 5 individuals of undetermined sex [169-260 cm]) (Fig. 2). Of these, vertebrae were removed from 317 specimens (182 males [113-215 cm], 132 females [111.5-234.9 cm], and 3 individuals of undetermined sex [169-242 cm]). Differences in the relationship between VR and TL between sexes were not found to be significant (P=0.81D. The regression for the overall sample showed a linear relationship: TL = 13.523V/? + 41.824 , indicating that vertebrae are suitable structures for age determination, and methods based on direct proportion are appropriate for back-calculation. The average percentage error, calculated between two readings, ranged from 098 to 4.5^ in vertebrae with 2 to 17 bands and the average IAPE for the overall sample was 2.4'-. Coefficient of variation (CV) between readings for total sample was 6.88' < . Monthly PMI analysis, for the entire sample, indicated that bands were formed from June to October, when high- Santana and Lessa: Age and growth of Carcharhinus signatus off the northeastern Brazilian coast 159 70 i 60 50 >, 40 CJ c CD => 30 i cr CD it 20- 10 ■ ii n i if 1 1 1 n=1055 \\\] Jl 1 r, t I . . Lengtl easter bars = muimmmLfiifimiflminmmifimifimcn OUCVJCvjCvjC\ic\JC\iC\iOT-cjpO'0.05) and likelihood ratio tests (Table 4). Data were then treated together, incorporat- ing individuals of undetermined sex. VBGFs derived from observed length at age were not tested because of missing values in different age classes. The method of Fabens for combined sexes, fitted to back-calculated data, provided L, and K, by using b = 0.781, L0= 62.5 cm (Compagno, 1984) and. At = 1 year (Table 2). Parameters from back-calculation were close to those derived from length-frequency analysis for 1055 specimens, whereas observed lengths and the Fabens method, provided the most varying parameters with lowest correlation and highest coefficients of variation (Table 2). The smallest specimen in the vertebral sample show- ing two complete bands in sections was 111.5 cm, close to the estimated mean back-calculated length at age two of 113.7 cm (Table 3). Size at maturity, 185-190 cm for males and 200-205 cm for females, corresponded to 8- and 10-year-old individuals, respectively (Fig. 6). The largest and oldest specimen whose vertebrae were used, was 242 cm, which corresponded to 17-year-old individual. A growth rate of 25.4 cm/yr was estimated from birth to the first band — a rate that corresponded to 389 of the birth 0 • ■ 0 □ 0.1-0.5 5! r 1 □ 0.6-1 33 45 8 ■ 51 6 ■ 15 2< \ 43 4 • 2 ■ 1 7 9 1 6 5 ' % -i 0 • -| i IJI 1 J ■ 1 -X --I-1- M A J J Month Figure 4 Categorization of edges by month for the night shark iC. signal its) off northeastern Brazil. length (the length at birth being 66.8 cm). Also, a mean rate of 8.55 cm/yr was calculated for 8- to 10-year-old individu- als, when maturity is achieved (Table 3). Considering mature individuals >185 cm. the age com- position for the vertebral samples («=317) indicated that 17.3% of specimens were adults (Table 5). Instead, for the total sample (ra=1055), where the age ranged between 2 to al7 years, adults corresponded to 25.3% of the total sample Santana and Lessa: Age and growth of Carcharhinus signatus off the northeastern Brazilian coast 161 0 1 0.2 0 3 0 4 0 5 0.6 0.7 08 0 9 1 12 10 - 8 6 4 2 0 ill 0 1 0 2 0 3 0 4 0 5 0.6 07 08 09 1 12 10 8 6 4 - 0 3,d Trimester n=36 I.. I 0 1 0 2 0 3 0.4 0 5 0.6 0 7 0 8 0 9 1 12 - 10 4lh Trimester Nihil. . 0 1 0 2 0 3 0 4 0.5 0.6 0 7 08 0 9 1 B r' Trimester n= 14 II I ■ - 2nd Trimester 12 - n = 24 10 - 8 6 4 2 ■ o - 01 0.2 0.3 0.4 0.5 0.6 0.7 08 09 1 12 10 - 3rd Trimester n= 14 ll 01 0.2 03 0.4 05 0.6 0.7 0.8 09 1 12 10 8 6 4 2 0 4m Trimester n=10 Jl 0 1 0.2 0 3 0 4 0.5 0 6 0.7 0 8 0.9 1 Ml Figure 5 Marginal increments (MI) by trimester for ages 4 and 5 (n = 139) (A) and a8 (Bl (n=54) for the night shark (C. signatus) from northeastern Brazil. (Fig. 7). According to the inverted back-calculated VBGF the oldest specimen in the sample was 31.7 years old (260 cm), whereas longevity was 31.5 years. Discussion Validating the time of band formation is considered critical when using hard parts for age estimates (Brothers, 1983), and validation is successful when growth zones are shown to form annually in all age groups of the population (Beam- ish and McFarlane 1983). Marginal increment analysis, carried out on younger and faster growing individuals, cannot always be used for validating older age groups, and therefore all ages must be ascertained (Brothers, 1983). In the present study, we obtained significant differences in marginal increments for the total sample. However, the significance level of the test (P=0.046) was close enough to 0.05 to cause us to suspect that the distributions could have been similar. The time of band formation varied when different age groups were analyzed separately, despite suggestions that bands are completed in the third and 162 Fishery Bulletin 102(1) E o 150 100 B \ 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 0 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 1 250 200 150 100 50 0 i ! V '■ ■ Back-calculated Observed Fabens 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Age (years) Figure 6 Growth curves generated from (A) females. (B) males, and (Cl sexes combined for the night shark (C. signatus) off the northeastern Brazil. Table 2 Von Bertalanffy parameters derived from back-calculated lengths (BC), observed lengths (OL), lengths the length-frequency data analysis (LFDA) package for the pooled database (SE is standard error; CV from the Fabens method, and is coefficient of variation i. Methods Sex L_, (cm) SE CV /f(/year) SE CV f0(year) SE CV r- BC Males 256.5 5.56 0.022 0.124 0.007 0.055 -2.538 0.119 0.047 0.999 Females 265.4 4.15 0.016 0.114 0.005 0.045 -2.695 0.127 0.047 0.999 Both 270 2.78 0.01 0.112 0.003 0.031 -2.705 0.099 0.037 0.999 OL Males 306.1 37.71 0.117 0.076 0.02 0.267 -4.663 0.882 0.189 0.995 Females 297.1 26.71 0.09 0.077 0.018 0.235 -4.853 0.977 0.201 0.99 Both 289.9 7.6 0.026 0.085 0.006 0.077 -4.395 0.348 0.079 0.998 Fabens Both 285.3 15.69 0.055 0.08 0.016 0.2 — — — — LFDA Both 270.9 — — 0.106 — — — — — — fourth trimesters (new bands begin to form in this period) in juveniles. Results were inconclusive for adults. For C. obscurus (Natanson et al., 1995), C. plumbeus (Sminkey and Musick 1995), C. porosus (Batista and Silva, 1995: Lessa and Santana, 1998), C. acronotus (Carlson et al., 1999). and /. oxyrhynchus (Lessa et al., 2000), inconclusive results for MI analysis were obtained. The inability to dem- onstrate the periodicity of band deposition in adult sharks Santana and Lessa: Age and growth of Carcharhinus signatus off the northeastern Brazilian coast 163 in the present study is similar to the outcome for C. limbatus older than four years (Wintrier and Cliff, 1996). For the last mentioned species, the problem was circumvented by restricting MI analysis to juveniles (Killam and Parsons, 1989). Age was assigned by assuming an annual pattern of deposition, as commonly occurs for most carcha- rhinids like C. brevipinna and C. limbatus, Rhizoprionodon terraeno- vae (Branstetter et al., 1987; Brans- tetter and Stiles, 1987), Negaprion brevirostris (Gruber and Stout, 1983), and C. longimanus (Seki et al, 1998; Lessa et al., 1999c). Three sources of bias generally occur with MI analysis: 1) sample sizes are small for any particular month or for any age class (Cailliet, 1990); 2) data are collected over a too long a period causing variability on ac- count of annual marks that are not formed at the same time ( Brothers, 1983 ) and 3 ) births occur over a long period (Brothers, 1983). All these may have biased MI analysis in the present study. Research carried out in the study area by Hazin et al. (2000) indi- cated that copulation takes places throughout the austral summer. Embryos measuring 10 to 40 cm were collected in February, whereas 31.8 to 37.2 cm embryos were found in June. This remarkable variability in embryo size during the gestation period suggests that birth period lasts several months. Furthermore, with an estimated back-calculated birth length of 66.8 cm, individuals measuring -40 cm in February will be born long before individuals that measured 37.2 cm in June. Such a protracted parturition period could lead to differences in MI of the same cohort. Thus, after an assumed -12 months gestation period, individu- als are born with birth dates vary- ing by several months. Moreover, no significant differences in MI analysis was found for C. porosus and /. oxyrhynchus, which also have a protracted birth seasons (Lessa et al., 1999a, 1999b). A comparison of growth model parameters by using known size information, such as size-at-birth and maximum observed size, can be 01 t- CO CO 1 CM O Hr-> 03 - — - 1 CM CN CO ed co XI 00 CO CN CM a o> -— CS lO [^ r- l> CO ■* 135 a > — 1 CO ^ CO CN c- CO CO «? 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CO u 05 CJ) t-; CN CN rH O) in CN CO IM Tf 05 in CO o ho 00 c— o t-' d 00 CO 00 00 d [> CO d CO CO d CN 05 CO 2 -c Oi CO rt cm o CO CO rH 00 "* 00 rH IN "* ■* 05 Tf [^ 00 I> c- Tf en in CO CM CN CN in CO CN CO CO — CN CN ~ iri - yi CN CN ri - ft c -c CU CN rH CO r~ O 05 in in CO (N CD 00 CN CO 00 in CO CM C^ 1 1 -a c rn 03 d CO CN rH CN (N CO CO 05 d rH CO CN CN 1 1 CJ '3 CO 05 05 05 Ol 05 05 05 05 05 05 (35 OS t o CO XI O CO X CU CO l> i-j t- 00 CO CO CN in CO i-i 05 CO 00 in CO X | | -a c "cO 3 o iri iri t> t> I> 00 00 t> r> d ■* d iri d CO r-i CO CO CO in CO CO CO CO CO CO CD CD CO CO X CO T> -a '> cj ■3 ~ "oj O CN 03 o CO CO CO m O) CO rH CN rH a 4J rH CN ■* OJ CO CN CN rH CO 3 HH o o 3 CJ T3 CO CJ Ji CJ CO CD CD cm "cO o -g cm CO CO CQ Tf in CO t- 00 05 = rH CI in C- c CO CJ - CO > e c « j3 S 3q o CQ "~" < 164 Fishery Bulletin 102(1) Table 4 Likelihood ratio tests comparing estimates of von Bertalanffy parameters for males (noted as 1 1 and females (noted as 2) for C. signatus in the linear constraints. Hypothesis Linear constraints Residual SS X2r df P HQ none 60536.4 Hwl £=oi = Lx2 10511 0.049 1 0.996 Hto2 A, = K2 10524.3 0.047 1 0.996 Hto3 '01 = '02 10205.6 0.122 1 0.999 HvA Same L^, A. and t0 24301.2 0.164 3 0.973 useful as a method of verification ( Cailliet et al., 1983). Although no specimens younger than 2-years-old were caught (perhaps due to the gear selection bias), the presumed size at birth was about 60-65 cm ( Compag- no, 1984), which is similar to the estimated size in the present study (66.8 cm). Also, the estimated Lr value (270 cm), derived from the back-calculated or observed VBGF is close to the maximum size of 276 cm men- tioned by Bigelow and Schroeder ( 1948), 280 cm off Cuba (Compagno, 1984), and 275 cm byGarrick(1985). Mean observed length-at-age is gener- ally higher than back-calculated mean length-at-age (Bonfil et al., 1993; Lessa and Santana. 1998), leading to lower values of La and higher values of K. However, in the present study, although mean observed length-at-age is higher than mean back-cal- culated lengths, parameters derived from back-calculation provided a lower Lx and a higher A' value. Inconsistency of the observed length-at-age set is attributed to the missing values in for ages 0, 1, 13, 14, and 16. This led to a VBGF which provided an unrealistic birth size of 90 cm and which present a flatter shape than the back-calculated curve. Von Bertalanffy growth parameters generated from both back-calculation and by the Fabens method were all consid- ered suitable and were of the same magnitude. However, taking into account 1 ) parameters close to those derived for length-frequency analysis, and 2) the best statistical fit, the back-calculated VBGF was chosen as best representing growth in the species. Comparisions of biological features such as maturity size and maximum sizes have been used for inferences in growth and to explain differences between sexes (Natanson et al., 1995; Natanson and Kohler, 1996; Lessa et al., 2000). Tin' studied species shows a disparity of -15 cm in matu- rity sizes between sexes (Hazin, et al., 2000), corresponding to ~2 years. In addition, the largest specimen, for which six was determined, was a 252-cm female and the largest male was 248 cm. These disparities, however, did not bring about differences in growth between sexes, as indicated by results of both tests used. Such a result can be explained by the number of juveniles used for age determination (-83' I 300 250 n =1055 >. 200 0 c S 150 O" 1 "- 100 ■ | 50 0 .ll Mil.. <1 3 5 7 9 11 13 15 >17 Age (years) Figure 7 Age composition for the night shark (C. signatus) collected off northeastern Brazil. Thus, the number of adults was not high enough to bring about any differences in the growth equation although differences frequently occur after maturity, caused by dif- ferent growth rates between sexes (Natanson et al., 1995; Sminkey and Musick, 1995). Assuming that the time elapsed between birth and the band corresponding to age 1 is one year, the species grows 38% of its birth length during the first year. This growth rate is close to that (50%) generally assumed (Branstetter 1990; Cortes, 2000). Furthermore, the estimated K value falls within the range suggested by the first author, and according to him, the night shark is a relatively fast grow- ing species, presenting a life strategy similar to that of C. falciformis, and apparently depending on rapid growth for adequate neonate survival due to vulnerability to preda- tion from large sharks. In summary, considering the increasing fishing effort on the night shark as a targeted species and that catches are mainly composed by juveniles (representing 74.7' i of specimens in landings), we believe that the A'-selected characteristics of the species (including late maturity, long gestation period, and low fertility 1 should be taken into account in determining the management of this resource. Demographic analyses will be required for the examination of consequences of current levels of exploi- Santana and Lessa: Age and growth of Carcharhtnus signatus off the northeastern Brazilian coast 165 c Q. C 3 2 pq LT> c u a) .Q » ,(B CO be 3 O bo tj, bf no ■" cm 2 I 2 I S 2? oi >5 CO «o CO !N I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 8 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I IS I I I I I ! I I I I I I II I I I I I I 12 I8S I M I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I CO CO [- o 1 ' CO -* CO ^ ^H CO I I I I I I CO iO lO ■* CO «-; ^ oj _j ~-; 00 t— tH CO CO t> CO I I I I I I I I I I I I I I I I co I co co t> in o 1 •* co' ~ oi "= I I I I h m -* co I I I I I I I I I I t> m tH CO CO C- I 1> I I I I I I « h ™ ^ n h ! cb I I I I I I I I H CO * —I llllllliquaiaoqwco-*. co«|||||||||| — i cm co co ■* m co I I I I | M10CB^^CDt-C5 CONHCtirtT|'dH h •-< co in in co cm I coocomcoin^-i^j- ' co'^co'cNcodcdiri CO CO CD CM CM ^H I I I I I I I I I I I I I I I I I I I I I I co in t> o — m 10 cri oci o o t-~ in 2 2 «o 7000 ■ ~ 6000 ■ £ 5000 ■ g> |j 4000 ■ □ Females UfkJ/^B wmales = o.oi 64 x u** 'jijr ° — w,emates = o.oi 79 * l* «* afljllo ° 3000 ■ 2000 J r2 (combined model) = 0.95 _^ffl^ 1000- ^«e**^ 0 20 40 60 80 100 Length (cm) Figure 5 Prowfish body weight (W) fitted by an exponential function of fish total length (/..land sex. Data for males (/i=83) are shown by diamonds and the fitted model by a dashed line. Data for females (n=88) are shown by squares and the fitted model by a solid line. Food habits Fish used for diet study averaged 63.8 cm in total length (range: 49-87 cm) in the Gulf of Alaska and 56.9 cm (range: 30-79 cm) in the Aleutian Islands. The contents of 18 prowfish stomachs from the Gulf of Alaska and 58 from the Aleutian Islands showed that jellyfish (999r and 31% Smith et al.: Distribution and biology of Zaprora silenus 175 100 90 80 70 E 60 £ 50 \ S 40 30 20 10 0 L = 89 33(1-e-°,8'<'-<-°554») r = 0.752 10 15 Age (years) 20 25 Figure 6 Prowfish total length \L) fitted by a von Bertalanffy function of age (f). Data for males («=71) are shown by diamonds; data for females (rc=67) are shown by squares. The fitted model is shown by a solid line. Table 1 Mean percent weight (%W) and mean percent freque icy of occurrence (%FO) of the prey items from 18 prowfish stomachs collected in the Gulf of Alaska (GOA; 1996; total prev weight= =299 g and 58 stomachs from the Aleutian Islands area (Al; 1997; total prey weight=1446.6 g). Sample prowfish had an average total length of 63.8 cm (range 49 -87 cm) from the GOA and and 56.9 cm (range: 30-79 cm) from the AI. Prey name GOA(n = 18) AI(n =58) %W %FO %W %FO Scyphozoa (jellyfish) 98.84 100 30.45 29.88 Ctenophora (comb jelly) 0.09 1.23 Polychaeta (worm) 0.03 5.8 Calanoida (copepod) 0.26 28.13 0.04 29.14 Thysanoessa raschii (euphausiid) 0.05 6.67 Mysidacea Mysida (mysid) 0.01 3.13 Hyperiidea (amphipod) 0.19 33.46 Gammaridea (amphipod) 0.12 30.49 Themisto sp. (amphipod) 0.32 28.57 0.14 36.91 Salpa sp. (pelagic salp) 34.06 46.79 Larvacea (pelagic tunicate 1 0.13 12.5 Sebastes sp. (rockfish) larvae, 5-8 mm long 0.43 42.86 Microsomus paeifieus (Dover sole) eggs 0.01 3.13 Unidentified organic material 34.84 32.59 by weight of total food in the two regions, respectively) and gelatinous pelagic tunicates (Salpa spp.; 34% in the Aleutian Islands area only) were the most important food (Table 1). Although calanoid copepods and Themisto sp. (amphipod) were both often present in GOA specimens (28.13% and 28.57% of stomachs, respectively), they were not important food in terms of weight. The same was true in the AI for calanoid copepods, Themisto sp., gammaridean amphipods, and hyperiidean amphipods (29.14%, 36.91%, 30.49%, and 33.46% respectively). Mysids and larvaceans from GOA specimens as well as ctenophors, polychaetes, and euphasiids from AI specimens occurred in trace amounts. Sebastes larvae (5-8 mm standard length), the only fish species found, were found in 43% of Gulf of Alaska stomachs but made up only 0.43% of prey weight. Some Dover sole [Microstomas paeifieus) eggs had also been consumed. 176 Fishery Bulletin 102(1) 0.8 H 0.6 0.4- 0.2- 0 t* 000000 2 -e- 311 2 12 000 000 Pmat = 1/(1+e3"'»-"'M 00 i 0 30 35 40 45 50 55 60 65 70 75 80 85 90 Length (cm) 5 4 1 3 1 1 0.8 - 0.6 - / 3 Jo 0.4 - P 1/(1 +e9«-,9°') 0.2 - 0- 1^2 6 0 i 10 15 Age (years) 20 25 Figure 7 Proportion of female prowfish mature (Pmat) as logistic functions of length (L) and age it). Data points based on 39 maturity-at-length and 27 maturity-at-age observations are shown by diamonds, and numbers of females of each cm-length and year-age class are shown next to the corresponding symbol. The fitted logistic models are shown by solid lines. The length and age at which Pmat = 0.5 with 95^ confidence limits are 57.0 ±0.4 cm and 5.1 ±0.7 years. Discussion Geographic distribution Historically occurring in the catch in AFSC bottom trawl surveys in areas of the eastern Bering Sea, Aleutian Islands, and Gulf of Alaska regions, prowfish were also observed more rarely farther south along the West Coast as far as the vicinity of San Miguel Island, California. This is the apparent southern limit of their range in the northeastern Pacific (Allen and Smith, 1988). They were most often encountered in the vicinity of the edge of the continental slope near 200 m depth (Fig. 2), although our data increase the maximum known depth of occurrence from 675 m (Allen and Smith, 1988) to 801 m. As indicated by survey CPUE, prowfish density was greatest between the depths of 100 m and 240 m (Fig. 3). Our distribution data show similarities with those of Tokranov ( 1999), who studied >300 bottom trawls executed in 1995-97 on the shelf and slope off the southern Kamchatka Peninsula and northern Kuril Islands, in which adult prowfish were taken at 100-480 m. Tokranov often found fish concentrated in areas of high-relief, rocky bottom — a common feature of the shelf edge in the Gulf of Alaska and Aleutian Islands regions. Such areas near the shelf break may be important prowfish habitat. Underwater videos taken in the north- east Gulf of Alaska by the Alaska Department of Fish and Game (Brylinsky5) show numerous adult fish resting on or just above this type of substrate. Density was greater in the AI than in the GOA, over all bottom depths combined and in most cases by individual depth interval (Fig. 3). One reason may be that preferred habitat comprises a larger proportion of the Aleutian Is- 5 Brylinsky, C. 2000. Pers. commun. Alaska Department of Fish and Game, 304 Lake Street, Sitka, AK 99835. Smith et al.: Distribution and biology of Zaprora stlenus 177 lands area. Because of the lack of a relatively broad shelf in the region, a larger proportion of trawls are in or near areas of steep seafloor gradient and therefore likely over rough bottom (Fig. 2). Length distribution In both the Gulf of Alaska and the Aleutian Islands, few prowfish <40 cm in length were captured (Fig. 4). This paucity of small prowfish is not due to size selection by the trawl net mesh because the codend is lined with small mesh (1.3 cm stretched measure) webbing that retains small individuals of other species. A different explanation, based on the observations of Brodeur (1998) and Scheffer (1940), is that pre-adult prowfish are pelagic, remaining in proximity with large coelenterates and thus avoiding bottom trawls. Thus, the minimum capture length may indicate the length at which prowfish recruit to a demer- sal habitat. Our data showed no statistically significant length difference between sexes, in contrast with the data of Tokranov (1999) who suggested a length dimorphism where females are generally longer than males. Weight-length relationship The best-fitting model of weight versus length predicts that for any length, female prowfish are, on average, 3.7% heavier than males (Fig. 5). It seems unlikely that this relationship exists over all developmental stages because our samples were almost all adults and such a (relative) difference might not remain constant during all ontoge- netic sexual divergence. What is more certain is simply the existence of some small degree of length-weight dimorphism (females slightly heavier at a given length). Also, this dimorphism is not likely to stem primarily from a sexual difference in gonad weight because the maximum proportion of total female body weight composed of ovarian tissue was only 2.7%. Thus the difference is due to other morphological or behavioral differences. Growth There was no significant difference between sexes in length versus age. The predicted length of a prowfish of given age based on our samples was higher than that indicated by Tokranov (1999). In our study 5-year-old and 9-year-old fish averaged 56.6 cm and 73.5 cm in length, respectively. Tokranov (1999) considered that prowfish growth deter- mined from otoliths of 102 specimens from the Northwest Pacific indicated a comparatively fast-growing species reaching an average length of 44.6 cm by 5 years of age and 67 cm after 9 years. These data suggest prowfish are indeed relatively fast growing and that growth rates for the Gulf of Alaska are faster than those for off southeastern Kamchatka and the northern Kuril Islands. Alternatively, size-dependent mortality from such elements as incidental capture by commercial fishing may affect the two popula- tions differently. Historically, two other prowfish have been aged from otoliths: a male 84 cm long taken near Eureka, CA (Fitch and Lavenberg, 1971), and a female 50.1 cm long ( standard length) from off Monterey (Cailliet and Anderson, 1975). The ages estimated were 12 and 3 years, respectively. Af- ter converting the standard length record to an estimate of total length for the second specimen of 58 cm by using a ratio described by Baxter,6 both lengths are slightly greater than our predictions for the same ages, albeit near the limits of our data range. This finding contrasts with the predictions of lesser length at a given age presented by Tokranov (1999). Maturity Little previous data exist with which to compare our obser- vations of female prowfish rate of maturation. Cailliet and Anderson (1975) examined the ovaries of their 50.1-cm 3- year-old female specimen for vitellogenesis and predicted an age at first spawning of 4 years, slightly less than the lower 95% confidence limit of 4.4 years for our expected average age at 50% maturity. Food habits Our observation that gelatinous zooplankton was the largest constituent in the contents of prowfish stomachs (Table 1) is supported by Tokranov ( 1999), who found that the two most common prey taxa among the contents of 102 stomachs of adult specimens from the northwestern Pacific were Scyphozoa (59.6-62.0% of stomachs) and Ctenophora (6.0-15.4% of stomachs). Anecdotal observations have also been made of the feeding behavior of an aquarium specimen over an approximate 2-year period (Carollo and Rankin, 1998). When first obtained, the fish ate only various jel- lyfish species, rejecting other food, including a variety of live invertebrates. In our food samples, we observed other taxa, such as invertebrates and small fish, but these were a minor part, possibly first captured by jellyfish and then sec- ondarily ingested by prowfish. Carollo and Rankin (1998) found that the aquarium specimen would ingest such items when eating the bells of Chrysaora melanaster in which such food had previously been placed, indicating the pos- sibility of this occurring naturally. Possibly more reflective of the unnatural circumstances, the specimen later began accepting such items outside the bells of jellyfish. Apparent adaptations of the prowfish to a diet of ge- latinous zooplankton include the small, sharp, close-set teeth in a single row attached only to the jaws, which are capable of a 180-degree gape, and the large rough-scaled lips (Clemens and Wilby, 1961; Hart, 1973; Carollo and Rankin, 1998). Acknowledgments We are grateful for the expert advice given by Alaska Fish- eries Science Center colleagues Delsa Anderl and Nancy 6 Baxter, R. 1990. Unpubl. manuscript. Annotated key to the fishes of Alaska, 803 p. [Available from Sera Baxter, Box 182, Seldovia, AK 99663.1 178 Fishery Bulletin 102(1) Roberson regarding age-reading of prowfish otoliths, and by AFSC colleagues Kathy Mier and Susan Piquelle regarding statistical analyses of data. Literature cited Allen, James M., and Gary B. Smith. 1988. Atlas and zoogeography of common fishes in the Bering Sea and Northeastern Pacific. NOAA Tech. Rep. NMFS 66, 151 p. Brodeur, R. D. 1998. In situ observations of the association between juve- nile fishes and scyphomedusae in the Bering Sea. Mar. Ecol. Prog. Ser. 163:11-20. Cailliet, G. M., and M. E. Anderson. 1975. Occurrence of prowfish Zaprora silenus Jordan, 1896 in Monterey Bay, California. Calif. Fish Game 61(l):60-62. Carollo, M., and P. Rankin. 1998. The care and display of the prowfish, Zaprora silenus. Drum and Croaker 29:3-6. Clemens, W. A., and G. V. Wilby. 1961. Fishes of the Pacific coast of Canada. Fish. Res. Board Can. Bull. 68, 2nd ed., 443 p. Chilton, D. E., and R. J. Beamish. 1982. Age determination methods for fishes studied by the groundfish program at the Pacific Biological Station. Can. Spec. Pub. Fish. Aquat. Sci. 60, 102 p. Doyle, M. J., K. L. Meir, M. S. Busby, and R. D. Brodeur. 2002. Regional variation in springtime ichthyoplankton assemblages in the northeast Pacific Ocean. Progress in Oceanography 53 (2-4):247-281 Eschmeyer, W. N, E. S. Herald, and H. Hammann. 1983. A field guide to Pacific Coast fishes of North America, 336 p. Peterson field guide ser. Houghton Mifflin, Boston, MA. Fitch, J. E., and R. J. Lavenberg. 1971. Marine food and game fishes of California, 179 p. U.C. Press, Berkeley, CA. Hart, J. L. 1973. Pacific fishes of Canada. Fish. Res. Board Can. Bull. 180, 740 p. Jordan, D. S. 1897. Notes on fishes little known or new to science. Proc. Calif. Acad. Sci., 2nd sen, 6:203-205. Kessler, D. W. 1985. Alaska's saltwater fishes and other sea life: a field guide, 358 p. Alaska Northwest, Anchorage, AK Kimura, D. K. 1980. Likelihood methods for the von Bertalanffy growth curve. Fish. Bull. 77:765-776. Martin, M. H. 1997. Data report: 1996 Gulf of Alaska bottom trawl survey. NOAA Tech. Memo. NMFS-AFSC-82, 235 p. Matarese, A. C, A. W. Kendall, D. M. Blood, and B. M. Vinter. 1989. Laboratory guide to early life history stages of north- east pacific fishes. NOAA Tech. Rep. NMFS 80, 652 p. Nelson, J. S. 1994. Fishes of the world, 3rd ed., 600 p. John Wiley, New York, NY.. Orlov, A. M. 1998. On feeding of mass species of deep-sea skates (Bathy- raja spp., Rajidae) from the Pacific waters of the Northern Kurds and Southeastern Kamchatka. J. Icthyol. 38(8): 635-644. Payne, S. A., B. A. Johnson, and R. S. Otto. 1999. Proximate composition of some north-eastern Pacific forage fish species. Fish. Oceanogr. 8(3): 159-177. Scheffer, V. B. 1940. Two recent records of Zaprora silenus Jordan from the Aleutian Islands. Copeia 1940(31:203. Seber, G. A. F 1973. The estimation of animal abundance, 506 p. Hafner Press, New York, NY. Tokranov, A. M. 1999. Some features of biology of the prowfish Zaprora silenus (Zaproridae) in the Pacific waters of the Northern Kuril Islands and Southeastern Kamchatka. J. Ichthyol. 39(61:475-478. Van Pelt, T I., J. F Piatt, B. K. Lance, and D. D. Roby. 1997. Proximate composition and energy density of some North Pacific forage fishes. Comp. Biochem. Physiol. 118A(4 1:1393-1398. Wakabayashi, K, R. G. Bakkala and M. S. Alton. 1985. Methods of the U.S.-Japan demersal trawl surveys. In Results of cooperative U.S.-Japan groundfish investiga- tions in the Bering Sea during May-August 1979 (R. G. Bakkala and K. Wakabayashi, eds.). p. 7-26. Int. North Pac. Fish. Comm. Bull. 44. Yang, Mei-Sun. 1993. Food habits of the commercially important ground- fishes in the Gulf of Alaska in 1990. NOAA Tech. Memo. NMFS-AFSC-22, 150 p. 179 Abstract— Our analyses of observer records reveal that abundance esti- mates are strongly influenced by the timing of longline operations in rela- tion to dawn and dusk and soak time — the amount of time that baited hooks are available in the water. Catch data will underestimate the total mortal- ity of several species because hooked animals are "lost at sea." They fall off, are removed, or escape from the hook before the longline is retrieved. For example, longline segments with soak times of 20 hours were retrieved with fewer skipjack tuna and seabirds than segments with soak times of 5 hours. The mortality of some seabird species is up to 45% higher than previously estimated. The effects of soak time and timing vary considerably between species. Soak time and exposure to dusk periods have strong positive effects on the catch rates of many species. In particular, the catch rates of most shark and billfish species increase with soak time. At the end of longline retrieval, for example, expected catch rates for broadbill swordfish are four times those at the beginning of retrieval. Survival of the animal while it is hooked on the longline appears to be an important factor determining whether it is eventually brought on board the vessel. Catch rates of species that survive being hooked (e.g. blue shark) increase with soak time. In contrast, skipjack tuna and seabirds are usu- ally dead at the time of retrieval. Their catch rates decline with time, perhaps because scavengers can easily remove hooked animals that are dead. The results of our study have impor- tant implications for fishery manage- ment and assessments that rely on longline catch data. A reduction in soak time since longlining commenced in the 1950s has introduced a systematic bias in estimates of mortality levels and abundance. The abundance of species like seabirds has been over-estimated in recent years. Simple modifications to procedures for data collection, such as recording the number of hooks retrieved without baits, would greatly improve mortality estimates. Manuscript approved for publication 22 September 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull. 102:179-195 (2004). Fish lost at sea: the effect of soak time on pelagic longline catches Peter Ward Ransom A. Myers Department of Biology Dalhousie University Halifax, B3H 4JI Canada E-mail address (for P Ward) wardiSmathstat.dal ca Wade Blanchard Department of Mathematics and Statistics Dalhousie University Halifax, B3H 44 Canada Our knowledge of large pelagic fish in the open ocean comes primarily from tagging and tracking experiments and from data collected from longline fish- ing vessels since the 1950s. Abundance indices for pelagic stocks are often derived from analyses that model catch as a function of factors such as year, area, and season. However, the amount of time that baited hooks are available to fish is likely to be another important factor influencing catch rates (Deriso and Parma, 1987). The activity of many pelagic animals and their prey vary with the time of day. Broadbill swordfish, for example, feed near the sea surface at night. They move to depths of 500 m or more during the day (Carey, 1990). Other species may be more active in surface waters during the day (e.g. striped marlin) or at dawn and dusk (e.g. oilfish). Longline fishing crews take a keen interest in the tim- ing of their fishing operations and soak time (the total time that a baited hook is available in the water). However, as- sessments have not accounted for those factors in estimating the abundance or mortality levels of target species or nontarget species. In many assessments that use pelagic longline catch rates, fishing effort is as- sumed to be proportional to the number of hooks deployed. The effects of soak time and timing may have been omit- ted because a clear demonstration of their effects on pelagic longline catch rates is not available. The few pub- lished accounts on soak time in pelagic longline fisheries have been based on limited data and a few target species. For example, in analyzing 95 longline operations or "sets" by research vessels Sivasubramaniam ( 1961) reported that the catch rates of bigeye tuna increased with soak time, whereas yellowfin tuna catch rates were highest in longline seg- ments with intermediate soak times. In contrast to the limited progress in empirical studies, theoretical approach- es are well developed for modeling fac- tors that may influence longline catch rates. Soon after large-scale longlining commenced. Murphy (1960) published "catch equations" for adjusting catch rates for soak time, bait loss, escape, hooking rates, and gear saturation. He suggested that escape rates could be es- timated from counts of missing hooks and hooks retrieved without baits. Unfortunately, such data are rarely col- lected from pelagic longline operations. More recently, hook-timers attached to longline branchlines have begun to provide information on the time when each animal is hooked and also whether animals are subsequently lost, e.g. Boggs (1992), Campbell et al.1-2 Such data are particularly useful to under- 1 Campbell, R., W. Whitelaw, and G. Mc- Pherson. 1997. Domestic longline fish- ing methods and the catch of tunas and non-target species off north-eastern Queensland (1st survey: October-Decem- ber 1995). Report to the Eastern Tuna and Billfish Fishery MAC. 71 p. Aus- tralian Fisheries Management Authority, PO Box 7051, Canberra Business Centre, ACT 26 10, Australia. 2 See next page. 180 Fishery Bulletin 102(1) standing the processes affecting the probability of capture and escape. The purpose of our study is to determine whether varia- tions in the duration and timing of operations bias abun- dance and mortality estimates derived from longline catch rates. We present a theoretical model that is then related to empirical observations of the effects of soak time on catch rates. The strength in our approach is in applying a random effects model to large data sets for over 60 target and non- target species in six distinct fisheries. We also investigate the survival of each species while hooked because prelimi- nary analyses suggested that the effects of soak time on catch rates might be linked to mortality caused by hooking (referred to as "hooking mortality"). Factors affecting catch rates To aid interpretation of our statistical analysis of soak time effects, we first developed a simple model to illustrate how the probability of catching an animal may vary with soak time. The probability of an animal being on a hook when the branchline is retrieved is a product of two probability density functions: first the probability of being hooked and then the probability of being lost from the hook.3 In- fluencing the probability of being hooked are the species' local abundance, vulnerability to the fishing gear, and the availability of the gear. Catches will deplete the abundance of animals within the gear's area of action, particularly for species that have low rates of movement. Movement will also result in variations in exposure of animals to the gear over time — for instance, as they move vertically through the water column in search of prey (Deriso and Parma, 1987). Other processes that will reduce the probability of be- ing hooked include bait loss and reduced sensitivity to the bait (Ferno and Huse, 1983). Longline baits may fall off hooks during deployment, deteriorate over time and fall off or they may lose their attractant qualities. They may be removed by target species, nontarget species, or other ma- rine life, such as squids. Hooked animals may also escape by severing the branchline or breaking the hook. Sections of the longline may become saturated when animals are hooked, reducing the number of available baits (Murphy. 1960; Somerton and Kikkawa, 1995). After an animal has been hooked, it may escape, fall off the hook, be removed by scavengers, or it may remain hooked until the branchline is retrieved. Some of the processes affecting the probability of an ani- mal being on a hook when the the branchline is retrieved 2 Campbell, R., W. Whitelaw, and G. McPherson. 1997. Do- mestic longline fishing methods and the catch of tunas and non- target species off north-eastern Queensland (2nd survey: May- August 1996). Report to the Eastern Tuna and Billfish Fishery MAC, 48 p. Australian Fisheries Management Authority, PC) Box 7051, Canberra Business Centre, ACT 2610, Australia. In discussing continuous variables we use the terms "proba- bility" and "probability density function" interchangeably. are species-specific, whereas other processes may affect all species. For example, bait loss during longline deployment will reduce the catch rates of all species. In contrast, the probability of a hooked animal escaping may be species-de- pendent; some species are able to free themselves from the hook whereas other species are rarely able to do this. Our simple model of the probability of an animal being on a hook is based on a convolution of the two time-related processes described above: 1) the decay in the probability of capture with the decline in the number of baits that are available; and 2) gains due to the increased exposure of baits to animals and losses due to animals escaping, falling off, or being removed by scavengers. The probability of an animal being on a hook when the branchline is retrieved is the integral of the probability density functions of capture and retention: rtT) = J P(t)PrlT-t)dt, (1) where jriT) = the "catch rate" or probability of an animal being on a hook when the branchline is retrieved at time T (T is the total soak time of the hook); P(it) = the probability density function of an animal being captured at time t; and Pr(t) = the probability density function of a cap- tured animal being retained on the hook for a length of time f. The probability density function of capture can be approxi- mated with an exponential function: Pit) = P0e-", (2) where P0 = the probability of capture when the hook is deployed (r=0); and o = a parameter determining the rate of change in capture probability over time. After the animal is hooked, the probability density function of an animal being retained after capture can be approxi- mated as PIt) = e-pw, (3) where /I = the "loss rate," a parameter determining the rate of change in the probability of an animal being retained after it has been captured. Substituting approximations 2 and 3 into Equation 1 gives 7l(T)= \P0e '"e <"' ' dt (4) /?-«' Ward et al.: The effect of soak time on pelagic longlme catches 181 Seabirds (-0.06) p c\i (XX) Skipjack (-0.04) () () (> <> ° () o () 0 O 5 10 15 20 25 0 5 10 15 20 25 <) <) O O o C) °<»' j (X) o o o o° Lancetfish (-0.02) in Swordfis h (M (+0.09) o . <>°o 0 e.g. swordfish Losses eventually exceed captures Soak time coefficient <0 e.g. skipjack Captures balance losses Soak time coefficient -0 eg lancetfish 20 Figure 2 Illustration of different patterns in the theoretical relationship between longline catch rates and soak time. The probability of an animal being on a hook when a branchline is retrieved (the "catch rate") is estimated from Equation 4 by using soak times iT) ranging from 0 to 20 hours and three different combinations of values forPn (probability of capture), « (capture rate), and /3 (loss rate). For seabirds, the probabilities were estimated from Equation 6. The probabilities are not on the same scale for all species. Another approach might be to fit separate logistic regres- sions to each operation and then to combine the parameter estimates. This would overcome the problems of normality and homogeneity of variance. However, the separate re- gressions would not incorporate information that is com- mon to all operations. Instead, we used a logistic regression with random ef- fects. The key advantage in using random-effects models in this situation is that they carry information on the cor- relation between longline segments that is derived from the entire data set of operations. Data and methods Fisheries We analyzed observer data from six different fisheries in the Pacific Ocean to determine the effects of soak time and timing on longline catch rates (Table 1, Fig. 3). These fisheries involve two different types of longline fishing operation: 1 ) distant-water longlining involves trips of three months or longer and the catch is frozen on board the vessel; and 2) fresh-chilled longlining, which involves small vessels (15-25 m) undertaking trips of less than four weeks duration, and the catch is kept in ice, ice slurries, or in spray brine systems. The fresh-chilled longliners deploy shorter longlines with fewer hooks (-1000 hooks) than the distant-water longliners (-3000 hooks per operation) (Ward, 1996; Ward and Elscot, 2000). The six fisheries share many operational similarities, such as the types of bait used and soak time. However, they are quite different in terms of targeting, which is determined by fishing practices, e.g. the depth profile of the longline, timing of operations and the area and season of activity. South Pacific bluefin tuna longliners operate in cold waters ( 10-16°C) in winter to catch southern bluefin tuna. In the South Pacific yellowfin tuna longliners tar- get tropical species, such as yellowfin and bigeye tuna, in warmer waters (19-22°C) (Ward, 1996). To target bigeye tuna, longlines in the Central Pacific bigeye fishery are deployed in the early morning with hook depths ranging down to about 450 m. The depths of the deepest hook are much shallower (-150 m) in the North Pacific swordfish fishery where the longlines are deployed late in the after- noon and retrieved early in the morning (Boggs, 1992). Ward et al.: The effect of soak time on pelagic longline catches 183 Observer data National authorities and regional organizations placed independent observers on many longliners operating in the six fisheries during the 1990s. The observer data consisted of records of the species and the time when each animal was brought on board. We restricted analyses to operations where the last hook that had been deployed was retrieved first ("counter- retrieved"), where there was no evidence of stoppages due to line breaks or mechanical failure, and where there was continuous monitoring by an observer. Combined with records of the number of hooks deployed and start and finish times of deployment and retrieval, the observer data allowed calculation of soak time and catch rates of longline segments. We aggregated catches and the number of hooks into hourly segments. The soak time was estimated for the midpoint of each hourly segment. The Central Pacific bigeye tuna and North Pacific sword- fish fisheries differed from the other four fisheries in the species that were recorded and the method of recording the time when each animal was brought on board. Observ- ers reported catches according to a float identifier in the Central and North Pacific fisheries. Therefore we estimated soak times for each longline segment from the time when each float was retrieved. For those fisheries, observers re- ported the float identifier only for tuna, billfish, and shark (Table 2). Data are available for protected species, such as seals, turtles, and seabirds but were not sought for the present study. We assumed a constant rate of longline retrieval throughout each operation. The number of hooks retrieved during each hourly segment was the total number of hooks divided by the duration of monitoring (decimal hours). For each species we analyzed only the operations where at least one individual of that species was caught. Longline segments that involved a full hour of monitor- ing had several hundred hooks. Segments at either end of the longline involved less than an hour of monitoring and had fewer hooks. Catch rates may become inflated in segments with very small numbers of hooks. Therefore we arbitrarily excluded segments where the observer moni- tored less than 25 hooks. For four of the fisheries, data were available on survival rates, allowing the investigation of the relationship be- tween soak time and hooking mortality. For the Western Pacific and South Pacific fisheries, observers reported whether the animal was alive or dead when it was brought on board. We calculated survival rate (the number alive divided by the total number reported dead or alive) for spe- cies where data were available on the life status of more than ten individuals. Generalized linear mixed model Logit model We applied a generalized linear mixed model to the observer data. The model is based on a logis- tic regression, with the catch (y) on each hook assumed to have a binomial distribution with y ~ b(ra, n). n is the expected value of the distribution for a specified soak time. We refer to it as the probability of catching an animal or •s ■$ CD CO c "2 ° CJ ■2 2 •- ° 5. o J! Or'* B a -S o ^H CO CO o o CO CJ 8. " lO CM CM CO ^f CM Cfi a "in o ^ o 2 O o X - ^ CJ o g co >-* o ° C O <3J tG •*-» ■ — t_ - • , — 1 05 , — I CN CM CM t- a. a> CM ,—1 CM CM CM CM o » "3 =3 CO .—1 cd & Q >> u CO -- ■m-h o O C c -C „ o co O en 3 6 £ S M u '43 CM lO o t> O CO cc OQ}C3 rH CD , C8 2 CD 3 2 bp cd a> CO en CO lO ■* a> CD IS i -C '.2 CO ■* rH CO ,— i CD £ +* 6 es P. lO CM Ol CM ■* CD cfl to ,o ~ CO ^ "cfl O QJ o u "-J3 b C nj ™ CM CM ^H ,H a; a» S O O O O a £ t3 o o o o r- t> cu _c c o CC ° x o "C CM 4 CM 4 CM 1 o 1 O <3i 1 CM 03 1 CM Ph O) en ai o> 05 OS o c « .2 to en o> o> Ol cn ai CO +J o CO „, CtJ u 2 cO c CO c CO C o en 3 3 3 c C M" a .2 +j -M cu ■5 c c CD CO CD Lh * * >> c -P a cd c X - CO C cC CO 0) "o , >> CO a CD _3 o CO" CO" CO" 3 m Table er oper ?rn Pac CD i-. H X CO cC •a u O c 3 CJ CD d 3 CD CU c 3 *-> CD >> CD 4^ c cC o X c u CD X +a 3 V r- CD -C o, .^ CU3 be bo w en CD O cC CO (Tj CO X X X >> CO ■g* CO he mean number of ho perations. For the two and SP = South Pacific. CO CO -c X t- u CO CO CO 1 CO CD 3 o S3 'cj CO cu X +J S- O 2: u S3 CO "3 a o CJ S3 o CO Oh 0 0) CO 1 CJ CC 'u CO Oh C CD *J CO 1 3 < C u CD -4-> CO CO CD X -tJ u o c 3 CO 5 CO o tj -" > c CD 2 cj X o -a J2 Cr CD CO c CO c CO ±3 fisheries the total : 1 Pacific; \ X CO CD a -e CO £2 ary of the er data, a: ; CP = Cen s a c X to cC ■a s- o "cO C 3 cu >, CO a 3 CU >> CD bo X Ph +3 c CO c 3 C CC •s o CO a 3 +3 c cC CD C cu CO _; Summ observ Pacific Ir 0) XI en E CO Oh 2 X cm O ■3 "CD >> Oh cn _3 X Oh CO 3 .2 184 Fishery Bulletin 102(1) North Pacific Swordfish o o eg Western Pacific Bigeye o CM I o I Western Pacific Bigeye Western Pacific Distant South Pacific Yellowfin South Pacific Bluefin — I — 140 T T 160 220 180 200 Longitude (degrees) Figure 3 Geographical distribution of the observer data analyzed for each fishery. 240 the expected number of animals per hook. For each longline segment (j) within each operation (£), we link jr to a linear predictor ( ?;( ) through the equation rjj is then modeled as a function of soak time: r?y = ft+ATy, (5) where TtJ = the hook's soak time (decimal hours) in long- line segment j; P0 = the intercept; and /3j = the slope coefficient, which we term the "soak time coefficient." Modeling the probability of a catch on each individual hook would result in large numbers of zero observations and thus test the limits of current computer performance. Therefore we aggregated hooks and catches into hourly segments for each longline operation. We assumed that each longline segment had the same configuration and that the probability of capture was the same for each segment within a longline operation. The assumption may be violated where segments pass through different water masses or where they differ in depth profile or baits. Saturation of segments with animals will also al- ter the capture probability between segments. The effects of water masses, depth profiles, baits, and gear saturation were not analyzed in the present study. Capture probability may also vary through the differen- tial exposure of segments to the diurnal cycle of night and day. The addition of dawn and dusk as fixed effects allowed us to model diurnal influences on catch rates. Fixed effects To explore factors that might affect the rela- tionship between soak time and catch rate, we added four fixed effects to the logit model: year, season, and, as men- tioned above, whether the segment was available at dawn or dusk. A full model without interaction terms would be iu = A. + /Wj + AA> + PAi + PAj + P*Yu + °- where 71, = the hook's soak time (decimal hours) in long- line segment j; At = an indicator of whether the hook was exposed to a dawn period; P = an indicator of whether the hook was exposed to a dusk period; S: , = the season (winter or summer); Y- = the year; Oi = the random effect for operation that we mod- eled as an independent and normally distrib- uted variable (see "Random effects" section); and )30-/34 are parameters (fixed effects) to be estimated. We refer to fix as the "soak time coefficient." Ward et al.: The effect of soak time on pelagic longline catches 185 Table 2 List of common and scientific names of the species analyzed. Also shown is the number of individuals of each species analyzed in each fishery. A dash indicates that the species was not analyzec in the present study it does not necessarily mean that the spe- cies was not taken in the fishery. In particular, observer data on the time of capture were not aval lable for 'other bony fish" in the North Pacific swordfish and Centra] Pacific bigeye tuna fisheries . NP = North Pacific; CP = Central Pacific WP = Western Pacific; SP = South Pacific; LN = long- nosed; and SN = short-nosed. Fishery CP WP SP SP NP bigeye bigeye WP yellowfin Bluefin Common name Species swordfish tuna tuna distant tuna tuna Tuna and tuna-like species Albacore Thunnus alalunga 9707 23,128 14,194 11,976 21,550 1399 Bigeye tuna 77? annus obesus 5409 45,476 9814 2581 1846 - Butterfly mackerel Gasterochisma melumpus — — — — — 533 Skipjack tuna Katsuwonus pelamis 546 13,882 1456 445 691 — Slender tuna Allothunnus fallai — — — — — 28 Southern bluefin Thunnus maccoyii — — — — 1030 10.537 Yellowfin tuna Thunnus albacares 2811 21,654 16,029 4689 12,454 — Wahoo Acanthocybium solandri 383 5508 1345 — 308 — Billfish Black marlin Makaira indica 25 41 353 226 160 — Blue marlin Makaira nigricans 981 2379 1467 529 179 — Sailfish Istiophorus platypterus 49 193 706 399 151 — Shortbill spearfish Tetrapturus angustirostris 543 5467 529 398 654 — Striped marlin Tetrapturus audax 1963 8332 681 182 724 — Swordfish Xiphias gladius 22,457 1680 1472 287 1173 92 Other bony fish Barracouta Thyrsites atun — — — — 53 — Barracudas Sphyraena spp. — — 707 153 — — Escolar Lepidocybium flavubrunneum 1208 3983 1343 878 1726 84 Great barracuda Sphyraena barracuda 32 743 303 442 92 — Lancetfish (LN) Alepisaurus ferox 2788 30,136 325 419 2868 610 Lancetfish(SN) Alepisaurus brevirostris — — 155 84 257 59 Lancetfishes Alepisaurus spp. — — 1431 98 — — Long-finned bream Taractichthys longipinnis — — — — — 292 Mahi mahi Coryphaena hippurus 17,463 19,090 1436 211 447 — Oilfish Ruvettus pretiosus 555 1091 420 456 653 900 Opah Lampris guttatus 68 4724 527 129 80 213 Pomfrets Family Bramidae — — 623 60 — 40 Ray's bream Brama brama — — — — 1074 10,547 Ribbonfishes Family Trachipteridae — — — — — 22 Rudderfish Centrolophus niger — — — — — 90 Sickle pomfret Taractichthys steindachneri — — 122 21 — — Slender barracuda Sphyraena jello — — — — 121 — Snake mackerel Gempylus serpens 1971 9881 256 44 — — Snake mackerels Family Gempylidae — — 456 10 — — Southern Ray's bream Brama spp. — — — — — 28 Sunfish Mola ramsayi — — — — 249 99 Sharks and rays Bigeye thresher shark Alopias superciliosus 149 1930 145 61 — — Blacktip shark Carcharhinus limbatus — — 445 125 — — Blue shark Prionace glauca 31,503 31,413 5601 1628 1689 12.797 Bronze whaler Carcharhinus brachyurus — — — — 116 — Crocodile shark Pseudocarcharias kamoharai 153 73 continued 186 Fishery Bulletin 102(1) Table 2 (continued) Fishery CP WP SP SP NP bigeye bigeye WP yellowfin Bluefin Common name Species swordfish tuna tuna distant tuna tuna Sharks and rays (continued) Dog fishes Family Squalidae — — — — — 60 Dusky shark Carcharhinus obscurus — 112 — — 20 — Grey reef shark Carcharhinus amblyrhynchos — — 282 64 — — Hammerhead shark Sphyrna spp. — — 142 191 22 — Long finned mako Isurus paucus — 83 108 15 — — Oceanic whitetip shark Carcharhinus longimanus 568 2373 2376 384 142 — Porbeagle Lamna nasus — — — — 27 1011 Pelagic stingray Dasyatis violacea 2374 2849 1212 248 534 109 Pelagic thresher shark Alopias pelagicus — — 77 34 — — School shark Galeorhinus galeus — — — — — 143 Short finned mako Isurus oxyrinchus 476 685 408 169 432 128 Silky shark Carcharhinus falciformis 25 1433 5396 2406 8 — Silvertip shark Carcharhinus albimarginatus — — 168 74 — — Thintail thresher shark Alopias vulpinus — 74 — — — 31 Thresher shark Alopias superciliosus — — 415 — 93 18 Tiger shark Galeocerdo cuvier — — 56 18 38 — Velvet dogfish Zameus squamulosus — — — — — 156 Whip stingray Dasyatis akajei — — 78 15 — — Seabirds Albatrosses Family Diomedeidae — — — — — 88 Petrels Family Procellariidae — — — — — 29 Other seabirds Family Procellariidae — — — — 38 200 All operations 104,054 238,340 73,212 30,222 51,699 40,343 To maintain a focus on the effects of soak time, the models were limited to simple combinations of fixed effects and interaction terms. Dawn and dusk were added to various models of each species in each fishery. To reduce complex- ity, year and season were limited to models of seven spe- cies (bigeye tuna, oilfish, swordfish, blue shark, albacore, southern bluefin tuna, long-nosed lancetfish) in the two South Pacific fisheries. The seven species represented four taxonomic groups and the full range of responses observed in preliminary analyses of the soak-time-catch- rate relationship. Random effects We added random effects to all models to allow catch rates of segments within each longline opera- tion to be related. The random effects model assumes that there is an underlying distribution from which the true values of the probability of catching the species, jt, are drawn. The distribution is the among-operation varia- tion or "random effects distribution." The operations are assumed to be drawn from a random sample of all opera- tions, so that the random effects (0() in the relationship between catch rate and soak time for each operation (i) are independent and normally distributed with 0~N(Q, a2). The random effects and various combinations of the fixed effects were added to the linear predictor presented in Equation 5. For each species in the South Pacific yellowfin tuna data set we compared the performance of models under an equal correlation structure with that of models under an autoregressive correlation structure. Under an au- toregressive structure, catch rates in the different hourly segments within the operations are not equally correlated. For example, the correlation between segments might be expected to decline with increased time between seg- ments. However, we used an equal correlation structure for all models because the Akaike's information criterion (AIC) and Sawa's Bayesian information criterion (BIO indicated that there was no clear advantage in using the autoregressive structure rather than an equal correlation structure. Implementation We implemented the models in SAS (version 8.0) using GLIMMIX, a SAS macro that uses iteratively reweighted likelihoods to fit generalized linear Ward et al.: The effect of soak time on pelagic longline catches 187 Seabirds Other fish Tuna Billfish Sharks i -0.2 SP Bluefin — « — Other seabirds (1 07) -Albatross (0-99) Petrel (1.17) — Lancetfish(SN)(1) "Opah(1 .03) Pomfret(1 16) -Lancetfish(LN)(1.02) Southern Ray's bream (0.96) "© — Long finned bream (111) ^Ray's bream (2.47) — © Sunfish (0.97) © Ribbonfish (0.93) Seabirds Other fish e Rudderfish (0.89) © Escolar (0.66) -e-Oilfish (0.98) -e-Albacore (0.94) Slender tuna (0.9) "-Butterfly mackerel (0.93) eSouthern bluefin (1.4) —©—Swordfish (0 9) — Thintail thresher shark (0.88) — Mako (0.93) ©Blue shark (1 87) -©"Porbeagle (0.92) -© Ray (0.89) Tuna Billfish - Sharks — I — 0.0 "I 0.2 SP Yellowfin © — ; Other seabirds (1.26) — © iBarracouta (0.99) — © — 'Slender barracuda (0.98) ©-?— Opah (0.99) -©iancetfish (LN) (1 14) -s-Mahi mahi (1.09) — «r-Lancetfish (SN) (0.96) © Great barracuda (0.95) :-e-Ray,s bream (1.71) ■ — e — Sunfish (0.99) ; — e— Oilfish (1.23) -©"Escolar (1.33) -©"Skipjack (1 .06) ©Yellowfin (2.33) —f3— Southern bluefin (2.2) ©Albacore(2.12) -r6— Wahoo (0.96) -®-Bigeye(1 16) — © — Sailfish (1 03) — 9 — Blue marlin (0.88) r-e-Shortbill spearfish (0.99) :— e — Black marlin (0.92) ■ -©-Striped marlin (0.94) -©"Swordfish (0.85) ©i Porbeagle (0.87) j-e Silky shark (0.86) Tiger shark (0.87) "Mako (1.06) -Ray (0 99) > — Bronze whaler (0.95) © — Oceanic whitetip (0.99) ©-Blue shark (0.99) © Hammerhead (0.93) © Dusky shark (0.85) -0.2 Soak time coefficient 0.0 0.2 Figure continued on next page. Figure 4 Coefficients for the effect of soak time on the catch rates of the most abundant species in each fishery. The coefficients are from random effects models where soak time is the only factor. Horizontal bars are 95% confidence intervals for the estimated coefficient. The dispersion parameter is shown in parentheses (it is 1.00 for species that are distributed as predicted by the model, but may be higher for species that have a more clumped distribution along the longline). mixed models (Wolfinger and O'Connell, 1993). To judge the performance of the various model formulations, we checked statistics, such as deviance and dispersion, and examined scatter plots of chi-square residuals against the linear predictor I rj) and QQ plots of chi-square residuals. We used the AIC and BIC to compare the performance of the various model formulations. Variance in the binomial model depends on only one pa- rameter, P. A dispersion parameter is therefore necessary to allow the variance in the data to be modeled. In effect, the dispersion parameter scales the estimate of binomial variance for the amount of variance in the data. The disper- sion parameter will be near one when the variance in the data matches that of the binomial model. Values greater than one ("over-dispersion") imply that the species may have a clumped distribution along the longline. Results Soak time For most species, soak time had a positive effect on catch rates (Fig. 4). In addition to being statistically significant, the effect of soak time made a large difference to catch rates at opposite ends of the longline. In the South Pacific yellowfin tuna fishery, for example, the expected catch rates of swordfish can vary from 0.6 (CI ±0.1) per 1000 hooks (5 hours) to 1.9 (CI ±0.3) per 1000 hooks (20 hours) (Table 3). A soak time of 5 hours and 3500 hooks (if that were possible) would result in a total catch of about two swordfish. In contrast, almost seven swordfish are expected from a longline operation of the same number of hooks with 20 hours of soak time. 188 Fishery Bulletin 102(1) WP Bigeye WP Distant Other fish Tuna Billfish - Sharks -0.2 Great barracuda (0.94) -Mahi mahi(1.15) -«— Lancetfish (LN) (0.99) Lancetfish (SN) (0.97) ■e — Snake mackerel (1.06) -Barracudas (0.96) *- Opah (11) ■9-Escolar (0.97) -9 Sickle pomfret (1 .2) -e-Pomfret (0.99) - &— Escolars (1.07) — ° — OHfish (1.12) -^Skipjack (1 12) ■«-Wahoo(1) ?Yellowfin (1.61) :eAlbacore(1.45) !eBigeye(1.18) tShortbill spearfish (0.98) -^Swordfish (1.04) _e_Stnped marlin (1.23) — e— Sailfish (1.51) — e — Black marlin (1.32) -o-Blue marlin (1.14) Hammerhead (0.87) Grey reef shark (1 .49) 1 Pelagic thresher shark (1.16) ■® Bigeye thresher shark ( 1 .06) -° Tiger shark (1) Other fish - Pomfret (1.43) -Mahi mahi (1.45) Lancetfish (LN) (1.02) e— Great barracuda (0.84) — e Opah (1.24) e Barracudas (0.97) — e — — Sickle pomfret (2.13) Escolar (1.15) -Lancetfish (SN) (0 84) Snake mackerel (3.56) -Ollfish (1.17) Tuna Billfish -e Sllvertip shark (1.17) ■Q-Silky shark (115) -6— Thresher sharks (0.88) -s— Short finned mako (0.91 ) "^Pelagic stingray (0.92) "Long finned mako (0 96) Sharks ■ -° — Blacktip shark (1.3) 9Blue shark (0.93) -^Oceanic whitetip (0.91) e Whip stingray (1 .37) e Crocodile shark (1 .2) 0.0 I 0.2 Skipjack (0.91) Wahoo (0.97) e-Yellowfin (2.02) &Albacore(1.51) -e"Bigeye(1.32) ■Black marlin (0.89) Striped marlin (1.19) — Shortbill spearfish (1.17) -°— Sailfish (1 .04) -° — Swordfish (0.89) — s— Blue marlin (0 98) Tiger shark (1.2) — Crocodile shark (0.95) Hammerhead (0 88) Whip stingray (1.01) Sllvertip shark (1 .46) ■Blue shark (0.95) Blacktip shark (0.91) -e-Silky shark (1.5) e Pelagic stingray (1) e Oceanic whitetip (1 .06) "Pelagic thresher shark (1.81) -Bigeye thresher shark (1.17) "Short finned mako (0.91) -0.2 0.0 0.2 Soak time coefficient Figure 4 (continued) For some species (e.g. seabirds, skipjack tuna, and mahi mahi), soak time had a negative effect on catch rates that was often statistically significant (Fig. 4). For skipjack tuna in the Western Pacific distant fishery, for example. catch rates decreased from 1.3 (CI ±0.2) per 1000 hooks for a soak time of 5 hours to 1.0 (CI ±0.1) per 1000 hooks (20 hours). Soak time had a small or statistically insignifi- cant effect on catch rates for several species, such as yel- lowfin tuna and shortbill spearfish. Fixed effects Exposure to dusk had a positive effect on the catch rates for most species (Fig. 5). Dusk often had a negative effect on the catch rates of billfish, such as striped marlin and sailfish. For most species, however, the effect of dawn was weaker, and it influenced the catch rates of fewer species. Like soak time, timing made a substantial difference to catch rates (Table 4). For a soak time of 12 hours in the South Pacific yellowfin fishery, for example, longlinc seg- ments exposed to both dawn and dusk have a catch rate of 2.0 (CI ±0.5) escolar per 1000 hooks. The catch rate is 0.8 (CI ±0.1) per 1000 hooks for segments that were not exposed to dawn or dusk. The effects of timing on catch rates were most pro- nounced in the South Pacific bluefin tuna fishery. The fishery also showed the greatest range in soak time coef- ficients, and the coefficients tended to be larger than those of other fisheries (Fig. 4). Separately, the fixed effects often had statistically signifi- cant relationships with catch rates of the seven species that we investigated in detail. However, the interaction between soak time and each fixed effect was less frequently signifi- cant. Season was significant, for example, in none of the six models that included a soak-time-season interaction term. By comparison, season was significant in six of the 18 models that included season as a factor but not with a soak-time-season interaction term. The effect of soak time was not significant for southern bluefin tuna in any model for the South Pacific bluefin tuna fishery. It was significant Ward et al.: The effect of soak time on pelagic longline catches 189 Tuna NP Swordfish hBlgeye (0 94) 3 Pacific bluefin (0.95) CP Bigeye Billfish Sharks Tuna *— Skipjack (0-84) -s-Yellowfin (0 87) ^Albacore (1.03) Slue marlin (0.91) — Shortbill spearfish (0.93) "Swordfish (0 96) -°-Striped marlin (0.88) e Sailfish (0 96) Billfish -Salmon shark (0.96) "° — Oceanic whitetip (0.98) "e Crocodile shark (0 89) -e — Short finned mako (0 95) eBlue shark (0.96) e Bigeye thresher shark (0.83) Sharks -0.2 0.0 — I 0.2 -0.2 Soak time coefficient Figure 4 (continued) ^Skipjack (0.85) eAlbacore (0.81) eYellowfin (0.93) bigeye (0.88) Black marlin (0.89) eStnped marlin (0 86) ■^Shortbill spearfish (0 89) "^Blue marlin (0.86) — e Sailfish (1.05) "^Swordfish (0.9) — Sandbar shark (1.24) e Bignose shark (1.19) _e— Short finned mako (0.94) eBlue shark (0 81) -e-Silky shark (0.93) -° — Pelagic thresher shark (0.88) "^Oceanic whitetip (1.01) "^Bigeye thresher shark (0.86) e Long finned mako (0.86) e Thintail thresher shark (0.9) 0.0 "Dusky shark (1.05) -°— Crocodile shark (0.89) I 0.2 in 36 of the 48 models for the other six species. We con- cluded that the fixed effects modified the intercept of the soak-time-catch-rate relationship, but they rarely altered the slope of the relationship. Akaike's information criterion (AIC) and Sawa's Bayes- ian information criterion (BIC ) both indicated that models with soak time as the only variable were the most or second most parsimonious model. This was the case for all models, except for several models of albacore and long-nosed lan- cetfish. Therefore the following discussion concentrates on the effects of soak time and timing on catch rates. Discussion In considering results of the random effects models, we examined patterns in the effects of soak time and timing among taxonomic groups, the mechanisms that may cause the patterns, and their implications. First, however, we investigated whether the effects were consistent for the same species between fisheries. Comparison of fisheries The effect of soak time was consistent for several spe- cies between the fisheries, despite significant differences in fishing practices and area and season of activity. For example, the soak time coefficients for species in the South Pacific yellowfin tuna fishery were very similar to those of the same species in the Central Pacific bigeye tuna fishery (r=0.79) (Fig. 6). Several species had a narrow range of soak time coef- ficients over all the fisheries analyzed. Estimates of the coefficient of yellowfin tuna, for example, ranged from 0.00 (CI ±0.01) in the South Pacific yellowfin fishery to 0.04 ( CI ±0.0 1 ) in the North Pacific swordfish fishery. A coefficient of 0.04 is equivalent to a difference of 1.3 yellowfin tuna per 1000 hooks between longline segments with soak times of 5 and 20 hours. The range in coefficients is also small for other abundant and widely distributed species, such as al- bacore (r=0. 00-0.05) and blue shark (r=0.01-0.05). For many species, however, the correlation between soak- time coefficients from different fisheries was poor (Fig. 6). 190 Fishery Bulletin 102(1) Table 3 Examples of the effect of soak time on expected catch rates of species in the South Pacific yellowfin tuna ishery. The expected catch rates i number per 1000 hooks I are predicted from the soak-time coefficient for each species for longline segments exposed to a dusk period with a soak time of 5 or 20 hours. Figu re 4 shows the 95% confidence intervals for soak-time coe fficients used to calculate the expected catch rates. LN = ong-nosed; SN = short-nosed. Species Soak time h) 5 20 Tuna and tuna-like species Albacore 15.5 13.4 Bigeye tuna 1.1 2.3 Skipjack tuna 1.3 1.0 Southern bluefin tuna 5.2 5.5 Yellowfin tuna 8.4 7.7 Billfish Black marlin 0.4 1.6 Blue marlin 1.2 0.4 Sailfish 0.8 1.0 Shortbill spearfish 1.0 1.6 Striped marlin 0.8 1.0 Swordfish 0.6 1.9 Other bony fish Barracouta 0.8 0.7 Escolar 0.8 3.1 Great barracuda 0.9 1.1 Lancetfish (LN) 2.7 2.4 Lancetfish (SN) 1.6 1.4 Mahi mahi 1.0 0.9 Oilfish 0.8 2.2 Opah 0.7 0.5 Ray's bream 1.8 2.0 Slender barracuda 1.7 1.6 Sunfish 0.6 1.3 Wahoo 1.0 1.1 Sharks and rays Blue shark 1.1 2.0 Bronze whaler 0.7 0.8 Dusky shark 0.4 0.8 Hammerhead 0.2 1.8 Mako 0.6 0.8 Oceanic whitetip 0.5 0.9 Porbeagle 1.2 1.1 Pelagic stingray 0.9 1.2 Thresher shark 0.6 1.0 Tiger shark 0.5 0.5 Table 4 Examples of the effect of timing on expected catch rates of species in the South Pacific yellowfin tuna fishery. The expected catch rates (number per 1000 hooks I are pre- dicted from the soak-time coefficient for each species for a longline operation with a soak time of 12 hours. The differ- ent catch rates are for longline segments exposed to nei- ther the dawn or dusk period, for dawn only, and for dawn and dusk periods. LN = long-nosed; SN = short-nosed. Period Neither Dawn Dawn Species period only + dusk Tuna and tuna-like species Albacore 12.3 14.0 16.5 Bigeye tuna 0.9 1.2 2.1 Skipjack tuna 1.4 1.2 1.0 Southern bluefin tuna 3.8 2.9 4.1 Yellowfin tuna 7.7 7.6 8.0 Billfish Black marlin 1.2 0.6 0.4 Blue marlin 0.4 1.0 1.4 Sailfish 0.8 0.7 0.7 Shortbill spearfish 1.3 0.9 0.9 Striped marlin 0.8 0.9 0.9 Swordfish 0.5 0.7 1.3 Other bony fish Barracouta 1.1 1.2 0.7 Escolar 0.8 1.0 2.0 Great barracuda 1.0 0.8 0.8 Lancetfish (LN) 2.8 2.7 2.5 Lancetfish (SN) 1.2 1.1 1.3 Mahi mahi 1.2 1.3 1.1 Oilfish 0.8 1.1 1.8 Opah 0.5 0.5 0.6 Ray's bream 0.8 0.7 1.6 Slender barracuda 2.0 1.5 1.2 Sunfish 0.8 0.6 0.7 Wahoo 1.2 1.3 1.1 Sharks and rays Blue shark 1.3 1.4 1.4 Bronze whaler 0.6 0.9 1.0 Dusky shark 0.1 0.1 0.6 Hammerhead 0.4 0.2 0.3 Mako 0.7 0.8 0.8 Oceanic whitetip 0.7 0.8 0.7 Porbeagle 1.0 0.6 0.6 Pelagic stingray 0.9 0.9 1.1 Thresher shark 0.6 0.6 0.7 Tiger shark 0.4 0.5 0.7 For a few species (e.g. tiger shark) the poor correlation may have been a function of small sample sizes and the wide confidence intervals of the estimates. For other species the estimates were well determined, yet poorly correlated, e.g. the coefficient for short-nosed lancetfish was 0.09 (CI ±0.05) in the Western Pacific distant fishery compared to 0.01 (CI ±0.04) in the Western Pacific bigeye tuna fishery. Therefore, we urge caution in applying our estimates to the same species in longline fisheries in other areas. Ward et a!.: The effect of soak time on pelagic longlme catches 191 SP Yellowfin WP Bigeye o dusk preference dawn & dusk ^ - dusk preference dawn & du>k • Raj 's bream • • Swordfish C3 • o Oilfish . Hammerhead 4 Blue martin Oiltish • Tiger shark * sk coefficient 0.0 • • • Tiger shark ;• L ' • • • * • Swordfish Q • • l • 3 • Black marlin 9 ' • Spnped marlin • • • Black marlin Hammerhead • c nol dawn or dusk dawn preference _ not dawn or dusk dawn preference ~~ -1.0 -0.5 0.0 0.5 10 -1.0 -0.5 0.0 0.5 10 Dawn coefficient Figure 5 Pair-wise comparison of coefficients for the effects of dawn and dusk on catch rates for two fisheries. The shading of each symbol represents the sum of the standard errors of the dawn and dusk estimates (heavy shading for the lowest standard errors; light shading for large standard errors). Not all species names are shown. Underlying mechanisms The broad taxonomic groups taken by longlme each rep- resent a wide range of life history strategies and feeding behaviors. Nevertheless, the results show a tendency for soak time to have a positive effect on catch rates of most shark species (Fig. 4). It also had a positive effect on catch rates of many billfish species, including striped marlin, black marlin, and swordfish. There is no clear pattern in the effect of soak time on catch rates of tuna or other bony fish. It had a negative effect on the four seabird groups. The results imply that the ability of a species to stay alive and to escape or avoid scavengers while hooked is important in determining the catch that is actually brought on board. The effect of soak time is significantly correlated with the ability of a species to survive while hooked on the longline in the four fisheries with data on survival (Fig. 7). Soak time has a strong, positive effect on catch rates of spe- cies like blue shark, which are almost always alive when branchlines are retrieved. Species like skipjack tuna and seabirds are usually dead. Soak time had a negative effect on their catch rates. The opposite trend would be expected if escape is a significant process that affects catch rates; if escape is important, soak time should have a negative af- fect on the catch rates of the most active species. Therefore removal by scavengers is likely to be more important than escape in determining catch rates for many species. Longline branchlines are usually 20-30 m in length, al- lowing considerable room for a live, hooked animal to evade predators or scavengers. Or, scavengers may be attracted by immobile and dead animals. The scavenger avoidance hypothesis is attractive, but it is difficult to test with ob- server data. Data from hook-timer experiments may help to estimate the total number of animals that are lost or removed from the longline. Data presented by Boggs ( 1992 ) showed a large number of hook-timers that were triggered but which did not hold an animal when the branchline was retrieved, e.g. his data show that 2-4"7r of hook-timers on 10,236 branchlines that had "settled" were activated but did not have an animal. It is unclear whether the trigger- ing of hook-timers was due to equipment malfunction or whether it represents high loss rates. Of particular signifi- cance to the question of loss rates is the fact that current hook-timer technology does not identify the species that were lost and whether they were alive or dead. We noticed that soak-time coefficients tended to be poorly correlated between fisheries and that the effects of soak time on catch rates were most pronounced in the South Pa- cific bluefin tuna fishery. Our scavenging hypothesis might explain those observations as evidence that the activities of scavengers vary between fisheries. For example, blue shark might be an important scavenger. They are most abundant in temperate areas (Last and Stevens, 1994). Our analyses showed a predominance of negative soak-time coefficients 192 Fishery Bulletin 102(1) CD a. =0.10* •>•' y -0 1 ( I 0 2 WP Distant coefficient m o. /-0.65* -o I o o ii I ii 2 SP Yellowfin coefficient Q. ,-=oo,s -ol I 0.2 CP Bigeye coefficient r=0.15* o u •ii Xj) and r^Xj) are the autocorrelations of X and Y at lagj, defined here with the additional weighting factor proposed by Pyper and Peterman ( 1998): ^(Xt-XKXl+j-X) r„U) = "-./ £«■,-*) (7) 2 Age-length keys. In Kanto Kinkai no Masaba ni tuite, Ap- pendix 1, vol. 30, 30 p. [In Japanese. Available from Kanagawa Prefectual Fisheries Research Institute. Jyogashima, Misaki. Miura. Kanagawa 238-0237. .Iapan.1 Growth model We used the modified von Bertalanffy growth model to incorporate the effects of population density and sea sur- Watanabe and Yatsu: Interannual variation in length at age of Scomber /aponicus 199 face temperature according to Millar and Myers,3 who nvestigated three formulations of the modified von Berta- lanffv equations: 1) a reversible effect on the growth constant k; 2) a revers- ible effect on the asymptotic length Lr; and 3) an irreversible effect on Lx or k. We tested two of the models, 1 and 2, to investigate the effect of population density and SST. We did not test model 3 because we did not consider that the environmental effects on growth were permanent. Mean length at age i of year-class y was estimated with the fol- lowing formulas: Model 1: reversible environmental effect on k L,v=L„(l-e"*°'-'°) (8) 4v = A-lv + ( L~ ~ A-i.v X 1 - e~* ■ ) ( 9 ) klv=k + PlT,fv+P2D:v. 110) Model 2: reversible environmental effect on L L0,, = L^v(l-e-*'») Llv = L,_ly+(L^-L,_lvn-e- Year Figure 3 Interannual fluctuations in mean fork length (FLl at age 0, age 1, and age 2 for chub mackerel iScomberjaponicus) in 1970-97. Horizontal lines show the 28 year mean FL at age 0, 1, and 2, respectively. Vertical bars show standard deviations. (Ill (12) (13) We ran the models with all possible combinations of explanatory variables (T, D, T, and D), and compared AIC with that obtained with the base parameters (L,, r0, k). Results where tr, XL, V D. = the age at length 0 (year); = the asymptotic length; and = the growth coefficient; = L, at age i of year-classy; = k at age ;' of year-class y; = the sea surface temperature in year i+y; and = a population density presented by the number of stock at age i of year-class y. These variables were z-score standardized. The model parameters ax and /32 were estimated to represent the effects of Tl+v and DI v on k or Lv. The parameters were estimated by maximizing the like- lihood function which is represented by and L(i,y) = L: v +£,, f, -MO.cr), UL,,k,t0,pvP2,o'f) = {L(/y)-L,v}2 nnM'-p 2a; (14) (15) (16) 3 Millar, R. B., and R. A. Myers. 1990. Modeling environmen- tally induced change in growth for Atlantic Canada cod stock. ICES CM 1990/G:24. Fork length at age Mean FL at age 0 varied substantially over the time series examined. For example, it ranged from 16.9 (Sd ±3.0) cm in 1975 to 25.9 (Sd ±1.0) cm in 1989. The mean FL for the 28 years period was 21.7 (±2.1) cm (coefficient of variation: CV=9.8%, Table 1, Fig. 3). The FL-at-age-0 values were smaller than the 28-year mean FL for the 1970s, varied around the mean in the early and mid 1980s, reached a maximum in 1989, and were at about 22-24 cm in the 1990s (Fig. 3). Mean FL at age 1 was similarly variable; it ranged from 24.3 (±1.9) cm in 1976 to 31.6 (±1.4) cm in 1995. The 28- year mean FL was 27.7 (±1.6) cm (CV=5.6%,Table 1). The trend in interannual variability was similar to that in age 0, i.e. it was smaller in the 1970s and larger in the 1990s (Fig. 3). In age-2 fish the 28-year minimum FL of 29.1 (±1.8) cm was observed in 1986 and the maximum of 34.5 (±1.3) cm was observed in 1990 (the 28-year mean FL=31.1 (±1.5) cm, CV=4.7%, Table 1, Fig. 3). In fish age 3 and older, mean FL varied year-to-year in a manner similar to that found in the younger ages ( Table 1 ). Annual mean FLs for 3-, 4-, and 5-year-olds were 33.7 (±1.3) cm (3.8%), 36.2 (CI ±1.4) cm (CV=4.0%), and 38.5 (CI ±1.5) cm (CV=3.8%), respectively (Table 1). The mean FLs for ages 0-5 of each year were significantly different among different years (one-way ANOVA, P<0.01 ). 200 Fishery Bulletin 102(1 Table 1 Total biomass, year class strength I stock number at age 1970 to 1997. Blanks show the lack of data. 0;Yats u, et al. ), SST, and mean fork length (FL> of Scomber japonicus from Year Total Biomass (103t) Stock number at age 0 (106 individuals I SST (°C)' Mean FL (SD) cm 0 1 2 3 4 5 1970 2833 10,199 11.5 19.2 12.6) 26.3 (1.8) 30.5 2.4) 34.2 (1.7) 37.7 (1.6) 40.5 1.4) 1971 3781 14.138 10.9 20.2 (2.3) 26.8 (1.9) 31.4 1.5) 34.3 (1.6) 37.7 (1.6) 40.4 1.3) 1972 4860 8342 13.2 19.3 il. 0) 27.2 (1.4) 31.1 1.6) 34.3 (1.5) 37.3 (1.7) 40.0 1.5) 1973 4650 7154 11.1 22.2 U.4) 27.9 (1.5) 29.4 1.6) 31.2 (1.8) 33.1 (2.0) 36.1 1.9) 1974 4048 7854 10.5 19.7 (1.4) 27.7 (2.5) 30.4 1.4) 31.9 (1.7) 33.9 (1.8) 37.6 1.7) 1975 3558 10,353 12.3 16.9 (3.0) 25.4 (1.8) 30.3 2.6) 32.7 (1.6) 33.8 (1.6) 35.5 1.7) 1976 3896 14,402 11.5 19.7 (2.0) 24.3 (1.9) 29.4 2.4) 33.7 (1.9) 35.3 (1.8) 38.1 1.8) 1977 5868 11.701 10.9 21.4 (1.3) 26.2 (1.8) 30.1 2.8) 33.5 (2.2) 35.7 (1.7) 37.4 1.4) 1978 5285 6249 10.0 21.5 (1.1) 28.5 (1.7) 29.8 1.6) 32.1 (2.3) 34.5 (2.D 36.1 1.9) 1979 3250 2931 12.3 19.5 (1.1) 27.1 (2.0) 30.2 2.0) 33.0 (1.7) 35.2 (1.6) 37.2 1.3) 1980 1898 2952 11.3 20.7 (1.1) 25.8 (2.6) 30.3 2.2) 32.4 (1.8) 33.9 (1.8) 35.6 1.6) 1981 1683 3374 9.4 22.7 (1.3) 27.2 (1.7) 30.5 1.5) 33.1 (2.1) 36.5 (1.8) 38.0 1.5) 1982 1567 2883 10.8 22.5 (1.8) 27.9 (1.6) 29.3 1.8) 33.6 (2.2) 36.6 (1.6) 38.3 1.4) 1983 1516 3175 11.5 19.6 (1.2) 26.7 (2.2) 30.8 1.6) 33.6 (1.5) 35.5 (2.0) 37.8 1.2) 1984 1759 3605 9.3 22.7 (1.3) 27.0 (2.4) 31.0 1.8) 34.8 (1.9) 36.6 (1.8) 38.2 2.0) 1985 1565 4998 11.4 20.1 (2.2) 27.3 (2.11 30.9 1.9) 33.3 (1.9) 37.4 (1.7) 39.0 1.8) 1986 1373 1833 9.7 21.5 (1.7) 26.4 (1.4) 29.1 1.8) 32.5 (2.4) 35.9 (2.1) 38.9 1.9) 1987 812 583 10.9 20.5 (2.1) 27.6 (1.7) 30.2 1.3) 32.8 (1.6) 36.4 (2.3) 39.2 0.8) 1988 555 236 11.4 24.9 (1.4) 28.1 (1.5) 30.5 1.4) 32.8 (1.7) 36.8 (1.6) 40.1 1.2) 1989 289 219 9.8 25.9 (1.0) 29.7 (2.3) 32.2 1.4) 34.6 (1.5) 35.7 (1.5) 39.2 1.5) 1990 185 356 11.7 24.4 (1.3) 30.3 (2.6) 34.5 1.3) 35.8 (1.5) 38.2 (1.1) 39.7 0.8) 1991 338 1017 12.2 24.1 (1.6) 28.9 (1.8) 33.5 1.9) 35.5 (1.2) 36.7 (1.9) 39.0 1.8) 1992 724 2839 9.7 24.0 (1.6) 29.0 (1.7) 32.1 1.4) 34.1 (1.5) 37.5 (1.6) 40.5 1.6) 1993 685 589 10.7 23.9 (0.9) 29.3 (1.3) 31.7 l.D 33.2 (0.5) 1994 343 547 11.3 23.7 (1.7) 28.8 (2.5) 32.8 1.0) 34.6 (0.8) 35.9 (0.7) 39.1 1.0) 1995 351 1183 11.3 22.0 (1.3) 31.6 (1.4) 32.9 1.8) 35.5 (1.8) 38.0 (1.3) 39.2 0.8) 1996 726 4452 9.9 22.5 (1.1) 28.7 (2.5) 34.1 1.2) 36.1 (1.1) 37.8 (0.9) 39.7 0.7) 1997 682 529 9.9 23.6 (1.4) 29.0 (1.5) 33.0 1.3) 35.4 (1.7) 37.6 (0.7) 38.6 0.5) 28-year mean of FLs at ages 21.7 (2.1) 27.7 (1.6) 31.1 1.5) 33.7 (1.3) 36.2 (1.4) 38.5 1.5) ' SST during ApriUJ une in the waters bounded by 38^tO°N ar d 141- 143°E. Mean growth increments I of each year class from age 0 (6 months old) to ages 1-5 (/0_,) showed significantly nega- tive correlations (Table 2). Correlations between the two variables tended to increase with age: -0.69 for /,, _,, -0.71 for I„_.,, -0.80 for /„_.,, and -0.77 for I0^. The relative FL at age 0 for any given year class was maintained throughout the life span. A correlation be- tween the mean FL at age 0 and age 1 within each year class (1970 to 1996 year class) was positive and statisti- cally significant (P<0.05, Fig. 4). Similarly, the positive cor- relations between the mean FL at age 0 and age 3 ( 1970 to 1994 year class, P<0.01, Fig. 4), and age 0 and age 4 (1970 to 1993 year class. P<0.05, Fig. 4) were significant (P<0.05. Fig. 4). Correlation between FL and population density Population densities represented by stock in number at age 0 and total biomass were negatively correlated to FL at age. Negative correlations between the logarithm of abundance of age 0 (ln/V0) and FL at ages were relatively high in age 0 to 3 (-0.69 to -0.83, Table 3) and low in age 4 and 5 (-0.63 and -0.64, Table 3). Correlations were statistically signifi- cant for ages 0, 2, and 3 (Table 3). Negative correlations between the logarithm of total biomass and FL at ages were relatively high at ages 0 to 2 (-0.73 to -0.75) and moderate for age 3 to 5 (-0.50 to -0.52, Table 4). However, the relationships were not statistically significant for all ages (Table 4). Watanabe and Yatsu: Interannual variation in length at age of Scomber /aponicus 201 32 E 3 29 26 -- 23 15 H h 20 25 FL at age 0 (cm) 38 36 S. 34 IB 32 -■ 30 B 15 H — H H — i- 20 25 FL at age 0 (cm) 20 25 FL at age 0 (cm) Figure 4 Scatter plots of FL at age 1 (A), age 3 (Bl and age 4 (C) on FL at age 0 for chub mackerel (Scomber japonicus). Correlations between FL at age 1 with age 0 (r=0.83. n=28, actual sample size n'=8, df=6), age 3 with age 0 (r=0.62, ;i=26. n =11. df =9) and age 4 with age 0 (r=0.67, u=24, n'=10) were all significant at P < 0.05. Table 2 Correlation of FL at age 0 and growth increment after age 0. n = actual sample size, n* and degree of freedom (df) show the effective ;? and df when the data were corrected for autocorrelation (Pyper and Peterman, 1998). Signifi- cance level: **, P<0.01. Growth increment Ages 0-1 Ages 0-2 Ages 0-3 Ages 0-4 Ages 0-5 df 0.69** 0.48 27 21 19 0.71** 0.51 26 25 23 0.80** 0.64 25 23 21 0.77** 0.59 23 24 22 0.78** 0.61 22 22 20 Table 3 Correlation between the natural logarithm of the abun- dance of age 0 and mean FL for each age. n = actual sample number, n* and degree of freedom (df) show the significant n and df when autocorrelation was considered (Pyper and Peterman, 1998). Significance levels: *, P < 0.05. Age r r2 n n df 0 -0.75* 0.57 28 8 6 1 -0.69 0.48 27 7 5 2 -0.83* 0.69 26 6 4 3 -0.71* 0.51 25 9 7 4 -0.63 0.40 23 8 6 5 -0.64 0.40 22 6 4 Correlation between FL and SST Growth in the first six months of life was correlated with SST. We detected significant negative correlation between FL-at-age 0 and SST between April and June in the waters bounded by 38-40°N and 141-143°E U--0.45, r2=0.20, n=28, n =27, df=25, P<0.05, Fig. 5). The SST between July and September of this area was also negatively correlated with FL at age 0 although the correlation coefficient was not significant at 5% level. Growth analysis Model 1 that incorporated SST ( T) and population density (D) gave a minimum Akaike's information criterion (AIC) of 457.68 (Table 5) and the model was expressed by L- = 43.98 1 - exp( -2.585 )exp -5X :0. 271-0. 008T -0.2LD, (17) (18) Table 4 Correlation between natural logarithm of total biomass and mean FL for each age. n = actual sample size, n* and dgree of freedom ( df ) show the effective n and df when the data were corrected for autocorrelation (Pyper and Peter- man, 1998). No correlations were significant (P>0.05). Age df 0.74 0.38 27 6 4 0.73 0.32 27 6 4 0.75 0.36 27 5 3 0.52 0.26 27 11 9 0.51 0.26 26 9 7 0.50 0.22 26 7 5 This model estimated the FL at ages 0-5 well (Fig. 6). The AIC of model 1 incorporating T and D was smaller than the AIC of model 2; therefore the environmental factors had an affect on k rather than LT. 202 Fishery Bulletin 102(1) 45 N 40 N 35 N 30 N 140N 145 N 150 B 25 -- 8> 20 + CD r =o.20 15 -I — l — l — l — i — I — i — i — i — i — I — i — i — i — i — I 8 10 12 14 Mean SST Figure 5 (A) Map to show correlation between sea surface temperatures (SST) and mean fork length (FLl at age 0 for chub mackerel (Scomber japonicus). The dotted area indicates the negative correla- tion coefficient r above 0.4. The contour interval is 0.1 of the correlation coefficient and positive contours are shown as dashes. (B) Relationship between mean SST for the area 38°-40°N and 141-143°E from April to June and mean FL at age 0. Correlation was significant at the 5^ level (r=-0.45, n=28, n "=27, df=25). To investigate the effect of T and D, we calculated the total effect on k for year-class v according to Sinclair et al. (2002): lA^ I ft A, for T, and for D. Discussion Estimated population abundance of age-0 fish and total biomass may explain density-dependent growth. FL at age 0, 2, and 3 of the Pacific stock of chub mackerel were negatively correlated with the number of age-0 recruits. Correlations between biomass and FL at ages 0-5 were low and not significant. Therefore, year-class strength is indi- cated to have a greater negative influence on the growth of the Pacific stock of chub mackerel than total biomass, as reported for the Atlantic mackerel (Scomber scombrus) (Agnalt, 1989; Overholtz, 1989; Neja, 1995) and Atlantic herring (.CI upea harengus) (Toresen, 1990). Density-dependent growth in fish populations seems to be a common phenomenon for pelagic fishes found in the temperate waters of Japan. The FL at age 0 of the 1963-69 year classes ranged from 16 to 20 cm, and were smaller than those of the 1970s, possibly indicating density-dependent growth ( Iizuka, 1974 ). According to Honma et al. ( 1987 ), the stock abundance of the Pacific stock of chub mackerel from 1963 to 1969 was larger than it was in the 1970s. Wada et al. (1995) and Hiyama et al. ( 1995) found negative relation- ships between total biomass and body length in the Pa- Table 5 Summary of statistics from the estimation of growth for chub mackerel (Scomber japonicus). AIC = Akaikf 's infor- mation criterion. No. of Log unknown likeli- Model Variables parameters hood AIC 1 L„, k, tn, (jj . . .a5 9 -280.20 578.40 L,.k,t0,a1 ...a5, /3, 10 -270.10 560.20 Lr, k, t0, CTj . . .a5, p2 10 -222.38 464.77 L„, k, t0, a, . . .cts,/S] ft 11 -217.84 457.68 2 La,k,t0,a1...as 9 -280.20 578.40 L,,k.t„.ax . . -cr5, jSj 10 -268.63 557.25 Lr, k, t0, at . . .ii-, />., 10 -224.01 468.02 Lx, k, tQ, (Jj . . .Or,, fix ft 11 -220.81 463.62 cific and Tsushima Current stock of the Japanese sardine (Sarclinops melanostictus). Kishida (1990) demonstrated a density-dependent relationship between the growth and total stock density (CPUE) of Japanese Spanish mackerel (Scomberomortis nipkonius). Our results do not agree with the positive effect of sea wa- ter temperature on somatic growth that has been shown for several species, including Japanese common squid (Kidokoro, 2001). Atlantic herring ( Moores and Winters, 1981; Toresen, 1990). and Atlantic cod (Gadus morhua ) (Brander, 1995; Du- til et al, 1999; Ratz et al. 1999; Otterson et al., 2002). Watanabe and Yatsu: Interannual variation in length at age of Scomber /aponicus 203 15 I i mini 20 I i 70 75 80 85 90 95 70 75 80 85 90 95 35 30 Age 2 35 30 -- Age 3 25 I I I I I I I I I I I I I I I I I I II I I I I I I I I I 25 I I I I I I I I I I I I II I I I I I I I I I I I I I I I 70 75 80 85 90 95 70 75 80 85 90 95 40 35 Age 4 .. Age 5 40 -- 35 30 I I I II I I II I I I I I I II II I I I I I I II I I 30 I I I I I I II I I I I I I III I I I I I I I I I I I I 70 75 80 85 90 95 70 75 80 85 90 95 Year Figure 6 Time series of observed (open circles) and modeled (solid line) values of mean fork length (FL) at ages 0-5 during 1970-97 for chub mackerel iScomber japonieus). There was a positive correlation between FL at age 0 and l°xl° block SST in the waters of 32-34°N and 144-149°E, located south of the Kuroshio Extension flowing eastward at the latitude of 35-37°N from April to June (Figs. 1 and 5A). But the correlation coefficient was not significant, and this area was not considered to be inhabited by juvenile mackerel (Watanabe, 1970). Thus, we considered that the SST in the waters of 32-34°N and 144-149°E was not a significant factor on the variation of FL at age 0. The low SST in the waters bounded by 38-40°N and 141-143°E is indicative of a large inflow of Oyashio Cur- rent waters (Hirai and Yasuda, 1988), which is a cold water current and has high productivity (Odate, 1994), into the Kuroshio-Oyashio transition zone, where is one of the main feeding grounds of mackerels (Odate, 1961; Watanabe, 1970; Watanabe and Nishida, 2002; Fig. 1). Thus, we hypothesized that the large inflow of Oyashio current waters into the Kuroshio-Oyashio transition zone improved the feeding condition and accelerated the growth of juvenile mackerel. Jobling ( 1988) suggested a parabolic relationship between water temperature and fish growth. The range of SST in this area, which was negatively cor- related with FL at age 0 of mackerel, was 9-13°C (Table 1 ). This temperature range is near the lowest nonstressful temperatures for mackerel ( 10-12°C, Schaefer, 1986). Thus, we do not consider that the negative relationship between growth and SST was the result of suppressed growth by the high ambient temperature. In mackerel, maximum egg production appears to have shifted to later in spring during the 1990s, as compared to the late 1970s and 1980s, resulting in a shorter period of growth and thus smaller fish (Fig. 8, Mori et al.4; Kikuchi and Konishi5; Ishida and Kikuchi6; Zenitani et al.7; Kubota et al.8). In the early 1970s, the main spawning period was 4 Mori, K., K. Kuroda, and Y. Konishi. 1988. Monthly egg production of the Japanese sardine, anchovy, and mackerels off the southern coast of Japan by egg censuses. Datum Collect. Tokai Reg. Fish. Res. Lab. 12:1-321. [In Japanese. Available from National Research Institute of Fisheries Science, 2-12-4 Fukuura, Kanazawa, Yokohama 236-8648, Japan.] 5 See next page. 6 See next page. 7 See next page. 8 See next page. 204 Fishery Bulletin 102(1) 0.015 T -0.015 0.015 i i i i i i i i i i i ii n i i 70 75 80 85 90 95 -0.015 I I I I I I I I I I I I I I 70 75 80 Year Figure 7 The total effect of I A) mean SST for the area of 38-40°N and 141-143"E from April to June, and (Bl population density on k for each year class of chub mackerel i Scomber japonicus). also in April (Kuroda9). Delayed spawning in the 1990s should have resulted in a reduction in the mean FL at ages during September-December in the 1990s compared to the 1970s and 1980s; however the present study showed the op- posite result (Table 1 ). We hypothesize that the effect of the shift of spawning period on the FL at ages may have been overwhelmed by the effect of population density (Fig.7). ■"' Kikuchi, H.,andY. Konishi. 1990. Monthly egg production of the Japanese sardine, anchovy, and mackerels off the southern coast of Japan by egg censuses: January, 1987 through December, 1988, 72 p. National Research Institute of Fisheries Science. Tokyo. [In Japanese. Available from National Research Insti- tute of Fisheries Science, 2-12-4 Fukuura, Kanazawa. Yokohama 236-8648, Japan.] 6 Ishida, M. and H Kikuchi. 1992. Monthly egg production of the Japanese sardine, anchovy, and mackerels off the southern coast of Japan by egg censuses: January, 1989 through December, 1990, 86 p. National Research Institute of Fisheries Science, Tokyo. [In Japanese. Available from National Research Insti- tute of Fisheries Science, 2-12-4 Fukuura, Kanazawa, Yokohama 236-8648, Japan.]. 7 Zenitani, H., M. Ishida, Y Konishi, T Goto, Y. Watanabe, and R. Kimura. 1995. Distributions of eggs and larvae of Japanese sardine, Japanese anchovy, mackerels, round herring, jack mack- erel and Japanese common squid in the waters around Japan. 1991 through 1993. Resources Management Research Report Series A-2, 368 p. National Research Institute. Japan Fisheries Agency, Tokyo. [In Japanese. Available from National Research Institute of Fisheries Science, 2-12-4 Fukuura, Kanazawa, Yoko- hama, 236-8648 Japan] s Kubota, H.,Y Oozeki, M. Ishida, Y Konishi, T Goto, H. Zenitani, and R. Kimura. 1999. Distributions of eggs and larvae of Japanese sardine, Japanese anchovy, mackerels, round her- ring, jack mackerel and Japanese common squid in the waters around Japan, 1994 through 1996, 352 p. Resources Manage- ment Research Report Series A-2., National Research Institute, Japan Fisheries Agency, Tokyo. [In Japanese. Available from National Research Institute of Fisheries Science, 2-12-4 Fuku- ura, Kanazawa, Yokohama 236-8648, Japan.] 9 Kuroda, K. 2002. Personal commun. 1-1-3-406. Kasumi. Narashino. Chiba 275-0022, Japan. Jun 6 May 5 - - Apr 4 Mar 3 H — l — I — I — I — I— 78 80 82 84 86 88 Year —l — l — l — l — l — i — i 90 92 94 96 Figure 8 Interannual variation in the peak period (weighted monthly means) of egg production for the Pacific stock of chub mackerel (Scomber japonicus), which includes a small portion from the eggs of spotted mackerel (Scomber australasicus) (Mori et al.4: Kikuchi and Konishi''; Ishida and Kikuchi1'; Zenitani et al.7; Kubota et al.8). The estimated FL at age from our growth model, with the use of AIC, fitted well to the observed FL at age (Fig. 6). Mean growth increments / of each year class from age 0 (6 months old) to ages 1-5 (/„_,) were signifi- cant and negatively correlated with FL at age 0 (Table 2), indicating that the growth rate of mackerel had changed from year to year for a given year class. This negative correlation indicated that the effects of population density and SST was temporal, and influenced k rather than L r. The negative correlation between FL at age 0 and growth increments also suggested that the FL at age of mack- erel approximated the asymptotic length. Thus, mackerel growth was best fitted to the modified von Bertalanffy growth model with the temporal environmental effect on k (Table 5). Watanabe and Yatsu: Interannual variation in length at age of Scomber /aponicus 205 The effect of population density on growth of mackerel was higher than the effect of SST (Fig. 7, Table 6). Our result agreed with the results for Japanese sardine ( Wada et al., 1995 ) and Atlantic cod ( Sinclair et al., 2002 ). Particu- larly, the effect of population density was significant in the late 1980s, which resulted in a remarkable increase in FL at age 0 (Figs. 3 and 7). The relative size at age 0 was carried over to older ages (Fig. 4), indicating that the cohorts that were small at age 0 could not compensate for this early small size. Iizuka (1974) reported that the trend of growth established at age 0 for chub mackerel was maintained until age 2 for the 1963-73 year classes. Toresen (1990) demonstrated from length data that a trend in rate of growth for a given year class of Norwegian herring was determined at the im- mature stage and was consistent after maturation. Total length of Hokkaido-Sakhalin herrings iClupea pallasii) at age 5 and older was positively correlated with the length at age 4 (Watanabe et al., 2002). Because fish first mature at age 4. this implied that the trend in total length of each year class was determined by the age at maturity. From these results we hypothesize that the variability in size at age in the Pacific stock of chub mackerel is largely attribut- able to growth before maturity, especially during the first 6 months after hatching. Acknowledgments We would like to thank K. Meguro of Chiba Prefecture Governmental Office and K. Kobayashi of Shizuoka Pre- fecture Governmental Office for providing insights into chub mackerel's growth and into age determination. We also thank T. Akamine, M. Suda, and N. Yamashita of the National Research Institute of Fisheries Science for advice on the statistical analysis. We also thank Y. Watanabe and C. B. Clarke of the Ocean Research Institute, University of Tokyo, for their constructive comments on this manuscript. Literature cited Agnalt, A. -L. 1989. Long-term changes in growth and age at maturity of mackerel. Scomber scombrus L.. from the North Sea. J. Fish Biol. 35 (suppl. A):305-311. Brander. K. M. 1995. The effect of temperature on growth of Atlantic cod iGadus morhua L.). ICES J. Mar. Sci. 52:1-10. Dutil. J. -D., M. Castonguay, D. Gilbert, and D. Gascon. 1999. Growth, condition, and environmental relationships in Atlantic cod iGadus morhua) the northern Gulf of St. Lawrence and implications for management strategies in the Northwest Atlantic. Can. J. Fish. Aquat. Sci. 56: 1818-1831. Haddon, M. 2001. Modelling and quantitative methods in fisheries, 406 p. Chapman&Hall/CRC, New York, NY. Hirai, M. 1991. Fisheries oceanographic study on purse-seine fish- ing-grounds for chub mackerel in the Sanriku coastal waters. Bull. Tohoku Natl. Fish. Res. Inst. 53:59-147. [In Japanese.] Hirai, M., and I. Yasuda. 1988. Interannual variability of the temperature field at 100 m depth near the east coast of Japan. Bull. Otsuchi Ocean Res. Center. Univ. Tokyo. 14:184-186. Hiyama, Y , H. Nishida. and T Goto. 1995. Interannual fluctuations in recruitment and growth of the sardine, Sardinops melanostictus, in the Sea of Japan and adjacent waters. Res. Popul. Ecol. 37(21:177-183. Honma, M., Y Sato, and S. Usami. 1987. Estimation of the population size of the Pacific mack- erel by the cohort analysis. Bull. Tokai Reg. Fish. Res. Lab. 121:1-11. [In Japanese.) Iizuka, K. 1974. The ecology of young mackerel in the north-eastern sea of Japan FY Estimation of the population size of the 0-age group and the tendencies of growth patterns on 0. I. and II age groups. Bull. Tohoku Reg. Fish. Res. Lab. 34: 1-16. [In Japanese.] Jobling, M. 1988. A review of the physiological and nutritional energet- ics of Cod, Gadus morpha L., with particular reference to growth under farmed conditions. Aquaeulture, 70:1-19. Kawasaki, T. 1966. Structure of the Pacific population of the mackerel. Bull. Tokai Reg. Fish. Res. Lab. 47:1-34. [In Japanese.] Kidokoro, H. 2001. 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[In Japanese.] Watanabe, T. 1970. Morphology and ecology of early stages of life in Japa- nese common mackerel. Scomber japonicus Houttuyn, with special reference to fluctuation of population. Bull. Tokai Reg. Fish. Res. Lab. 62:1-283. [In Japanese.] Watanabe, Y, Y Hiyama. C. Watanabe, and S. Takayanagi. 2002. Inter-decadal fluctuations in length-at-age of Hok- kaido-Sakhalin herring and Japanese sardine in the Sea of Japan. PICES Scientific Report 20:63-67. Yatsu, A., and H. Kidokoro. 2002. Coherent low frequency variability in biomass and in body size of Japanese common squid, Tadorodes padificus. during 1964-2002, 89 p. Abstracts of PICES 11th annual meeting. 207 Latitudinal and seasonal egg-size variation of the anchoveta (Engraulis ringens) off the Chilean coast Alejandra Llanos-Rivera Leonardo R. Castro Laboratorio de Oceanografia Pesquera y Ecologia Larval Departamento de Oceanografia Universidad de Concepcion Casilla 160-C, Concepcion, Chile E-mail address (for L. R Castro, contact author) lecastro@udeccl occur among populations of E. ringens along its distribution. In this study, we 1 ) report changes in egg size through- out the anchoveta spawning season as well as for the peak months of the spawning season, 2) evaluate whether egg size varies with respect to latitude, and 3 ) evaluate whether differences in larval length and yolksac volume occur in hatching larvae from the two major spawning stocks along Chile (central and southern stocks). The anchoveta Engraulis ringens is widely distributed along the eastern South Pacific (from 4° to 42°S; Serra et al., 1979) and it has also supported one of the largest fisheries of the world over the last four decades. However, there are few interpopulation comparisons for either the adult or the younger stages. Reproductive traits, such as fecundity or spawning season length, are known to vary with latitude for some fish species (Blaxter and Hunter, 1982; Conover, 1990; Fleming and Gross, 1990; Castro and Cowen, 1991). and latitudinal trends for some early life history traits, such as egg size and larval growth rates, have been reported for others clupeiforms and other fishes (Blaxter and Hempel, 1963; Ciechom- ski. 1973; Imai and Tanaka, 1987, Conover 1990, Houde 1989). However, there is no published information on potential latitudinal trends during the adult or the early life history of the anchoveta, even though this type of information may help in understand- ing recruitment variability, especially during recurring large scale events ( such as El Nino or La Nina) that affect the entire species range. Egg volume has been found to vary widely among species and among popu- lations of the same species. For fish that broadcast planktonic or benthic eggs, egg size often varies as the spawning season progresses (Bagenal, 1971), and the magnitude of this variation depends on the species. For instance, the egg vol- ume of the pelagic spawners Engraulis anchoita and Solea solea decreases 23% and 38%. respectively, throughout the spawning season (Ciechomski, 1973; Rijnsdorp and Vingerhoed, 1994). Ma- ternal and environmental factors may also affect egg volume (Bagenal, 1971; Thresher, 1984; Rijnsdorp and Vinger- hoed, 1994; Chambers and Waiwood. 1996; Chambers, 1997). Variations in size of the spawning females and shifts in energy allocation from reproduction to growth as the spawning season pro- gresses may influence the egg volume (Wootton, 1990). Alternatively, seasonal variations in photoperiod, seawater temperature, and food supply during the spawning season may affect the reproductive output (Wootton, 1990). Scarce information exists on the variability of egg sizes for fishes in the Humboldt Current. In this extensive area, the heavily exploited anchoveta Engraulis ringens is the dominant small pelagic species. Throughout this range, three major stocks are recog- nized: the northern stock off northern Peru ( the largest ); the central stock off southern Peru and northern Chile (mid- size), and the southern stock off central Chile (the smallest of the three). For the entire distribution of anchoveta, the main spawning season is from July through September, but may extend to December or January (Cubillos et al., 1999). The wide latitudinal range and prolonged spawning period suggest the possibility of egg-size variation, as ob- served in other clupeifoms (Blaxter and Hempel, 1963; Ciechomski, 1973; Imai and Tanaka, 1987). Egg size correlates with larval characteristics such as lar- val length at hatching, the time to first feeding, and time before irreversible starvation (Shirota, 1970; Ware, 1975; Hunter, 1981; Marteinsdottir and Able, 1992). To explore whether differences in potential early-life-stage survival would exist among populations and (or seasons ), the objective of our research was to determine whether variations in some early-life-stage characteristics Materials and methods We collected anchovy eggs from four locations along the coastal zone (<20 nmi offshore ) off northern and central Chile during the austral winter and spring spawning seasons 1995-97 (Fig. 1). Eggs were collected with a Calvet net (150 urn mesh) in Iquique and Antofagasta (northern Chile), with a standard conical net (330 um) in Valparaiso and with either a Tucker trawl (250 ; 73 Longitude West Figure 1 Areas where anchoveta eggs were collected to determine egg-size variations along the Chil- ean coast. Arrows show the locations depicted in Table 2. egg sizes measured in Iquique (<0.19 mm3) did not occur in Talcahuano. Similarly, the largest sizes determined in Talcahuano (>0.30 mm3) did not occur in Iquique. at the lowest latitude. Larval length at hatching determined in the rearing experiments at normal field temperatures was greater for the southernmost population (Talcahuano) (Table 3). The mean larval size for the southern location (2.70 mm noto- chord length) was 8.2% greater than the larvae hatched from eggs collected at the northern experimental location (Antofagasta, 2.50 mm). Furthermore, the yolksac volume in the recently hatched larvae in Talcahuano (0.130 mm3) Note Llanos-Rivera and Castro: Egg-size variation of Engraul/s ringens 209 80 60 40 20 0 „ 80 & 60 c S -+- 0 10-0 14 0 15-0 19 0 20-0 24 0.25-0.29 0.30-0.34 0.35-0.39 0.40-0.44 Size interval (mm3) Figure 3 Latitudinal variation in egg size of the anchoveta (£. ringens ) along northern and central Chile during the peak months of the spawning season. Y-axis is frequency over the total number of eggs measured at each locality. of these factors co-occur. For instance, changes in growth rates for yearly cohorts during the spawning season (low at the beginning, fast at the end) have been documented for the southernmost population (Cubillos et al., 2001). Alter- natively, variations in the population age structure during the spawning season have also been reported as the 1.5 year-old new recruits begin to spawn in early summer (late December-January, Cubillos et al., 1999, 2001 ). Changes in environmental factors affecting the spawning adults also correlate with the egg-size variations. The photoperiod and nearsurface temperatures increase as the spawning season progresses from mid-winter to late spring. Larger egg size at the beginning of the spawning season in winter may be advantageous for these offspring because the chances of survival increase with the larger sizes of the hatching larvae. According to Cushing (1967), larger size larvae should be favored over smaller larvae in seasons with variable environmental conditions. In theTalcahuano area, strong fluctuations in the hydrographic regime occur during winter as strong north wind storms alternate with short periods of south winds, and also because of the in- creased river flow to the coastal zone (Castro et al., 2000). Larval food, although variable, seems to be sufficient to support most of the larval growth demands for larger exogenous feeding larvae during winter (Hernandez and Castro, 2000). For recently hatched larvae, however, the picture might be slightly different because, in addition to food supply variability, the strong turbulent environmental conditions may jeopardize first feeding success. In these highly variable areas, therefore, larger larval size at hatch- ing and larger yolk reserves may be even more important than in other less hydrographically variable areas and seasons. A remarkable increase in egg size at the peak spawn- ing season occurred with respect to latitude. Egg from the northernmost (20°S) latitude were at a maximum 559c larger than eggs from the southernmost (36°S) lati- tude. Latitudinal variations in egg size have been previ- ously reported for other anchovies (i.e. Engraulis anchoita; Ciechomski, 1973). However, egg-size variations for fishes Note Llanos-Rivera and Castro: Egg-size variation of Engraulis nngens 211 Table 2 Width, length, and volume of anchoveta eggs collected at different latitudes along the Chilean coast during the peak months of the spawning season. SD = - standard deviations, n = number of eggs measured. Latitude and area Width (mm) Length (mm) Volume (mm3] mean SD mean SD mean SD ?! 20° Iquique 0.563 0.032 1.201 0.076 0.201 0.031 1670 23° Antofagasta 0.597 0.030 1.293 0.083 0.243 0.034 425 33° Valparaiso 0.643 0.023 1.373 0.064 0.298 0.026 62 36° Talcahuano 0.657 0.027 1.377 0.063 0.312 0.030 1833 Table 3 Morphological characteristics of recently hatched Engraulis ringens larvae from rearing experiments at normal field tempera- tures in Antofagasta (15°C) and Talcahuano (12°C). SD = standard deviations. N = number of eggs measured Exp. = expe riment. Egg volume Larval length Yolksac size ( mm3 ) at hatching (mm) at hatching (mm3) Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 Antofagasta 15°C Mean 0.264 0.260 2.49 2.50 0.099 0.096 SD (0.023) (0.023) (0.170) (0.1041 (0.012) (0.0121 n 358 325 30 30 30 30 Talcahuano 12°C Mean 0.302 0.292 2.71 2.69 0.126 0.134 SD (0.023) (0.026) (0.111) (0.103) (0.016) (0.017) n 254 66 30 30 30 30 are not necessarily always associated with latitude (i.e. north Atlantic herring stocks) because local environmen- tal conditions that trigger spawning (i.e. specific tempera- ture or others) may have a stronger effect in some species (Chambers, 1997). Because of the extremely wide distri- bution range of the anchoveta (4-42°S) and its residence along an almost linear coast oriented exactly north-south, we proposed that any potential differences in egg size due to specific local conditions is probably over-driven by the larger scale changes in environmental conditions associ- ated with latitude. The strong latitudinal gradient in egg size of the ancho- veta may be an adaptive measure if different egg sizes are favored at different latitudes or if there is a correlation between egg size and adult life history traits that maximize net reproductive output. Unfortunately, an analysis of the anchoveta in which fecundity, age of first reproduction, longevity, or other adult traits are compared in relation to latitude has not yet been carried out. The timing and length of the spawning season seem to be similar for the northern (Iquique, 20°S) and southern (Talcahuano, 36°S) stocks along Chile, despite the different temperatures at which anchoveta spawn (Castro et al., 2001 ). The decrease in egg size coincides with known temperature effects on physiological rates (Houde, 1989) and on ecological factors related to the need of anchoveta at early life stages to re- main in nearshore environments (Bakun. 1996). At lower latitudes, the sea temperature is higher and the seaward surface Ekman transport is stronger and therefore eggs and larvae in such conditions would likely develop rapidly. Alternatively, anchovy egg and larvae at higher latitudes are retained nearshore in winter (because the Ekman transport is negative, Castro et al., 2000) but are exposed to lower temperatures and to strong turbulence that may not facilitate the first feeding of recently hatched larvae and subsequent rapid larval development. Larger eggs, larger larvae at hatching, and more energy reserves may be the favored early life history strategy in southern popu- lations. How the latitudinal variations in environmental characteristics affect the rest of the life history traits of the different populations of Engraulis ringens, one of the most important fish species in the world in terms of catches, remains to be assessed. Acknowledgments We acknowledge help from R. Escribano (U. Antofagasta), G. Claramunt (U. Arturo Prat), and F. Balbontin (U. of Valparaiso) who facilitated ichthyoplankton collections. 212 Fishery Bulletin 102(1) H. Moyano (U. of Concepcion) allowed the use of his labora- tory and optical material. This study was financed by the project FONDECYT 1990470 to L. R. Castro. E. Tarifeno, and R. Escribano. Alejandra Llanos-Rivera was also par- tially supported by the Graduate School of the Universidad de Concepcion. Literature cited Bagenal, T.B. 1971. The interrelation of the size of fish eggs, the date of spawning and the production cycle. J. Fish Biol. 3: 207-219 Bakun, A. 1996. Patterns in the ocean. Ocean processes and marine population dynamics, 233 p. California Sea Grant College Publ., NOAA in Cooperation with the Centro de Investiga- ciones Biologicas del Noreste, La Paz, BCS. Mexico. Blaxter, J., and G. Hempel. 1963. The influence of egg size on herring larvae iClupea harengus L.). J. Con. Perm. Int. Explor. Mer 28: 211-240. Blaxter, J. H. S., and J. R. Hunter 1982. The biology of clupeoid fishes. Adv. Mar. Biol. 20: 3-194. Castro, L. R., and R. K. Cowen 1991. Environmental factors affecting the early life history of bay anchovy (Anchoa mitchilli) in Great South Bay, New York. Mar. Ecol. Prog. Ser. 76:235-247. Castro, L. R., G. R Salinas, and E. H. Hernandez. 2000. Environmental influences on winter spawning of the anchoveta, Engruulis ringens, off Central Chile. Mar. Ecol. Prog. Ser. 197:247-258. Castro. L. R., A. Llanos, J. L. Blanco, E. Tarifeno, R. Escribano, and M. Landaeta. 2001. Latitudinal variations in spawning habitat charac- teristics: influence on the early life history traits of the anchoveta, Engraulis ringens, off northern and central Chile. GLOBEC Report 16:42-45. Chambers. R. C. 1997. Environmental influences on egg and propagule sizes in marine fishes. In Early life history and recruitment in fish populations (R. C. Chambers and E. A. Trippel, eds.), p. 63-102. Chapman & Hall, London. Chambers, R. C, and K. Waiwood. 1996. Maternal and seasonal differences in egg sizes and spawning characteristics of captive Atlantic cod, Gadus morhua. Can. J. Fish. Aquat. Sci. 53:1986-2003. Ciechomski, J. de 1973. The size of the eggs of the Argentine anchovy, Engraulis anchoita (Hubbs & Marinil in relation to the season of the year and the area of spawning. J. Fish Biol. 5:393-398. Conover, D. 1990. The relation between capacity for growth and length of the growing season: evidence for and implications of coun- tergradient variation. Trans. Am. Soc. 119:416-430. Cubillos, L. A., D. Arcos, D. Bucarey, and M. Canales. 2001. Seasonal growth of small pelagic fish off Talcahuano, Chile (37°S, 73°W): a consequence of their reproductive strategy to seasonal upwelling? Aquat. Living Resour. 14:1-10. Cubillos, L. A., M. Canales, D. Bucarey, A. Rojas. and R. Alarcon. 1999. Reproductive period and mean size at first maturity for Strangomera bentincki and Engraulis i-ingens from 1993 to 1997, off central-southern Chile. Invest. Mar. 27:73-85. (In Spanish.] Cushing, D. H. 1967. The grouping of herring populations. J. Mar. Biol. Assoc. U.K. 47:193-208 Escribano, R., L. Rodriguez, and C. Irribarren 1995. Temporal variability of sea temperature in bay of Antofagasta, Northern Chile ( 199 1-1995 1. Estud. Oceanol. 14:39-47. [In Spanish.] Fisher. W. 1958. Huevos, crias y primeras prelarvas de la anchoveta Engraulis ringens Jenyns. Rev. Biol. Mar. 8 (1-2-31:111- 124. Fleming. I. A., and M. Gross. 1990. Latitudinal clines: a trade-off between egg number and size in pacific salmon. Ecology 71(11:1-11. Hernandez, E. H.. and L. R. Castro. 2000. Larval growth of the anchoveta, Engraulis ringens, during the winter spawning season off central Chile. Fish. Bull. 98:704-710. Houde, E. D. 1989. Comparative growth, mortality and energetics of marine fish larvae: temperature and implied latitudinal effects. Fish. Bull. 87:471^195. Hunter, J. R. 1981. Feeding ecology and predation of marine fish larvae. In Marine fish larvae (R. Lasker. ed.l. p. 34-77. Univ. Washington Press, Seattle, WA. Imai, Ch., and S. Tanaka. 1987. Effect of sea water temperature on egg size of Jap- anese anchovy. Nippon Suisan Gakkaishi 53(12): 2169- 2178. Marteinsdottir, G., and K. Able. 1992. Influence of egg size on embryos and larvae ofFundu- lus heteroclitus (L.l. J. Fish Biol. 41:883-896. Rijnsdorp, A., and B. Vingerhoed. 1994. The ecological significance of geographical and sea- sonal differences in egg size in sole Solea solea (L.). Neth. J. Sea Res. 32(3/41:255-270. Serra J., O. Rojas, M. Aguayo. F. Inostroza, and J. Canon. 1979. Anchoveta {Engraulis ringens). In Estado actual de las principales pesquerias nacionales. Bases para un desarrollo pesquero. 36 p. Corporacibn de fomento de la production. In- stituto de Fomento Pesquero. Santiago, Chile. Shirota, A. 1970. Studies on the mouth size offish in the larval and fry stages. Bull. Jap. Soc. Sci. Fish. 36:353-368. Thresher, R. E. 1984. Reproduction in reef fishes, 399 p. T.F.H. Publica- tions. Inc. Ltd. .The British Crown Colony of Hong Kong. Ware, D. M. 1975. Relation between egg size, growth, and natural mortal- ity of larval fish. J. Fish. Res. Board Can. 32: 2503-2512. Wootton. R. 1990. Ecology of teleost fishes, 404 p. Chapman & Hall. London. 213 Molecular methods for the genetic identification of salmonid prey from Pacific harbor seal (Phoca vitulina richardsi) scat Maureen Purcell Greg Mackey Eric LaHood Conservation Biology Molecular Genetics Laboratory Northwest Fisheries Science Center National Marine Fisheries Service. NOAA 2725 Montlake Blvd. E. Seattle, Washington 98112-2097 Harriet Huber National Marine Mammal Laboratory Alaska Fisheries Science Center National Marine Fisheries Service, NOAA 7600 Sand Point Way NE Seattle, Washington 98115 Linda Park Conservation Biology Molecular Genetics Laboratory Northwest Fisheries Science Center National Marine Fisheries Service, NOAA 2725 Montlake Blvd. E. Seattle, Washington 98112-2097 E-mail address (for L. Park, contact author): linda parkig'noaa gov Twenty-six stocks of Pacific salmon and trout [Oncorhynchus spp.), rep- resenting evolutionary significant units (ESU), are listed as threatened or endangered under the Endangered Species Act (ESA) and six more stocks are currently being evaluated for listing.1 The ecological and economic consequences of these listings are large; therefore considerable effort has been made to understand and respond to these declining populations. Until recently. Pacific harbor seals (Phoca vitulina richardsi) on the west coast increased an average of 5% to 1% per year as a result of the Marine Mammal Protection Act of 1972 (Brown and Kohlman2). Pacific salmon are season- ally important prey for harbor seals (Roffe and Mate, 1984; Olesiuk, 1993); therefore quantifying and understand- ing the interaction between these two protected species is important for biologically sound management strat- egies. Because some Pacific salmonid species in a given area may be threat- ened or endangered, while others are relatively abundant, it is important to distinguish the species of salmonid upon which the harbor seals are prey- ing. This study takes the first step in understanding these interactions by using molecular genetic tools for spe- cies-level identification of salmonid skeletal remains recovered from Pacific harbor seal scats. Most studies of harbor seal food hab- its rely on morphological identification of indigestible parts (e.g. otoliths and bones) from scat. Otoliths can be used to identify fish species (Ochoa-Acuna and Francis, 1995) but are not always present in scats, which can result in an underestimate of the number of species and the number offish consumed (Har- vey, 1989). Skeletal remains in scat are much more common and generally bones can be identified to the species level (Cottrell et al., 1996). Morpho- logical identification is possible to the family level only with Pacific salmonid bones; however, genetic markers have the ability to discriminate between species, and the feasibility of extracting DNA from bones has been clearly dem- onstrated (Hochmeister et al., 1991). Mitochondrial DNA (mtDNA) has been widely employed in systematic studies (reviewed by Avise, 1994) mak- ing it ideal for animal species identifi- cation. In this study, we explored three regions of the mitochondrial genome that have been previously character- ized in Pacific salmonids (Shedlock et al., 1992; Domanico and Phillips, 1995: Parker and Kornfield, 1996). DNA sequencing of these regions provided an unambiguous way to de- termine species identity. Because high throughput sequencing can be prohibi- tively expensive for laboratories with limited facilities, restriction fragment length polymorphism (RFLP) analysis was also explored as an alternative for species identification. A previous study had established a species-specific poly- merase chain reaction (PCR) test for Pacific Northwest salmon and coastal trout species (McKay et al., 1997). The PCR test is based on the initial ampli- fication of an approximately 1000-bp fragment of the nuclear growth hor- mone 2 gene. The degraded state of the DNA isolated from bones recovered from scat has generally limited suc- cessful PCR to amplicons of 300 bp or less (data not shown). Furthermore, the amount of DNA isolated from bone fragments can be quite small; mtDNA is present in higher copy number per cell than is nuclear DNA. Thus, we considered mtDNA it to be a more 1 http://www.nwr.noaa.gov/lsalmon/salmesay specprof.htm. [Accessed June 17, 2003.] - Brown, R. F. and S. G. Kohlman. 1998. Trends in abundance and current status of the Pacific harbor seal tPhoca vitulina richardsi) in Oregon: 1977-1998. ODFW (Oregon Department of Fish and Wildlife i Wildlife Diversity Program Technical Report, 98-6-01. 16 p. [Available from ODFW, 7118 NE Vandenberg Ave. Corval- lis, OR 97333.] Manuscript approved for publication 9 October 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull. 102:213-220 (2004). 214 Fishery Bulletin 102(1) appropriate target for our assay. We chose to explore smaller regions of the mitochondrial genome, including the d-loop (Shedlock et al., 1992), a portion of the 16s ribosomal gene (Parker and Kornfield, 1996), and a region spanning the cytochrome oxidase III, t-RNA glycine, and ND3 genes (hereafter, referred to as COIII/ND3) (Domanico and Phil- lips, 1995 ). Significant interspecific variation but not intra- specific variation was observed in the COIII/ND3 region among salmonid species in previous studies, making it a particularly good candidate region for the development of diagnostic markers (Domanico and Phillips, 1995). In the first phase of the study, we developed and vali- dated the genetic tools for species identification by using frozen or ethanol-preserved tissues collected from known species and populations. In the second phase, we applied these tools to the identification of bone remains from har- bor seal scats collected at the Umpqua River (Oregon). A number of Pacific salmonid species are present in the Umpqua River but of particular concern were the sea- run cutthroat (Oncorhynchus clarki) that were listed as endangered under the ESA during 1996 (Johnson et al., 1999). Here we report the method associated with these two phases of the project. The salmonid bones that were identified genetically were incorporated into a larger study of the harbor seal diet and are reported in a companion paper (Orr et al., 2004). Materials and methods Salmonid tissue samples of known species have been collected over the past decade by geneticists from the Conservation Biology Molecular Genetics Laboratory (NOAA/NMFS/NWFSC) or generously donated by others (see "Acknowledgments" section) and maintained either frozen at -80°C or preserved in 95% ethanol. Reference populations were chosen to represent the geographic range of chinook salmon (O. tshawytscha), coho salmon (O. kisutch), sockeye salmon (O. nerka). pink salmon (O. gorbuscha), chum salmon (O. keta), steelhead (O. mykiss), coastal cutthroat trout (O. clarki clarki), and Yellowstone cutthroat trout ( O. clarki bouvieri ) ( collection information is listed in Table 1 ). Tissues were extracted with either a stan- dard phenol and chloroform extraction (Sambrook et al., 1989) or by using the DNAeasy 96-well tissue kit (Qiagen, Valencia, CA), following the manufacturer's instruction for tissue preparations. PCR primers were either taken directly from the published studies or designed from the reported sequences (Table 2). All primers were cycled with 2.5 mM MgCl2, 0.8 mM dNTPs, 0.04 ,«M primers, 0.25 units of Taq DNA polymerase (Promega, Madison, WI), 20-40 ng of DNA, and cresol red loading buffer (final concentration 2' < sucrose and 0.005% cresol red) for 35-45 cycles of 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 1 minute. A single individual of each salmonid species listed in Table 1 was sequenced for both the 16s rRNA and COIII/ ND3 regions. For DNA sequencing, the PCR products were purified with an Ultrafree MC column (Millipore, Beverly, MA i and resuspended in 20 ,uL of sterile water. The puri- fied product (1-10 uL depending on band intensity) was manually sequenced by using the USB ThermoSeque- nase cycle sequencing kit (Cleveland. OH), following the manufacturer's instructions. MACDNASIS (Miraibio Inc., Alameda. CA) and SEQUENCHER (Gene Codes Corp., Ann Arbor. MI) were used for sequence alignment and identifi- cation of diagnostic restriction enzyme cut sites. RFLP analysis of the unpurified COIII/ND3 PCR product was performed in the presence of a cresol red loading buf- fer. Restriction digests were incubated for 6 to 12 hours at 37°C for Dpn II, Sau 961, Fok I, Ase I, at 50° for Apo I, and at 60°C for Bst NI with the supplied buffers (NEB, Beverly, MA) and 1-5 units of enzyme. Restricted products were electrophoresed in a 47c 3:1 high-resolution and medium- resolution agarose gel (Continental Laboratory Products, San Diego, CA). DNA bands on the agarose gels were visualized with SYBR Gold, following the manufacturer's instructions (Molecular Probes, Eugene, OR). Personnel from the National Marine Mammal Laboratory (NMML) collected and processed harbor seal scat samples from the Umpqua River (Orr et al., 2004). NMML research- ers identified bone remains to either family or species level by using morphological characteristics of skeletal remains (Orr et al., 2004). From 39 harbor seal scats, 116 bones were identified morphologically to the genus Oncorhynchus and subjected to DNA analysis for species identification. For a positive DNA extraction control, we simulated digestion by treating coastal cutthroat bones (collected from Cowlitz Trout Hatchery, Winlock, WAi in a mixture of laboratory- grade trypsin (a digestive enzyme), baking soda, and water for 1 to 2 days. These trypsin-treated bones from a coastal cutthroat trout were used as positive DNA extraction and amplification control. To prepare samples for DNA extraction, bones were soaked in 107c sodium hypochlorite for 10 minutes to destroy any contaminating DNA that may have adhered to the outside of the bone and were rinsed twice in sterile water. Bones ranged in weight from 0.1 to 105.6 mg and included teeth, vertebrae, gillrakers, radials, and bone fragments (hereafter, all bony parts and teeth will be re- ferred to as "bone"). The bones were decalcified overnight in 0.5M EDTA solution (Hochmeister et al., 1991); fragile or small fragments were not decalcified. The EDTA was removed and the decalcified samples were extracted with the QIAamp tissue extraction kit (Qiagen. Valencia. CA) according to the manufacturer's instructions with the following modifications: 1) samples were proteinase K digested overnight or until completely digested; 2) 10 mg/«L yeast t-RNA carrier was added to the extractant before placement on the QIAQuick column; and 3) DNA was eluted in a reduced volume (50-100 «L) of buffer AE. Negative controls containing no tissue were simultane- ously processed to verify that the extraction was free of contaminating DNA. The trypsin-treated coastal cutthroat bones were used as positive extraction and PCR controls. Five to ten microliters of the extracted DNA were used in each amplification reaction. Amplification success was determined by electrophoresis through a 27c agarose gel followed by staining with ethidium bromide or the more sensitive SYBR Gold i Molecular Probes). Species identifi- NOTE Purcell et al.: Genetic identification of salmonid prey from scat of Phoca vitulina nchardsi 215 Table 1 Species, locations, and sampl ; sizes (n 1 examined for RFLP analysis. Species Population Location 71 Chinook Walker Creek Upper Frasier River. British Columbia 10 Grovers Creek Hatchery Puget Sound, Washington 12 Lookingglass Hatchery Snake River. Oregon 12 Carson Hatchery Columbia River, Washington 12 Abernathy Hatchery. Columbia River, Washington 11 Upper Sacramento Mainstem Sacramento River. California 10 Coho Edison Creek Oregon Coast 13 Sandy River Columbia River, Oregon 15 North Fork Moclips River Washington Coast 15 Minter Creek Hatchery Puget Sound, Washington 15 Yakoun River Queen Charlotte Island, British Columbia 7 Sockeye Nehalem Ponds Oregon Coast 4 Redfish Lake Snake River, Idaho 4 Alturas Lake Snake River, Idaho 2 Ozette Lake Washington Coast 14 Lake Wenatchee North Cascades.Washington 10 Babine Lake Central British Columbia 2 Kamchatka River Kamchatka Peninsula, Russia 9 Chum Hamma Hamma River Hood Canal. Washington 11 Frosty Creek Alaskan Peninsula 12 Utka River Chucotka Peninsula, Russia 9 Miomote River West Honshu. Japan 11 Pink Nisqually River South Puget Sound. Washington 6 Snohomish River Even Year North Puget Sound, Washington 12 Skagit River North Puget Sound, Washington 7 Hood Canal Hatchery Hood Canal, Washington 9 Steelhead Gaviota Creek South California Coast 4 Coquille River Oregon Coast 8 Upper Tucannon River Snake River, Washington 12 Finney Creek Puget Sound, Washington 12 Quinault Hatchery Washington Coast 12 Tigil River Kamchatka Peninsula. Russia 12 Cutthroat' Alsea River Oregon Coast 2 Alsea Hatchery Oregon Coast 3 Duwamish River Puget Sound Washington 12 Yellowstone River Yellowstone River. Montana 5 ' Cutthroat trout from the Yellowstone River are a different subspecies (O. clarki bouvieri) from the Washington and Oregon coastal cutthroat trout (O. clarki clarki). cation was accomplished by sequencing of either the d-loop or the COIII/ND3 region. RFLP analysis was performed as described above with the following modifications: Bst NI was excluded because it is redundant with Dpn II, the enzyme amount was reduced to 0.4-1.0 units per reaction, and incubation time did not exceed 2 hours. The COIII/ND3 primers are specific to the family Salmonidae. To test the possibility that the failure to obtain amplifica- tion with the COIII/ND3 primers was due to morphologi- cal misidentification of an Oncorhynchus species we used the 16s primers that are conserved across a broad set of taxa from Platyhelminthes through Chordata ( Parker and Kornfield, 1996). Results The COIII/ND3 and 16s sequences were confirmed for all seven salmonid naturally present in the Pacific Northwest (Figs. 1 and 2) and deposited in Genbank (COIII/ND3: AF294827-AF294833; 16S: AF296341-AF296347). Two chinook salmon were sequenced representing two Dpn II 216 Fishery Bulletin 102(1) Primer sequences size of amplified product in base Table 2 Dairs, and references for mitochondria] loci used in this study. Locus Primer sequences (5' to 3') Product size Reference d-loop COIII/ND3 16sV P2: tgt taa ace cct aaa cca g P4: gec gaa tgt aaa gca tct ggt F: tta caa teg ctg acg gcg R: gaa aga gat agt ggc tag tac tg F: tac ata aca cga gaa gac c R: gtg att gcg ctg tta tec 230 368 260 Shedlocketal.. 1992 Domanico and Phillips Parker and Kornfield, 1995 1997 Table 3 Restriction fragment length polymorphisms of the cytochrome oxidase III a id ND3 region digested w ith six restriction ?nzymes. The "A" haplotype does not cut with the enzyme, "B" cuts with the enzyme, and "C" cuts with the enzyme but at a different site than "B." Species Dpn II Sau 961 Fok I Asel Apo I Bst NI Chinook A/B; B B A A A Coho A A B A A A Sockeye A A A A C B Chum A A A B C A Pink C A A B C C Steelhead A A A B B A Cutthroat A A A A A A 1 Spring-running chinook from the Columbia and Snake Rivers were polymorphic foi the Dpn II cut site. Spring chinook from Carson Hatchery (derived from the upper Columbia River spri ng-running ESU [evolutionary significam unit] I had the "A" haplotype at a frequency of 0.91 ( n=12) and spring chinook from Lookingglass Hatchery (Snake River spring-summer- ■unning ESU) had the "A" haplotype at a frequency of 0.83 (n = 12). All other chinook samples from Table 1 were invariant for the "B" h£ plotvpe. haplotypes (A and B) and their sequences are presented in Figure 1; the chinook salmon individuals were from the Upper Columbia River summer and fall ESU (Methow River, WA). A second intraspecific polymorphism in chi- nook salmon was observed at position 341 between our ND3 sequence and the published sequence (Domanico and Phillips, 1995) (Fig.l). Sufficient nucleotide varia- tion exists in the d-loop (Shedlock et al., 1992) and in the COIII/ND3 region ( Fig. 1 ) to distinguish among the salmon species by sequencing; both regions were used for bone identification. Six restriction enzymes were selected from the COIII/ ND3 sequence that appeared to distinguish among all the species (Dpn II, Sau 961, Fok I, Ase I, Apo I, and Bst NI) (Fig. 1). The Dpn II and Bst NI cut patterns are redundant in that only one of these enzymes is required for species identification when used in conjunction with the other four enzymes (however, only Dpn II exhibits the intraspecific chinook polymorphism, see below). Haplotype patterns for all species are listed in Table 3. The haplotypes were scored with a simple alphabetic system: "A" was uncut (368 base- pair (bp) band) and "B" was cut (the size differed depending on enzyme). A few of the enzymes had an alternative cut site, and the resulting haplotype we labeled "C." The "B" haplotype produced by Apo I occurs in steelhead and the bands migrate at 300 and 68 bp, whereas the bands of the "C" haplotype in sockeye, chum, and pink salmon migrate at 250 and 118 bp. The enzyme Bst NI also has two cut pat- terns: the sockeye salmon "B" haplotype bands migrate at 282 and 87 bp and the "C" haplotype bands in pink salmon migrate at 271 and 98 bp. The Dpn II "B" haplotype in chinook salmon creates two fragments, 290 and 80 bp; the "C" haplotype in pink salmon creates three fragments, 292, 53, and 24 bp. To confirm that the restriction enzyme polymorphisms were diagnostic within each species, we surveyed all seven Pacific salmon species representing multiple populations spanning a large geographic range (Table II. No intra- specific polymorphisms were detected among populations with the exception of chinook salmon (Tables 1 and 3). A single intraspecific polymorphism was found with the Dpn II enzyme in chinook salmon lineages in the Columbia and Snake River basins (Tables 1 and 3). Chinook salmon from the Snake River spring-summer run (Lookingglass NOTE Purcell et al.: Genetic identification of salmonid prey from scat of Phoca vitulina nchardsi 217 Chinook A Chinook B Coho Sockeye Chum Pink Steelhead Cutthroat Chinook A Chinook B Coho Sockeye Chum Pink Steelhead Cutthroat Chinook A Chinook B Coho Sockeye Chum Pink Steelhead Cutthroat Chinook A Chinook B Coho Sockeye Chum Pink Steelhead Cutthroat Chinook A Chinook B Coho Sockeye Chum Pink Steelhead Cutthroat Chinook A Chinook B Coho Sockeye Chum Pink Steelhead Cutthroat Chinook A Chinook B Coho Sockeye Chum Pink Steelhead Cutthroat 20 DpnII 40 60 * * * * TTACAATCGCTGACGGCGTGTACGGCTCTACTTTCTTTGTCGCCACCGGATTCCATGGCC . . . . A. .T. .A. .T. .A. DpnII 80 100 Apol/ Sau96I TACACGTGATTATTGGCTCAACCTTTCTAGCCGTTTGCCTTCTGCGACAGGTCCAATACC A . . . . A. . . . . A. .T. .A. :. .G. . . .G. .T.G. .A. .T. . AA . T . . AA.T. . AA.T. Fokl 140 160 180 ********** ACTTTACATCCGAACATCATTTTGGCTTTGAAGCTGCTGCTTGATATTGACACTTTGTAG .T. . . . .T. . . . .T. .G. 200 220 start tRNA glycine --> ACGTTGTGTGACTCTTCCTATACGTCTCTATTTACTGATGAGGCTCATAATCTTTCTAGT .A. .G. . A. . . . Asel ****** 260 BSTNI 280 Start ND3 — > ATTAACACGTATAAGTGACTTCCAATCACCCGGTCTTGGTTAAAATCCAAGGAAAGATAA . .G . TGA . TTA .TTA. . .CG. .T Apol DpnII 340 360 ****** **** TGAACTTAATTACAACAATCATCACTATTACCATCACATTRTCCGCAGTACTAGCCACTA .CG. .C.G. .CG. . . .A. . . .G. TTTCTTTC Figure 1 Aligned sequences of the 3' region of the cytochrome oxidase III gene (COM I, the tRNA glycine gene, and the 5' region of the ND3 gene for seven species of the genus Oncorhynchus. The cutthroat trout sequence is represented by the coastal cutthroat subspecies (O. clarki clarki). Chinook "A" refers to the "A" Dpn II haplotype; chinook "B" refers to the "B" Dpn II haplotype. Sequence identity relative to the chinook salmon "A" sequence is denoted by dots; nucleotide substitutions are indicated. The arrow at basepair (bp) 230 is the start of the tRNA glycine gene and the arrow at bp 300 is the start of the ND3 gene. Stars above the sequence correspond to restriction enzyme cut sites used in this study. At position 341 in chinook. the R represents an A or G. 218 Fishery Bulletin 102(1) Chinook Coho Sockeye Chum Pink Steelhead Cutthroat Chinook Coho Sockeye Chum Pink Steelhead Cutthroat Chinook Coho Sockeye Chum Pink Steelhead Cutthroat Chinook Coho Sockeye Chum Pink Steelhead Cutthroat 20 40 60 GGAGCTTTAGACACCAGGCAGATCACGTCAAACAACCTTGAATTAACAAGTAAAAACGCAGT G 80 100 120 GACCCCTAGCCCATATGTCTTTGGTTGGGGCGACCGCGGGGGAAAATTAAGCCCCCATGTGG 140 160 180 ATGGGGGCATGCCCCCACAGCCAAGAGCCACAGCTCTAAGCACCAGAATATCTGACCAAAAA T T...A 200 220 TGATCCGGCAAACGCCGATCAACGGACCGAGTTACCCTAG. . . Figure 2 Aligned sequences of a variable portion of the 16s gene for seven species of the genus Oncorhynchus. Sequence identity in relation to the chinook salmon "A" sequence is denoted by dots; nucleotide substitutions are indicated. Hatchery) and hatchery stocks descended from the Upper Columbia River spring run (Carson Hatchery) had the "A" (uncut) haplotype at a frequency of 83% and 91%, respec- tively, whereas those from the Lower Columbia River ESU were invariant for the "B" (cut) haplotype. The "B" hap- lotype was also invariant in the other lineages examined (Sacramento River, CA; Puget Sound, WA; and the Fraser River, BC). Despite this Dpn II polymorphism, the haplo- type patterns were still chinook-specific. Extractions from the trypsin-treated cutthroat trout bones, used as positive controls, were amplified consis- tently, but of the 116 salmonid bones from harbor seal scats, only 78 (67%) were amplified. Failed samples were repeated several times with all possible primer sets. Be- cause each scat contained multiple bones, we were able to amplify bones representing 35 of the 39 scats (90%). The smallest bone we successfully amplified was a O.'2-mg tooth and the largest was a 21.8-mg vertebra. There did not appear to be a relationship between bone size and DNA extraction success; no significant difference in mean bone size was detected between 32 bones that either amplified or failed (P=0.280; unpaired t-test; SYSTAT 8.0 [Chicago, IL| ). The bone samples that failed to amplify repeatedly were also tested by using the evolutionarily conserved 16s primers. Some samples were still refractory to PCR, indicating that the overall DNA quality or quantity was insufficient for this assay; however, those samples that did amplify were identified by sequencing as salmon. In an un- related study using river otter bones (data not presented), one bone sample morphologically identified as salmonid yielded a sequence with 100% identity to the published 16s sequence available for Northern squawfish {Ptychocheilus oregonensis) (Simons and Mayden, 1998). After verifying the specificity of the RFLP analysis for differentiating the Pacific salmon species, the assay was applied to the bone samples. Restriction enzyme digestion required some modification when applied to bone. On occa- sion, the restriction enzyme protocol developed for the fresh tissue resulted in degradation of the amplified bone PCR product. Enzyme amount and digestion times were scaled back for the analysis of the bone samples. The Fok I enzyme proved the most difficult for the bone samples, which was likely due to nonspecific restriction that occurs when the enzyme is present at a high concentration in relation to its target or if the reaction is allowed to digest for more than two hours. In some cases, only very weak amplification was achieved with the bone samples and it was difficult to get digestion without degradation. Although sequencing was the main technique used for bone identification. 23 bones in this study were identified by using the RFLP technique. Fourteen of these 23 bones were additionally confirmed by sequencing and the two techniques gave matching results. NOTE Purcell et al.: Genetic identification of salmonid prey from scat of Phoca vitulina nchardsi 219 Discussion This study focused on the development of tools for the genetic identification of Pacific salmon skeletal remains recovered from harbor seal scats. These tools help to deter- mine the diet of marine mammals and can also be used to address direct management questions regarding interspe- cific interactions in rivers such as the Umpqua River where salmonid species of concern (cutthroat trout (occur with pro- tected marine mammal species. The harbor seal diet in the Umpqua River consisted of nonsalmonid fish and chinook. coho, and steelhead; no cutthroat trout were observed in the scat samples (Orr et al., 2004). The majority of salmonid species identifications were possible only by using genetic methods because very few otoliths were recovered in the Umpqua River scats. A number of other sites exist were this technology may also be applicable. In Hood Canal ( WA) the summer chum salmon run is listed as threatened under the ESA. A report of seal diets in Hood Canal determined that 2T7c of the fish consumed by harbor seals were salmonids (Jeffries et al.3). The study used both bones and otoliths, but only 25% of the samples contained otoliths that allowed species-level identification. In the Alsea River (OR), coho salmon are listed as threatened. A report by Riemer et al.4 indicated that 69r of fish consumed by pinnipeds in the Alsea River are salmonids; none of the salmonid remains were morphologically identifiable to species. Extraction of DNA from bones can be done with a com- mercially available kit with minor modifications. In our study, only 67% of the bone DNA extracts could be ampli- fied by PCR. PCR failure could be due to DNA degradation during the digestive process or to environmental exposure after defecation. However, multiple bones are often present in scats and we were able to amplify DNA from at least one bone representative from 35 out of the 39 scats examined. Sequencing or RFLP analyses of the COIII/ND3 locus are both viable methods of identifying the seven common On- corhynchus species. This study used manual sequencing with radioactivity and we did have better results using this method compared to the RFLP method. A recently published study also identified restriction enzymes in the cytochrome B gene that distinguish among the salmonid species (Russell et al., 2000). The study reported diagnostic RFLP differences among these species but did not confirm the lack of intraspecific variation in a wide geographic sur- vey of each species. The goal of the cytochrome B RFLP as- say designed by Russell et al. (2000) was to identify salmon species found in processed food products but the primers 3 Jeffries, S. J., J. M. London, and M. M. Lance. 2000. Obser- vations of harbor seal predation on Hood Canal summer chum salmon run 1998-1999. Annual progress report to Pacific States Marine Fisheries Commission, 39 p. [Available from WDFW, Marine Mammals Investigations, 7801 Phillips Rd. SW, Tacoma, WA 98498.] 4 Riemer, S. D., R. F. Brown, B. E. Wright and M. I. Dhruv. 1999. Monitoring pinniped predation on salmonids at Alsea River and Rogue River, Oregon: 1997-1999. Oregon Depart- ment of Fish and Wildlife, Marine Mammal Research Program, Corvallis, OR, 36 p. [Available from ODFW, 7118 NE Vanden- berg Ave., Corvallis, OR 97333.] may also prove useful in species identification of bone re- mains. The 16s primer set is also valuable for bones that are morphologically unidentifiable. However for salmonid species identification, the 16s region contains fewer diag- nostic nucleotide substitutions in relation to the d-loop and the COIII/ND3 region. Overall, the techniques established here would be useful for further study of marine mammal diets and may have the potential for forensic application. Acknowledgments The authors acknowledge Robert Delong for suggesting this study. Jon Baker at the Northwest Fisheries Science Center and Paul Spruell at the University of Montana kindly provided cutthroat DNA. James Shaklee at the Washington Department of Fish and Wildlife kindly provided pink salmon samples. Sam Wasser and Virginia Butler provided advice on the recovery of DNA from scat and bone samples. Gail Bastrup assisted in technical aspects of this study. Literature cited Avise, J. C. 1994. Molecular markers, natural history, and evolution, p. 44-91. Chapman and Hall, New York, NY. Cottrell, P. E.. A. W Trites, and E. H. Miller. 1996. Assessing the use of hard parts in faeces to identify harbour seal prey: results of captive-feeding trials. Can. J. Zool. 74:875-880. Domanico, M. J., and R. B. Phillips. 1995. Phylogenetic analysis of Pacific salmon (genus Oncorhynchus) based on mitochondrial DNA sequence data. Mol. Phylogenet. Evol. 4:366-371. Harvey. J. T. 1989. Assessment of errors associated with harbour seal {Phoca vitulina) faecal sampling. J. Zool. 219:101-111. Hochmeister, M. N, B. Budowle. U. V. Borer, U. Eggmann, C. T. Comey, and R. Dirnhofer. 1991. Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains. J. Forensic Sci. 36: 1649-1661. Johnson, O., M. Ruckelshaus, W. Grant. F. Waknitz, A. Garrett, G Bryant, K. Neely, and J. Hard. 1999. Status review of coastal cutthroat trout from Washing- ton, Oregon, and California. NOAA Tech. Memo. NMFS- NWFSC-37, p 138-139. McKay, S. J., M. J. Smith, and R. H. Devlin. 1997. Polymerase chain reaction-based species identifica- tion of salmon and coastal trout in British Columbia. Mol. Mar. Biol. Biotechnol. 6:131-140. Ochoa-Acuna, H., and J. M. Francis. 1995. Spring and summer prey of the Juan Fernandez fur seal, Arctocephalus philippii. Can. J. Zool. 73:1444-1452. Olesiuk, P. F. 1993. Annual prey consumption by harbor seals (Phoca vitulina) in the Strait of Georgia, British Columbia. Fish. Bull. 91:491-515. Orr, A., A. Banks, S. Mellman, and H. Huber. 2004. Examination of Pacific harbor seal (Phoca vitulina richardsi) foraging habits to describe their use of the 220 Fishery Bulletin 102(1) Umpqua River, Oregon, and their predation on salmonids. Fish. Bull.l02:108-117. Parker, A., and I. Kornfield. 1996. An improved amplification and sequencing strategy for phylogenetic studies using the mitochondrial large sub- unit rRNA gene. Genome 39:793-797. Roffe, T., and B. Mate. 1984. Abundance and feeding habits of pinnipeds in the Rogue River, OR. J. Wildl. Manag. 48:1262-1274. Russell, V. J., G. L. Hold, S. E. Pryde, H. Rehbein, J. Quinteiro. M. Rey-Mendez, C. G. Sotelo, R. Perez-Martin, A. T. Santos, and C. Rosa. 2000. Use of restriction fragment length polymorphism to distinguish between salmon species. J. Agricult. Food Chem. 48:2184-2188. Sambrook, J., E. F Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, p. 9.17-9.19. Cold Spring Harbor Laboratory. Cold Spring Harbor. NY. Shedlock, A. M., J. D. Parker, D.A. Crispin, T. W. Pietsch, and G. C. Burmer. 1992. Evolution of the salmonid mitochondrial control region Mol. Phylogenet. Evol. 1:179-192. Simons, A. M., and R. L. Mayden. 1998. Phylogenetic relationships of the western North Amer- ican phoxinins (Actinopterygii: Cyprinidae I as inferred from mitochondrial 12S and 16S ribosomal RNA sequences. Mol. Phylogenet. Evol. 9:308-329. 221 Diel vertical migration of the bigeye thresher shark (Alopias superci/iosus), a species possessing orbital retia mirabilia Kevin C. Weng Barbara A. Block Tuna Research and Conservation Center Hopkins Marine Station of Stanford University 120 Oceanview Boulevard Pacific Grove, California 93950 E-mail address (for K. C. Weng): kevin cm wengia'stanford edu The bigeye thresher shark {Alopias superciliosus, Lowe 1841) is one of three sharks in the family Alopiidae, which occupy pelagic, neritic, and shallow coastal waters throughout the tropics and subtropics (Gruber and Compagno, 1981; Castro, 1983). All thresher sharks possess an elongated upper caudal lobe, and the bigeye thresher shark is distinguished from the other alopiid sharks by its large upward-looking eyes and grooves on the top of the head (Bigelow and Schroeder, 1948). Our present under- standing of the bigeye thresher shark is primarily based upon data derived from specimens captured in fisheries, including knowledge of its morpho- logical features (Fitch and Craig, 1964; Stillwell and Casey, 1976; Thorpe, 1997), geographic range as far as it overlaps with fisheries (Springer, 1943; Fitch and Craig, 1964; Stillwell and Casey, 1976; Gruber and Compagno, 1981; Thorpe, 1997), age, growth and maturity (Chen et al., 1997; Liu et al., 1998), and aspects of its reproductive biology (Gilmore, 1983; Moreno and Moron, 1992; Chen et al.. 1997). Limited information on the move- ment patterns of bigeye thresher sharks has been obtained from mark- recapture studies by using conven- tional tags. The longest straight-line movement of a conventionally tagged bigeye thresher shark to date is 2767 km from waters off New York to the eastern Gulf of Mexico (Kohler and Turner, 2001). The bigeye thresher shark has been captured on longlines set near the surface at night (0 m to 65 m, Fitch and Craig, 1964; Stillwell and Casey, 1976; Thorpe, 1997; Buencuerpo et al., 1998) and at 400 m to 600 m during the day (Nakamura1). There is no published information available regarding its habitat and behavior, al- though Francis Carey tracked a bigeye thresher with an acoustic tag for six hours (Carey2). Endothermy is a rare trait in fishes and has been demonstrated only in tunas (Thunnini), billfishes (Xiphiidae, Istiophoridae), and lamnid sharks (Lamnidael (Carey and Teal, 1969; Carey, 1971, 1982a; Block, 1991). In all endothermic fishes, the blood supply to aerobic tissues such as slow-twitch swimming muscle, visceral organs, extraocular muscles, retina, and brain occurs by counter-current heat exchangers known as retia mirabilia. The vascular supply reduces heat loss to the environment and enables heat conservation in metabolically active tissues (Carey, 1971). Lamnid sharks have retia mirabilia in the circulatory anatomy supplying the slow-oxidative swimming muscles, viscera, brain, and eyes (Burne, 1924; Block and Carey, 1985; Tubbesing and Block, 2000). In many lamnid species, tissue tempera- tures significantly above ambient have been recorded from freshly captured specimens and through telemetry stud- ies of swimming animals (Carey, 1971; Carey et al., 1981, 1982, 1985; McCos- ker, 1987; Goldman, 1997; Tubbesing and Block, 2000). The anatomy of alopiid sharks sug- gests that endothermy may occur in this family. The bigeye thresher and the common thresher (Alopias vulpinus) have centrally located slow-oxidative muscle and primitive retia mirabilia supplying blood to them (Carey, 1982b: Bone and Chubb, 1983). Burne (1924) noted a coiling of the pseudobranchial artery supplying the orbit and cranial regions in the common thresher. No internal tissue temperature measure- ments have been taken for free-swim- ming thresher sharks to ascertain whether heat is conserved in oxidative tissues. A freshly caught bigeye thresh- er shark was found to have a body-core thermal excess of 4°C (Carey, 1971); thus the species may have the ability to conserve metabolic heat. In this study we present electronic tagging data on the movements, div- ing behavior, and habitat preferences of the bigeye thresher shark based on two individuals studied with pop-up satellite archival tags. In addition, we provide a brief description of the orbital rete mirabile of the species. The presence of this highly developed rete mirabile within the orbital sinus suggests a physiological mechanism to buffer the eyes and brain from the large temperature changes associated with diel vertical migration, potentially conferring enhanced physiological per- formance. Materials and methods The movements of two bigeye thresher sharks were monitored with pop-up satellite archival tags (PAT tag version 2.00, Wildlife Computers, Redmond, WA; Gunn and Block, 2001; Marcinek et al., 2001). The first shark was cap- tured on a longline set in the Gulf of Mexico at 26.5°N, 91.3°W on 12 April 1 Nakamura. I. 2002. Personal commun. Institut National des Sciences et Technolo- gies de la Mer. 28 rue 2 Mars 1934, 2025 Salammbo. Tunisia. 2 Carey. F. G. (deceased). 1990. Personal commun. Woods Hole Oceanographic Institution, Woods Hole, MA 02543. Manuscript approved for publication 15 August 2003 by Scientific Editor. Manuscript received 20 October 2003 at NMFS Scientific Publications Office. Fish. Bull. 102:221-229 (2004). 222 Fishery Bulletin 102(1) 2000 in waters with a surface temperature of 21.9°C. The longline set contained 184 hooks set at depths between 70 m and 90 m and was made at 06:00 h and retrieved at 09:00 h. Circle hooks (L2045 20/0 circle hook, Eagle Claw, Denver, CO) were used to avoid hooking of the gut, and the shark in this study was hooked in the corner of the jaw. Hooks were baited with squid, and chemical light sticks were attached to every other line. The mass of the shark was visually estimated at 170 kg by an experienced commercial longline fisherman, which corresponds to a fork length of 229 cm, and a total length of 377 cm, based on the weight-length relationship of Kohler et al. (1995). According to this size estimation and the published size-at- maturity data (Chen et al., 1997; Liu et al., 1998), the shark was mature. The sex of the shark was not determined. The second shark was captured by hook-and-line gear near Hawaii at 19.5°N, 156.0°W on 13 May 2003 in waters with a surface temperature of 25.5°C. A baited circle hook set at a depth of 40 m was taken by the shark at 02:00 h. The mass of the shark was estimated at 200 kg by an experienced sportfishing captain, which corresponds to a fork length of 242 cm, and a total length of 400 cm (after Kohler et al., 1995). Given this size, the shark was mature (Chen et al., 1997; Liu et al., 1998), but its sex was not determined. Each pop-up satellite archival tag was attached to a tita- nium dart (59 mm x 13 mm) with a 17 cm segment of 136- kg monofilament line ( 300-lb test extra-hard Hi-Catch, Mo- moi Fishing Net Mfg. Co. Ltd., Ako City, Hyogo prefecture, Japan). The dart was inserted into the dorsal musculature of the shark at the base of the first dorsal fin, such that the tag trailed behind the fin. Following attachment of each tag, the fishing line was cut near the hook and both sharks swam away vigorously. Tagging locations were recorded by using the vessel's global positioning system. After the Gulf of Mexico shark was tagged, a depth-temperature recorder (ABT-1, Alec Electronics, Kobe, Japan) was used to deter- mine the temperature-depth profile of the upper 200 m of the ocean at the release site, at a resolution of 1 m. The pop-up satellite archival tag deployed in the Gulf of Mexico was programmed to collect pressure and tem- perature data at two-minute intervals, which the on-board software (PAT software version 1.06, Wildlife Computers, Redmond, WA) summarized into six-hour bins. This version of PAT software did not permit light-based geolocation. The summary data for each time interval comprised percentage distributions of time-at-depth and time-at-temperature, and profiles of temperature-at-depth. Temperature-depth profiles for this generation of software were recorded at intervals by measuring a single temperature at depths of 0, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, and 400 me- ters for the deepest dive. A mean temperature-depth profile was obtained by calculating the mean temperature at each specified depth for all profiles taken during the track. The endpoint position of the shark's track was obtained from the tag's radio transmissions to the Argos satellites. The six-hour bins were later combined into 12-hour bins repre- senting day (06:00 to 17:59 h local time) and night ( 18:00 to 05:59 h local time). At the time and place of tag deployment, sunrise occurred at 05:45 h and sunset at 18:28 h; whereas at the popup time and position, sunrise occurred at 05:02 h and sunset at 18:55 h (U.S. Naval Observatory), such that the day and night bin cutoffs were always within one hour of true sunrise and sunset. The pop-up satellite archival tag deployed off Hawaii col- lected data at 30-second intervals and summarized them into four-hour bins (PAT software version 2.08e, Wildlife Computers, Redmond, WA). The data were later combined into day and night bins as for the first tag, and the actual sunrise and sunset times were within one hour of 06:00 h and 18:00 h, respectively (U.S. Naval Observatory). The tag measured the minimum and maximum temperature at the surface, maximum depth, and six intermediate depths, for the deepest dive in each time interval. Temperature-depth profiles for each time interval were later constructed by us- ing the maximum temperature at each depth for all profiles taken during the track, and a curve was fitted by using a LOWESS (locally weighted regression smoothing) function (Cleveland, 1992). Version 2.08e PAT software collected light data for geolocation; however the diel dive pattern of the shark prevented the calculation of accurate positions. The vascular circulation to the brain and eyes was exam- ined in two bigeye thresher sharks: one common thresher shark and one pelagic thresher shark iAlopias pelagicus). A female bigeye thresher (1.5 m fork length) was captured off Cape Hatteras, North Carolina, and a male (1.4 m fork length) was captured in the Gulf of Mexico. The circula- tory systems of the bigeye threshers were injected with latex to aid in identifying the blood vessels. A male com- mon thresher (1.3 m fork length) was captured off Cape Hatteras, North Carolina, and was examined without being frozen or preserved. An immature female pelagic thresher shark (1.37 m fork length) was captured in the Indian Ocean. The orbital retia mirabilia were prepared from casts of the vascular circulation that were removed from the orbit. Results One bigeye thresher shark was tracked in the Gulf of Mexico for 60 days, and another in the Hawaiian Archi- pelago for 27 days, by using pop-up satellite archival tags. Both tags released from the sharks as programmed and transmitted summary information to Argos satellites. The tag deployed in the Gulf of Mexico popped up on 10 June 2000 at 27.95°N, 89.54°W (Fig. 1A). The shark moved a straight-line distance of 320 km during the track, start- ing from the central Gulf in depths exceeding 3000 m and moving to waters 150 km south of the Mississippi Delta where depths were approximately 1000 m. The second shark was tagged off the Kona coast of Hawaii and the tag released on 9 June 2003 at 24.2°N, 165.6°W. northeast of French Frigate Shoals, a straight-line distance of 1125 km from the deployment position (Fig. IB). The depth and temperature distributions of the bigeye thresher sharks showed a strong diel movement pattern (Fig. 2). The Gulf of Mexico shark spent the majority of the daytime (84f* (±2.39H. mean [±1 SE]) below the ther- mocline between 300 m and 500 m and the majority of nighttime (809? [±4.7%], mean (±1 SE] ) in the mixed layer NOTE Weng and Block: Diel vertical migration in Alopias superaliosus 223 30° N 82°W 24°N 166 164 162 160 158 156°W Figure 1 Deployment (A) and end-point (•) positions for the two pop-up satellite archival tags attached to bigeye thresher sharks. Both tags surfaced on the programmed dates and transmitted data to Argos satellites. Pressure sensors in the tags confirmed that the tags remained attached to the sharks for the duration of the tracks. (A) In the Gulf of Mexico a shark was tagged and released on 12 April 2000 and the tag surfaced on 10 June 2000. The shark moved a straight-line distance of .320 km during the 60-day track. (B) In the Hawaiian Archipelago a shark was tagged on 13 May 2003 off Kona, Hawaii, and the tag surfaced on 9 June 2003 northeast of French Frigate Shoals. The shark moved a straight-line distance of 1125 km during the 27-day track. and upper thermocline between 10 m and 100 m (Fig. 2A). The shark spent most of the daytime in deeper waters of 6°C to 12°C (70% [±4.4%], mean [±1 SE]), and most of the nighttime in shallower waters from 20°C to 26°C (70% [±2.7%], mean [±1 SE]) (Fig. 2B). A temperature-depth profile taken by the tag during the first day of the shark's track closely matched a profile taken from the vessel with a bathythermograph (Fig. 3A). The mean temperature-depth profile for the 60-day track (Fig. 3B), when compared with the shark's depth preferences (Fig. 2A), indicated that 224 Fishery Bulletin 102(1) Percent time 00 75 50 25 0 25 50 75 100 0-5 5-10 10-50 50-100 ~ 100-150 g 150-200 200-250 250-300 300-500 500-700 700-1000 ,r Percent time 50 25 0 25 50 75 45 25 Percent time 5 25 28-30 26-28 24-26 22-24 20-22 18-20 16-18 14-16 10-14 10-12 6-10 <6 _i i . i i_ 45 j i Percent time 50 25 0 25 50 75 Figure 2 Depth and temperature distributions of two bigeye thresher sharks showing diel vertical migration. The tags recorded depth and temperature at two-minute (A, B> or 30-second (C, Di intervals; data are summarized into a series of bins for the full duration of each track. (Al Depth distribution for the Gulf of Mexico shark is shown as the percentage of day (□> and night ■ spent within depth bins ranging from the surface to 1000 m. Error bars are 1 SE. (B) Temperature distribution for the Gulf of Mexico shark is shown as the percentage of day (□) and night ■ spent within temperature bins ranging from 6°C to 30°C. The shark occupied cool waters during the day and warm waters during the night, a consequence of its deep daytime and shallow nighttime habitats. Error bars are 1 SE. (Cl Depth distribution for the Hawaii shark showing diel vertical migration. The shark spent most of the daytime at the base of the thermocline and must of the nighttime in the mixed layer and upper thermocline. iD) Temperature distribution for the Hawaii shark showing cool daytime and warm nighttime water temperatures. NOTE Weng and Block: Diel vertical migration in Alopias superaliosus 225 10 15 20 25 Temperature (C) 10 15 20 25 5 10 15 20 25 30 50 100-1 -jT 150 n Q_ q 200 -I 250 300 -I c 350 Figure 3 Temperature-depth profiles characterizing the thermal habitat of two bigeye thresher sharks. (Al Profiles of the Gulf of Mexico taken with a bathythermograph ( ) sampling at 1-m intervals deployed from the fishing vessel after the tagging event, and by the pop-up satellite archival tag (O) during the first day it was attached to the bigeye thresher shark. The two profiles are similar, indicating that the pop-up satellite archival tag is capable of characterizing thermal habitat. (B) Average temperature-depth profile for the 60-day track of the bigeye thresher shark in the Gulf of Mexico, showing a mixed layer shallower than 50 m and a thermocline extending beyond 400 m where waters were 10°C. The curve was fitted by using a LOWESS function and error bars are 1 SD, because 1 SE bars are invisible at this scale. (C) Average temperature-depth profile for the 27-day track of the bigeye thresher shark in the Hawaiian Archipelago, showing a shallow mixed layer a thermocline extending to approximately 600 m where waters were 6°C. Curve was fitted by using a LOWESS function and error bars are 1 SD, because 1 SE bars are invisible at this scale. the shark spent most of the daytime below the maximum gradient of the thermocline where temperatures were ap- proximately 10°C. On 25 April and 25 May 2000 the shark spent two hours of the day in waters between 4°C and 6°C. The Hawaii shark showed a similar diel vertical migration, with a lesser contrast between day and night (Fig. 2, C and D). The shark's modal nighttime depth was between 10 m and 50 m, whereas its modal daytime depth was between 400 m and 500 m (Fig. 2C). The temperature-depth profile for the Hawaii shark ( Fig. 3C ) indicated that it spent night- time above the thermocline and daytime below it. The bigeye thresher shark possesses a large arterial plexus between the posterior part of the eye and the wall of the orbital sinus, which appears to be a rete mirabile (Fig. 4). The orbital rete is bathed in venous blood from the orbital sinus and its anterior surface is contoured to the posterior surface of the eye. The sources of venous input to the orbital sinus remain unknown but are most likely within the surrounding extraocular muscles, which are large and comprise numerous aerobic muscle fiber types, and the retina. The rete shown in Figure 4 measures 72 mm by 49 mm by 19 mm. A reduced structure of similar form is also found in the pelagic thresher shark, but is not present in the common thresher. The orbital rete of the bigeye and pelagic threshers is larger in absolute size and occupies a greater cross sectional proportion of the orbital sinus than the lamnid orbital rete noted by Burne (1924). The arterial vessels form a finer and more orderly mesh- work than those in the lamnid sharks (Block and Carey, 1985; Tubbesing and Block, 2000) and appear similar in physical structure to the mammalian carotid rete used for brain cooling (Baker, 1982). Discussion Observations of the biological features of the bigeye thresher shark are rare and our knowledge of the species is based primarily on incidental catches in fisheries. Using pop-up satellite archival tags we were able to record behav- ior for a total of 87 days, and for individual periods up to 60 days without recapturing or following the study animals. We observed a pronounced diel alternation between warm shallow waters and cool deep waters and a rete mirabile that may confer physiological benefits during deep dives by stabilizing brain and eye temperatures. The depth data obtained for the bigeye thresher shark shows a striking pattern of diel vertical migration. The big- eye thresher shark's vertical movement pattern is distinct from those of most other sharks for which observations 226 Fishery Bulletin 102(1) Figure 4 Orbital rete of a bigeye thresher shark, showing the highly developed arterial network. The rete was injected with latex so that the arterial structure (72 mm by 49 mm by 19 mm) could be photographed. The structure of the rete and its position in the orbital sinus suggest that it may be a heat exchanging vascular plexus. Retention of metabolic heat in the eyes and brain would buffer these sensitive organs from the large ambient temperature swings that occur as a result of the bigeye thresher shark's diel vertical migrations. A smaller but similar structure is found in A. pelagicus but not in A. vulpinus. exist. In satellite or acoustic tracks, diel vertical migra- tion was not observed for white sharks (Carcharodon car- charias; Carey et al., 1982; Goldman and Anderson, 1999; Boustany et al., 2002), salmon sharks (Lamna ditropis; Block et al.3), shortfin mako (Isurits oxyrhynchus; Carey, 1982b; Holts and Bedford, 1993), blue (Prionace glauca, Carey, 1982b; Carey and Scharold, 1990), sixgill (Hexan- chus griseus; Carey and Clark, 1995), tiger (Galeocerdo cuvier; Tricas et al., 1981; Holland et al., 1999), Pacific angel (Squatina californica; Standora and Nelson, 1977), whale [Rhincodon typus; Gunn et al., 1999), or scalloped hammerhead sharks (Sphyrna lewini; Klimley, 1993). Diel vertical migration has been observed in the sword- fish (Xiphias gladius; Carey and Robison, 1981; Carey4), the megamouth shark (Megachasma pelagios; Nelson et al., 1997), and the school shark iGaleorhinus ga/eus; West Block, B.A., K.G.Goldman, and J. A. Musick. 1999. Unpubl. data. Hopkins Marine Station of Stanford University. 120 Oceanview Boulevard, Pacific Grove, CA 93950. Carey, R G. 1990. Further acoustic telemetry observations of swordfish. In Planning the future of billfishes; proceedings of the second international billfish symposium, 1-5 August 1988, Kailua-Kona, Hawaii (R. H. Stroud, ed.), p. 103-122. National Coalition for Marine Conservation, 3 North King St., Leesburg, VA 20176. and Stevens, 2001). Carey and Robison ( 1981) and Carey4 studied swordfish in both the Pacific and Atlantic Oceans, acoustically tracking fish that moved from the surface at night to over 600 m during day. A megamouth shark showed a strong diel vertical migration when tracked acoustically off southern California (Nelson et al., 1997) with shallow nighttime and deep daytime distribution in a vertical range of 20 m to 160 m. West and Stevens (2001) studied school sharks in southern Australia using archival tags and noted that they ascended in the water column at night. The ambient temperature at the modal day- and night- time depths of the two bigeye thresher sharks differed by 15° to 16°C, requiring them to be eurythermal. The sharks spent most of the nighttime in shallow waters warmer than 20°C and commonly spent 8 or more hours during the daytime in deep waters cooler than 10°C. The coolest waters occupied had temperatures between 4°C and 6°C. The bigeye thresher sharks tracked in our study spent a higher proportion of their time in waters below 10°C than did white sharks (Carey et al., 1982; Boustany et al., 2002) and mako sharks (Carey and Scharold, 1990; Klimley et al.,2002). The presence of a rete mirabile in the cranial region may indicate a mechanism for heat conservation. Heat conservation in the brain and eyes would enable the big- NOTE Weng and Block: Diel vertical migration in Alopias superaliosus 227 eye thresher shark to prolong its foraging time beneath the thermocline, as we observed for both of the sharks tagged in our study. The retina and brain are extremely temperature sensitive in most vertebrates and the large changes in depth and temperature recorded would impose significant effects on the biochemical processes occurring in these tissues (Block and Carey, 1985; Block, 1994). Delayed responses to retinal stimulation can be caused by cooling, whereas increased noise and random firing of neurons can be caused by warming — both responses having adverse affects on sensory function (Konishi and Hickman, 1964; Friedlander et al., 1976; Prosser and Nelson, 1981). Anatomical and physiological adaptations to warm the brain and eyes have evolved independently in divergent pelagic fish lineages, including the lamnid sharks (Block and Carey, 1985), billfishes of the Xiphiidae and Istiophori- dae (Carey, 1982a; Block. 1983) and some scombrid fishes (Linthicum and Carey, 1972). A cranial rete mirabile also has been identified in mobulids (Schweitzer and Notarbar- tolo di Sciara, 1986) and is thought to be a heat exchanger (Alexander, 1995, 1996). Although it is premature to sug- gest that the orbital rete of the bigeye thresher shark is a heat exchanger without direct evidence of elevated tissue temperatures in the brain and eyes, the structure is larger than the rete mirabile of lamnid sharks, for which elevat- ed brain and eye temperatures have been demonstrated (Block and Carey, 1985). The anatomical arrangement of an arterial plexus in an orbital sinus is correlated with heat conservation strategies in other vertebrates (Baker, 1982). The phylogenetic relationships of the alopiid and lamnid sharks (Compagno, 1990; Naylor et al., 1997) sug- gest that endothermic traits evolved independently in the two families. This note presents new information on the depth and ambient temperature preferences of the bigeye thresher shark based on observations of two individuals, as well as the anatomy of the orbital rete mirabile, which appears to function as a vascular heat exchanger. Behavior of many organisms varies with ontogeny, season and location; therefore the present study should be considered as only the beginning of an understanding of the bigeye thresher shark's physical habitat preferences and adaptations to temperature change. Further studies on individuals of different sizes and in different regions will enhance our understanding of the behavior, and morphological and physiological adaptations, of the bigeye thresher shark to variations in temperature. Acknowledgments This research was supported by grants from the National Marine Fisheries Service, the National Fish and Wildlife Federation and the Packard Foundation. The authors wish to thank Captain David Price and crew of the FV Allison, and Captain John Bagwell and crew of the FY Silky. Shana Beemer provided scientific assistance on the cruise and Captain McGrew Rice assisted in tagging and releasing the Gulf of Mexico shark. This research was conducted under Scientific Research Permit TUNA-SRP-2000-002, issued by the Office of Sustainable Fisheries, National Marine Fisheries Service, Silver Spring, MD 20910. Literature cited Alexander, R. L. 1995. Evidence of counter-current heat exchanger in the ray, Mobu la tarapacana (Chondrichthyes: Elasmobranchii: Batoidea: Myliobatiformes). J. Zool. (Lond) 237:377-384. 1996. Evidence of brain-warming in the mobulid rays, Mobula tarapacana and Manta birostris (Chondrichthyes: Elasmobranchii: Batoidea: Myliobatiformes). Zool. J. Linn. 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W. R. Strong Jr., C. G. Lowe, J. A. Sisneros, D. M. Schroeder. and R. J. Lavenberg. 1997. An acoustic tracking of a megamouth shark, Mega- chasma pelagios: A crepuscular vertical migrator. Envi- ron Biol. Fishes 49:389-399. NOTE Weng and Block: Diel vertical migration in Alopias superahosus 229 Prosser, C. L., and D. O. Nelson. 1981. Role of nervous systems in temperature adaptation of poikilotherms. Annu. Rev. Physiol. 43:281-300. Schweitzer. J., and G. Notarbartolo di Sciara. 1986. The rete nurabile cranica in the genus Mobula: a com- parative study. J. Morphol. 188:167-178. Springer, S. 1943. A second species of thresher shark from Florida. Copeia 1943:54-55. Standora, E. A., and D. R. Nelson. 1977. A telemetric study of the behavior of free swimming Pacific angel sharks Squatina californica. Bull. South. Calif. Acad. Sci. 76:193-201. Stillwell, C. E., and J. G. Casey. 1976. Observations on the bigeye thresher shark, Alopias superciliosus, in the western North Atlantic. Fish. Bull. 74:221-225. Thorpe, T. 1997. First occurrence and new length record for the bigeye thresher shark in the northeast Atlantic. J. Fish Biol. 50: 222-224. Tricas, T. C. L. R. Taylor, and G. Naftel. 1981. Diel behavior of the tiger shark Galeocerdo cuvier at French Frigate Shoals Hawaiian Islands USA. Copeia 1981:904-908. Tubbesing, V. A., and B. A. Block. 2000. Orbital rete and red muscle vein anatomy indicate a high degree of endothermy in the brain and eye of the salmon shark. Acta Zool. (Stockh.) 81:49-56. West, G. J., and J. D. Stevens. 2001. Archival tagging of school shark, Galeorhinus galeus. in Australia: Initial results. Environ. Biol. Fishes 60: 283-298. Fishery Bulletin 102(1) 231 Superintendent of Documents Publications Order Form *5178 I I YrLo, please send me the following publicatic tions: Subscriptions to Fishery Bulletin for $55.00 per year ($68.75 foreign) The total cost of my order is $ . 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It is also available limited numbers to libraries, research institutions, Stale and Federal agencies, and in exchange for other scientific publicat ii U.S. Department of Commerce Seattle, Washington Volume 102 Number 2 April 2004 Fishery Bulletin Contents The conclusions and opinions expressed in Fishery Bulletin are solely those of the authors and do not represent the official position of the National Marine Fisher- ies Service (NOAAi or any other agency or institution. The National Marine Fisheries Service iNMFS) does not approve, recommend, or endorse any proprietary product or pro- prietary material mentioned in this pub- lication. No reference shall be made to NMFS, or to this publication furnished by NMFS. in any advertising or sales pro- motion which would indicate or imply that NMFS approves, recommends, or endorses any proprietary product or pro- prietary material mentioned herein, or which has as its purpose an intent to cause directly or indirectly the advertised product to be used or purchased because of this NMFS publication. Articles 233-244 Archer, Frederick, Tim Gerrodette, Susan Chivers, and Alan Jackson Annual estimates of the unobserved incidental kill of pantropical spotted dolphin (Stenella attenuata attenuata) calves in the tuna purse-seme fishery of the eastern tropical Pacific 245-250 Chernova, Natalia V., and David L. Stein A remarkable new species of Psednos (Teleostei: Liparidae) from the western North Atlantic Ocean 251-263 Chiang, Wei-Chuan, Chi-Lu Sun, Su-Zan Yeh, and Wei-Cheng Su Age and growth of sailfish Ustiophorus platypterus) in waters off eastern Taiwan 264-277 Clark, Randall D., John D. Christensen, and Mark E. Monaco, Philip A. Caldwell, Geoffrey A. Matthews, and Thomas J. Minello A habitat-use model to determine essential fish habitat for juvenile brown shrimp (Farfantepenaeus aztecus) in Galveston Bay, Texas 278-288 Delgado, Gabriel A., Claudine T. Bartels, Robert A. Glazer, Nancy J. Brown-Peterson, and Kevin J. McCarthy Translocation as a strategy to rehabilitate the queen conch (Strombus gigas) population in the Florida Keys 289-297 Lage, Christopher, Kristen Kuhn, and Irv Kornfield Genetic differentiation among Atlantic cod (Gadus morhua) from Browns Bank, Georges Bank, and Nantucket Shoals 298-305 Lenihan, Hunter S., and Charles H. Peterson Conserving oyster reef habitat by switching from dredging and tonging to diver-harvesting Fishery Bulletin 102(2) 306-327 Macewicz, Beverly J., John R. Hunter, Nancy C. H. Lo, and Erin L. LaCasella Fecundity, egg deposition, and mortality of market squid (Loli/go opalescens) 328-348 Orr, James W., and James E. Blackburn The dusky rockfishes (Teleostei: Socrpaeniformes) of the North Pacific Ocean resurrection of Sebastes variabilis (Pallas, 1814) and a redescnption of Sebastes ci/iatus (Tilesius, 1813) 349-365 Powers, Joseph E. Recruitment as an evolving random process of aggregation and mortality 366-375 Szedlmayer, Stephen T., and Jason D. Lee Diet shifts of juvenile red snapper (Lut/anus campechanus) with changes in habitat and fish size 376-388 Webb, Stacey, and Ronald T. Kneib Individual growth rates and movement of juvenile white shrimp (Litopenaeus setiferus) in a tidal marsh nursery Notes 389-392 Forsythe, John, Nuutti Kangas, and Roger T. Hanlon Does the California market squid (Loligo opalescens) spawn naturally during the day or at night? A note on the successful use of ROVs to obtain basic fisheries biology data 393-399 Kotas, Jorge E., Silvio dos Santos, Venancio G. de Azevedo, Berenice M. G. Gallo, and Paulo C. R. Barata Incidental capture of loggerhead (Caretta caretta) and leatherback (Dermochelys conacea) sea turtles by the pelagic longline fishery off southern Brazil 400-405 Yang, Mei-Sun Diet changes of Pacific cod (Gadus macrocephalus) in Pavlof Bay associated with climate changes in the Gulf of Alaska between 1980 and 1995 406 Subscription form 233 Abstract— We estimated the total number of pantropical spotted dolphin (Stenella attenuata) mothers killed without their calves ("calf deficit") in all tuna purse-seine sets from 1973-90 and 1996-2000 in the eastern tropical Pacific. Estimates were based on a tally of the mothers killed as reported by color pattern and gender, several color-pattern-based frequency tables, and a weaning model. Over the time series, there was a decrease in the calf deficit from approximately 2800 for the western-southern stock and 5000 in the northeastern stock to about 60 missing calves per year. The mean deficit per set decreased from approxi- mately 1.5 missing calves per set in the mid-1970s to 0.01 per set in the late-1990s. Over the time series exam- ined, from 75% to 95% of the lactating females killed were killed without a calf. Under the assumption that these orphaned calves did not survive with- out their mothers, this calf deficit rep- resents an approximately 14% increase in the reported kill of calves, which is relatively constant across the years examined. Because the calf deficit as we have defined it is based on the kill of mothers, the total number of mis- sing calves that we estimate is poten- tially an underestimate of the actual number killed. Further research on the mechanism by which separation of mother and calf occurs is required to obtain better estimates of the unob- served kill of dolphin calves in this fishery. Annual estimates of the unobserved incidental kill of pantropical spotted dolphin {Stenella attenuata attenuato) calves in the tuna purse-seine fishery of the eastern tropical Pacific Frederick Archer Tim Gerrodette Susan Chivers Alan Jackson Southwest Fisheries Science Center National Marine Fisheries Service 8604 La Jolla Shores Dr. La Jolla, California 92037 E-mail address (for F Archer): enc.archeriainoaa.gov Manuscript approved for publication 7 January 2004 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Sceintific Publications Office. Fish. Bull. 102:233-244 (2004). In the eastern tropical Pacific (ETP), yellowfin tuna (Thunnus albacares) are frequently found swimming under schools of pantropical spotted ( Stenella attenuata) and spinner (S. longirostris) dolphins. For the past four decades, the ETP yellowfin tuna fishery has made use of this association by chasing the more visible dolphins at the sur- face and using purse-seines to encircle the schools "carrying" the tuna (NRC, 1992). The large bycatch of dolphins in this fishery has become widely known as the "tuna-dolphin issue" (Gerro- dette, 2002). During the 1960s, the number of dolphins killed by the fishery was estimated to be 200,000-500,000 per year (Wade, 1995), and two stocks of spotted and spinner dolphins were reduced to fractions of their previous sizes (Smith, 1983; Wade et al.1). Along history of technological innovations by fishermen, laws and fishing regula- tions, dolphin quotas, eco-labeling of "dolphin-safe" tuna, and a comprehen- sive international observer program (Gosliner, 1999; Hall et al, 2000; Ger- rodette, 2002) has reduced the dolphin bycatch to less than 1% of its former level. The reported bycatch in recent years is less than 2000 dolphins per year for all species combined (IATTC, 2002). Although the reported kill has dra- matically decreased, recent studies suggest that there is little evidence that the stocks are growing close to expected rates (Wade et al.1). One hy- pothesis for this lack of recovery has been that there are unobserved kills of dolphins during tuna purse-seine sets. Archer et al. (2001) presented evidence of an under-representation of suckling spotted and spinner dolphin calves in a sample of tuna purse-seine sets in the eastern tropical Pacific. Given that some of these missing calves are still dependent on their mothers for nutri- tion, it is likely that once separated they would die and this under-repre- sentation represents some degree of unobserved kill. In Archer et al. (2001), the sample of sets examined was limited to those sets in which all of the animals killed had biological data collected by techni- cians aboard the tuna vessel. Calves still dependent on their mothers in the kill were identified by five intervals of body length, chosen to cover a range of 1 Wade, P. R.. S. B. Reilly. and T. Gerro- dette. 2002. Assessment of the popula- tion dynamics of the northeastern offshore spotted and the eastern spinner dolphin populations through 2002. National Oceanographic and Atmospheric Admin- istration Administrative Report LJ-02- 13. 58 p. Southwest Fisheries Science Center. 8604 La Jolla Shores Dr., La Jolla, CA 92037. 234 Fishery Bulletin 102(2) calf sizes. Because of this approach, it was not possible to derive a single estimate of the number of missing calves or to extrapolate their estimate to sets not used in this analysis. In the current study, we present a different method of estimating the number of missing calves in each set where offshore spotted dolphins (S. attenuate! attenuata) were killed. For brevity, we call the shortage of calves in the kill in relation to the number of lactating females in the kill the "calf deficit." We examined the western-southern and northeastern offshore stocks separately according to the geographic boundaries described by Dizon et al. (1994). As they age, spotted dolphins change color through five color phases (Perrin, 1970). We used the color-phase frequency distribution of the kill in conjunction with age- and color-based frequency distributions from a sample of the kill to estimate the total number of missing calves in each stock, along with confidence intervals derived from bootstrap replications. This method also allowed us to examine the calf deficit from sets in recent years from which we did not have biological samples and to examine the time series of available years for evidence of a trend in the calf deficit. Methods Since 1973, observers have been randomly placed on tuna purse-seine vessels. For each spotted dolphin killed during an observed set, observers attempted to record the sex and the color phase of the dolphin ( neonate, two-tone, speckled, mottled, and fused, see Perrin, 1970). From the National Marine Fisheries Service (NMFS) set log database, we obtained the number of northeastern and western-south- ern offshore spotted dolphins (by gender and color phase) killed in every observed set from 1973 to 1990. The Inter- American Tropical Tuna Commission (IATTC) provided the same data from 1996 to 2000. Proration In each set, color phase or gender (or both) may not have been recorded for some dolphins. Assuming that the distri- bution of the demographic composition of this missing data is equivalent to the overall demographic composition of the kill, we allocated the number of dolphins cf unknown color phase (nu) to unknown gender in each color phase (jigu) according to the following formula, ngu: = ngu, + N. I", (1) where c = one of the five color phases (neonate to fused I; Nc = the total number of dolphins in each color phase in the entire data set; and ngu\. = the new number of dolphins in each color phase where gender is unknown, including the indi- viduals of prorated unknown color phase The number of male (nm'c) or female (nf'c) dolphins in a color phase was calculated as nm,. = nm + ngu, ■ Nm. Nni + Nf\ j nfc'=nft + ngu w Nmc+Nfr (2) (3) where Nmc and Nfc are the total number of males and females, respectively, observed in that color phase in the entire data. Table 1 gives the sample size of sets for both stocks by year, as well as the fraction of the kill of unknown gender and color phase that were prorated as described above. Number of suckling calves As time permitted, NMFS observers would also collect biological data from a subset of the kill. For this study, we used ages estimated from teeth collected for a study of spotted dolphin growth and reproduction (Myrick et al., 1986 ). The specimens used were a random sample of all male and female spotted dolphins collected between 1973 and 1978 for which total body length was recorded and teeth were collected. However, additional specimens with lengths less than 150 cm were selected in order to match as closely as possible the length distribution of the aged sample to the underlying length distribution of the spotted dolphins in the kill. This was necessary because observ- ers did not generally collect teeth from smaller, younger animals. Later, another sample of female spotted dolphins was selected from specimens collected in 1981. Specimens were aged as described in Myrick et al. ( 1986 ). The final data set used in our analyses included age estimates for 1094 female spotted dolphin specimens and 798 male specimens. Of these, 649 females and 457 males belonged to the northeastern stock and had color phase re- corded. These 1 106 dolphins were used to generate the age frequency distribution for each color phase (F , Table 2). (4) '""Is,, where Sac = the number of samples of age a in color phase c. The oldest age recorded was 36 years. To derive an age distribution for the dolphins killed in each tuna set, we estimated the number of dolphins in each age class (na) as »,,=x^„ i 5) where n' the sum of nm'c and «/' (the number of males and females in each color phase after prora- tion from Equations 2 and 3). Archer et al.: Estimates of the incidental kill of Stenella attenuata attenuata calves in the tuna purse-seine fishery 235 Table 1 Sample sizes of NMFS (1973-1990) and IATTC (1996-2000) observed sets with spotted dolphin kill made on two stocks of pan- tropical spotted dolphins iStenella a ttenuata) by yea r. Northeastern stock Western-southern stock Fraction of Fraction of Fraction of Fraction of kill of kill of kill of kill of Number of Observed unknown unknown Number of Observed unknown unknown Year sets with kill kill color phase gender sets with kill kill color phase gender 1973 332 5242 0.09 0.31 75 1199 0.17 0.34 1974 515 5864 0.16 0.23 92 1715 0.10 0.31 1975 554 8073 0.31 0.19 75 1702 0.30 0.20 1976 239 2376 0.24 0.25 356 6293 0.27 0.23 1977 467 2146 0.23 0.26 528 3358 0.18 0.32 1978 224 1016 0.18 0.41 329 3998 0.37 0.34 1979 218 1045 0.38 0.27 168 1262 0.40 0.14 1980 165 1132 0.45 0.28 106 1206 0.73 0.13 1981 121 815 0.46 0.13 112 1346 0.48 0.12 1982 171 1696 0.51 0.22 159 1966 0.37 0.38 1983 12 177 0.80 0.08 35 148 0.32 0.35 1984 43 294 0.37 0.25 71 961 0.48 0.15 1985 186 2625 0.39 0.40 54 381 0.49 0.13 1986 150 1816 0.48 0.28 132 1818 0.60 0.22 1987 630 3327 0.25 0.31 175 1768 0.62 0.14 1988 207 1142 0.18 0.27 107 479 0.36 0.34 1989 293 1096 0.29 0.25 323 2793 0.48 0.14 1990 157 515 0.16 0.31 121 829 0.35 0.13 1996 273 724 0.27 0.44 161 374 0.18 0.54 1997 163 393 0.15 0.42 274 738 0.24 0.48 1998 161 260 0.21 0.51 125 236 0.19 0.46 1999 189 317 0.18 0.58 88 159 0.11 0.56 2000 146 291 0.23 0.47 115 250 0.20 0.61 In Equation 4, an age distribution was generated for each color phase, and then the number of dolphins in each age class was summed across all color phases. To estimate the number of calves in each set, we used this age distribution in conjunction with a weaning model developed from a study of the stomach contents and ages of calves (Archer and Robertson, in press). The model predicts the probability that an animal of a given age (a) will be suckling: Pi milk) (6) 1 + e1 The estimated number of calves (JV„„;f) in a set is then N. calf IK calf Pmilk). (7) In our estimate of Ncal? we chose to use only the first four age classes (0 to 3) because P(milk)4 was extremely small (2xl0~4). These age classes allowed us to decrease computational time without significantly affecting the estimates. Number of lactating females Observers visually examined the mamillaries of the 649 females used in the age distribution above (Eq. 4) for the presence of milk as part of the suite of biological data collected. Using these data in conjunction with the color phase of these females, we calculated the fraction of lactat- ing females in each color phase (Flacv), Flac ■S/ac Sfem, (8) where Slacv and Sfemc the number of females that were lactating and the total number of females in color phase c of the samples examined. Flacc was 0.00, 0.01, 0.04, 0.22, and 0.50 for neonate, two- tone, speckled, mottled, and fused specimens, respectively. The estimated number of lactating females (Nlac) in a set was then Nlai. = ^(nf; Flacv (9) 236 Fishery Bulletin 102(2) Calf deficit As described in Archer et al. (2001), the calf deficit (D) in each set was calculated by subtracting the number of calves (iVca;J from the number of lactating females (N[ac). If this value was zero or less, then D was set to zero to indicate that there were enough calves to account for all lactating females killed ( Fig. 1 1, D- 0 if7V„„ EstLacKill (11) (12) (13) where ZD = the total observed calf deficit in each year; ObsSets = the number of observed sets used in the analysis, including those sets without a dolphin kill; ObsKill = the number of dolphins killed in the observed sets; and EstLacKill = the total estimated number of lactating females killed. The above analysis was conducted each year. Estima- tion error was evaluated with 20,000 bootstrap replicates for each year. For each replicate, the sets within that year were randomly resampled. The frequency tables Fni. and Flact were also recalculated by resampling the list of bio- logical specimens. The parameters for the weaning model, P(milk)a, were estimated again by resampling the 29 calves and by fitting the logistic model to the new data set as described in Archer and Robertson (in press). All resa- mpling was done with replacement. Nralp Nlac, and D were estimated as described above for each set, and Ds, Dh, and Dj were calculated for the replicate. The 95°; confidence intervals for Af n//, Nlac, D, Ds, Dk, and D/ were estimated from the 2.5'; and 97.5% quantiles of the distributions of the bootstrap replicate values. The total calf deficit (Dtotal) was estimated as the deficit per dolphin killed (Dk) multiplied by the total number of dolphins killed [NkiUed) by stock each year, Table 2 Age- class frequency distribution for e ich color phase CFac). Age Two- (yr) Neonate tone Speckled Mottled Fused 0 0.80 0.12 0 0 0 1 0.20 0.32 0 0 0 2 0 0.31 0.04 0 0 3 0 0.16 0.18 0.01 0 4 0 0.05 0.14 0.02 0 5 0 0.02 0.13 0.03 0 6 0 0 0.13 0.04 0.01 7 0 0 0.06 0.05 0 8 0 0 0.10 0.06 0 9 0 0 0.06 0.07 0.01 10 0 0 0.01 0.10 0.01 11 0 0 0.01 0.14 0.03 12 0 0 0.01 0.08 0.02 13 0 0 0.04 0.07 0.03 14 0 0 0.03 0.07 0.03 15 0 0 0 0.06 0.06 16 0 0.01 0.01 0.06 0.07 17 0 0 0.01 0.03 0.07 18 0 0 0 0.01 0.07 19 0 0 0 0.03 0.09 20 0 0 0 0.03 0.07 21 0 0 0 0.01 0.08 22 0 0 0 0 0.06 23 0 0 0.01 0 0.07 24 0 0 0 0.01 0.04 25 0 0 0 0.01 0.04 26 0 0 0 0 0.04 27 0 0 0 0.01 0.03 28 0 0 0 0.01 0.02 29 0 0 0 0 0.01 30 0 0 0 0.01 0.02 31 0 0 0 0 0.01 32 0 0 0 0 0 33 0 0 0 0 0.01 34 0 0 0 0 0 35 0 0 0 0 0 36 0 0 0 0 0.01 For the period 1973-84, annual values of Nhlllcil for each stock were provided by the IATTC (Joseph2). For 1984-90 and 1996-2000. values were published by IATTC (2002). In the bootstrap estimation of the 959? CI around Dlntal, for the 1973-90 period, each replicate was randomly sampled from a normal distribution by using the estimated total kill standard error. For 1996-2000, the total kill was reported to be exact; therefore the total kill was used without variance in all replicates. D total Dl; * Nkmd (1 li - Joseph. J. 1994. Letter of September 6 to Michael Tillman. 2 p. Southwest Fisheries Science Center. 8604 La Jolla Shores Dr., LaJolla. CA 92037. Archer et al.: Estimates of the incidental kill of Stene/la attenuata attenuate/ calves in the tuna purse-seine fishery 237 Fraction of females lactating, by color (1973-78, 1981): Flac Number of lactating females killed: Nh„ Tally of females, by color: nj ' Tally of dolphins killed, by color and sex, from set log ( 1973-90. 1996-2000) Tally of dolphins, by color: n\ Fraction of dolphins in age class, by color (1973-78, 1981): Fat Number of dolphins killed, by age: Na Probability of suckling, by age (1989-91): P(milk)a Calf deficit: D Number of suckling calves killed: Ncay Figure 1 Diagram of the analytical method used to estimate the spotted dolphin (Stenella attenuata attenuata) calf deficit in each set as described in the text. Boxes identify original data that were bootstrapped to produce confidence intervals. Values in parentheses are years for which data were available. In a subset of the sets that we examined, every indi- vidual killed had been examined and biological samples had been collected from it; therefore, we knew the actual number of lactating females killed. There were 1108 of these "100% sampled" sets on the northeastern stock, and 697 on the western-southern stock from 1973 to 1990. We evaluated the accuracy of our frequency-based method by conducting a paired /-test between our estimate of the number of lactating females and the number observed in each of these sets. Stomach-content data were not available for every animal in these 100% -sampled sets; therefore, we did not know the actual number of suckling calves. However, we also used paired /-tests to compare our estimate of the number of suckling calves in each set with the number of animals smaller than 122 cm, which was the estimated length at which the probability of milk in the stomach was 0.5, given the weaning model of Archer and Robertson (in press). Likewise, our estimate of the calf deficit was compared with the deficit as estimated by using a cutoff length of 122 cm. These tests were done to determine if the method in the present study would produce significantly different results from the method used in the previous study Paired /-tests were conducted for each year sepa- rately, as well as for all years combined. A power analysis was also performed for these paired /-tests to determine the minimum detectable difference at which we could re- ject the null hypothesis of no difference between methods given observed sample sizes and variability. Results The calf deficit as a fraction of the number of dolphins killed (Dk) increased slightly during the mid-1970s but remained relatively constant throughout the rest of the time series at approximately 0.14 missing calves per dol- phin killed for both stocks (Fig. 2). The total calf deficit (Dtotal) as estimated from the annual kill decreased from highs of approximately 5000 in the mid-1970s down to 2000-3000 by the early 1980s (Fig. 3). In the late 1980s, this value increased to approximately 5000 in northeast- ern spotted dolphins (Table 3A) and approximately 2800 in the western-southern stock (Table 3B), reflecting an increase in the reported kills. In the last five years of the time series (1996-2000), the estimated total deficit was approximately 60 missing calves. The mean deficit per set (Z),) for northeastern spotters over all years was 1.03 missing calves per set, and the me- dian was 0.30 (Fig. 4). For western-southern spotted dol- phins, the mean was 1.28 missing calves per set, and the median was 0.33. The estimated mean deficit per set was approximately 1.5 in the mid-1970s and decreased over time to 0.01-0.02 at the end of the time series (Fig. 4). For both stocks, 75- 95% of lactating females killed were not killed with their calf (Fig. 5). In the sets that were 100% -sampled, for all years com- bined, there was no significant difference between the observed and the estimated number of lactating females killed in either stock (Table 4). The results of paired /-tests 238 Fishery Bulletin 102(2) 0.3- Northeastern 0.2- •:.[•• " "»"..| 1" 0.1 - \\- |t " "" f ^T ll' o.o- 0.3- Western-southern 0.2- 1 1 I " }ll|ttftf ll . i 0.1 - o.o- — 1 1 1 1— 1970 1980 1990 2000 Year Figure 2 Calf deficit per spotted dolphin (Stenella attenuata attenuata) killed (D/; ) by year. Vertical lines indicate 95% confidence intervals. 12000 8000 -- 4000 0 = 12000 ■■ 8000 4000 "- +■ Northeastern ••At Western-southern »t Air * -+- -i- 1970 1980 1990 Year 2000 Figure 3 Total estimated calf deficit ^Dlotal) by year. Vertical indicate 95% confidence intervals. by year indicated that the observed number of lactating females in each set was significantly greater (P<0.05) than the estimated number in 1977 for the northeastern and the western-southern stocks and in 1979 for the west- ern-southern stock. The difference was significantly less in 1984 for the western-southern stock. Using 0.1 as our type-2 error level, we determined through power analysis that the minimum detectable difference («=0.05) between the mean observed and estimated number of lactating females per set across all years was approximately 0.08 and 0.09 in the northeastern and western-southern stocks respectively. The observed number of calves per set, defined as the number of dolphins less than 122 cm, was significantly greater for both stocks, for all years combined, than the values estimated in this paper ( Table 5 ). The overall mean difference was 0.17 calves per set for the northeastern stock and 0.12 for the western-southern stock. About half of the years showed a significant difference for each stock. In the comparison of the calf deficit by year, only a few years showed significant differences in either stock (Table 5). However, the estimated deficit tended to be larger than the observed deficit. The paired t-test for all years combined was significant for the northeastern stock, although the mean difference was only -0.06 missing calves per set. The minimum detectable difference from the power analysis for the mean number of calves per set and mean calf deficit per set across all years was 0.06 and 0.08 respectively for both stocks. Discussion In the present study, we present an estimate of the number of missing dependent northeastern and western-southern offshore spotted dolphin calves in the tuna purse-seine kill from 1973 to 1990 and from 1996 to 2000. The total number of missing calves decreased through the time series, which, because we estimated the calf deficit as a function of the size of the kill, was a direct result of the large reduction in the annual dolphin kill by the fishery. Between 1973 and 2000, the shortage of calves in the kill remained at a relatively constant fraction of the kill, about 14ri , for both stocks of pantropical spotted dolphins (Fig. 2). On the assumption that suckling calves do not survive separation from their mother ( Archer et al., 2001; Archer et al.: Estimates of the incidental kill of Stenella attenuata attenuate calves in the tuna purse-seine fishery 239 3.0 2.5 2.0 1.5 1.0 2° 0.5 CD if) I 0.0 I 3.0 a 1 2.5 c a | 2.0 f 1.5 1.0 0.5" 0.0" -+- Northeastern •(.tt It • * Western-southern 1 1 ■+- -H -t- 1970 1980 1990 Year 2000 Figure 4 Mean calf deficit per set (Ds) by year. Vertical lines indicate 959? confidence intervals. 1.0" 1 0.9" ■ ,, .1 ",, " ii ii ti ' ■ 0.8" '< " 0.7- Q § 0.6" 5 Northeastern CD ;? 0.5- CD "5. 10- c J? 0.9 - i ■ 1 ' ,, Deficit per o co , ". ti ,, 0.7 1 0.6" Western-southern 0.5- 970 1980 1990 2000 Year Figure 5 Calf deficit per lactating female killed (D,) by year. Vertical lines indicate 95^ confidence intervals. Edwards3!, the estimated calf deficit represents an approx- imately 147c underestimate of the reported kill. The calf deficit in the present study was estimated from the number of dependent calves and lactating females killed by using age-color frequency tables and data on the stomach contents of weaning calves. Specimens used to derive the age and color table were collected from 1973 to 1978 and 1981, and specimens used for the weaning model were collected between 1989 and 1991. If the distributions of these samples were not representative of all years that we examined, then our results may be biased. However, the results of a study to construct the annual age distribution of the kill (Archer and Chivers4 ) indicated that there is no sig- nificant difference in the age-color frequency table across years. The sample size for the stomach data ( 29 calves) was too small to examine differences between years. Our finding of no significant difference between our esti- mates of the number of lactating females and the observed tally of lactating females in sets where the entire kill was sampled validates this portion of our estimation proce- dure. However, because the number of suckling calves present in these 100% -sampled sets was not recorded, we were unable to validate the method used to generate these estimates in a similar manner. The results of our paired Ntests indicated that the ob- served number of animals smaller than 122 cm tended to be greater than the number we estimated. This is most likely a result of the difference between how calves were counted in each method. Archer et al. ( 2001 ) considered all animals under a series of cutoff values to be calves that were dependent on suckling for survival. In the present study, the weaning model that we used (Archer and Rob- 3 Edwards, E. F. 2002. Behavioral contributions to separa- tion and subsequent mortality of dolphin calves chased by tuna purse-seiners in the eastern tropical Pacific Ocean. National Oceanographic and Atmospheric Administration Administra- tive Report LJ-02-28, 34 p. Southwest Fisheries Science Center, 8604 La Jolla Shores Dr., La Jolla, CA 92037. 4 Archer. F.. and S. J. Chivers. 2002. Age structure of the northeastern spotted dolphin incidental kill by year for 1971 to 1990 and 1996 to 2000. National Oceanographic and Atmo- spheric Administration Administrative Report LJ-02-12, 18 p. Southwest Fisheries Science Center, 8604 La Jolla Shores Dr., La Jolla. CA 92037. 240 Fishery Bulletin 102(2) Table 3 Estimated calf deficit per kill (Dt) and total calf deficit Total number of spotted dolphins killed reported by the I ATTC ( 2002 ) and Joseph (footnote 2 in the general text). Values in parentheses are 95% lower and upper confidence intervals. Mean calf Total number Estimated calf deficit of NE spotted Estimated Stock and deficit in Observed per kill dolphins killed total calf year observed sets dolphin kill (Dk) (±SE) deficit A Northeastern (NE) stock 1973 599 5242 0.11 49928 ±8899 5709 (464,964) (3947,6820) (0.10,0.16) (3972,9532) 1974 634 5864 0.11 37410 ±4222 4046 (583,10271 (4943,6916) (0.10,0.16) (3573,6708) 1975 1014 8073 0.13 49399 ±8809 6206 1618,12691 (6578,9965) (0.08,0.14) (3297.8254) 1976 300 2376 0.13 20443 ±4721 2583 (196,408) (1786.3079) (0.09,0.15) (1284.3903) 1977 341 2146 0.16 5937 ±690 943 (249,416) (1743.26221 (0.13,0.18) (656,1167) 1978 148 1016 0.15 4226 ±827 616 (83,209) (684,1431) (0.11,0.16) (336,8361 1979 138 1045 0.13 4828 ±817 640 (96.226) (680,1629) (0.11,0.17) (428,963) 1980 178 1132 0.16 6468 ±962 1016 (107,239) (724.1637) (0.12,0.18) (622,13001 1981 137 815 0.17 8096 ±1508 1366 (84,173) (560,1122) (0.12,0.18) (753,1774) 1982 212 1696 0.12 9254 ±1529 1155 (155,347) (1126,23951 (0.11,0.17) (833,1840i 1983 27 177 0.15 2460 ±659 377 (7,59) (35,410) (0.11,0.23) (169.678) 1984 38 294 0.13 7836 ±1493 1017 (26,57) (191,417) (0.10,0.17) (608,1602) 1985 337 2625 0.13 25975 ±3210 3338 (235,508) (1839.3529) (0.11,0.16) (2447,4748) 1986 290 1816 0.16 52035 ±8134 8297 H19.478) (859.3440) (0.10,0.17) 14496,9935) 1987 497 3327 0.15 35366 ±4272 5280 (397,667) (2777,4002) (0.13.0.18) (3949.71061 1988 182 1142 0.16 26625 ±2744 4234 (122,215) (880,1462) (0.12.0.17) (2825.4907) 1989 165 1096 0.15 28898 ±3108 4357 (120.217) (871,1371) (0.12.0.17) (3186,54921 1990 65 515 0.13 22616 ±2575 2875 (53.90) (421,632) (0.11,0.17) (2176,4085) 1996 88 724 0.12 818 99 (76.142) (568,926) (0.12.0.17) (96.139) 1997 49 393 0.13 721 91 (42,69) (331,461) (0.11,0.17) (81,121) 1998 33 260 0.13 298 38 (26,41) (230,296) (0.10.0.16) (30,46) 1999 36 317 0.11 358 40 (30,48) (282,357) (0.10.0.15) (35,53) 2000 43 291 0.15 295 44 (32.58) (247.342) (0.12,0.18) (35.541 continued Archer et al.: Estimates of the incidental kill of Stenella attenuata attenuate calves in the tuna purse-seine fishery 241 Table 3 (continued) Stock and year Mean calf Total number Estimated calf deficit of NE spotted Estimated deficit in Observed per kill dolphins killed total calf observed sets dolphin kill CD*) (±SE) deficit 141 1199 0.12 51,712 ±10.721 6076 (110,229) (836,1638) (0.10,0.17) (3993-10,633) 254 1715 0.15 35,499 ±10.309 5254 (100,318) (939,2733) (0.07.0.15) (1554,6890) 197 1702 0.12 48,837 ±10,055 5664 (123.322) 11104,2434) (0.09,0.15) (3285,9121) 795 6293 0.13 52,206 ±8883 6595 (524,1036) (4925,7860) (0.09,0.15) (3833,9223) 491 3358 0.15 11.260 ±1186 1647 (345,563) (2860,3906) (0.11.0.16) (1098,1959) 660 3998 0.17 11.610 ±2553 1917 (342,949) (2508,5922) (0.12.0.18) (932.2614) 157 1262 0.12 6.254 ±1229 776 1104.216) (939.1643) (0.09,0.15) (438.1138) 144 1206 0.12 11.200 ±2430 1339 (59.3441 (411.2542) (0.10,0.17) (831,2320) 191 1346 0.14 12.512 ±2629 1775 (90,340) (577.2416) (0.11.0.17) (1010,2682) 306 1966 0.16 9869 ±1146 1536 (198,474) (1337,2734) (0.13,0.19) (1156,2088) 23 148 0.16 4587 ±928 724 (15.33) (99.206) (0.12.0.20) (418,1087) 114 961 0.12 10.018 ±2614 1183 (80.224) (526,1513) (0.12,0.18) (712,2352) 52 381 0.14 8089 ±951 1105 (32,791 (225.5701 (0.11.0.17) (781,1524i 275 1818 0.15 20,074 ±2187 3037 (143,373) (1065.2784) (0.10.0.17) (1776,3617) 271 1768 0.15 19,298 ±2899 2959 (147,374) (1068,2661) (0.11,0.16) (1754,3695) 75 479 0.16 13,916 ±1741 2166 (51,96) (368,605) (0.12,0.18) (1453,2785) 392 2793 0.14 28,560 ±2675 4011 (242,589) (1819,4277) (0.11,0.16) (2861.4977) 123 829 0.15 12,578 ±1015 1864 (78,160) (582.1128) (0.11,0.17) (1283,2236) 53 374 0.14 545 77 (42,711 (308.448) (0.12,0.18) (64,97) 89 738 0.12 1044 126 (72,132) (598.931) (0.11.0.16) (112,165) 31 236 341 44 0.13 (25,42) (192.288) (0.11,0.17) (38,58) 22 159 0.14 253 35 (16.32) (123,209) (0.11,0.18) (28,44) 28 250 0.11 435 48 (22.44) (189,330) (0.10.0.15) (42.67) B Western- 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1996 1997 1998 1999 2000 southern (WS) stock 242 Fishery Bulletin 102(2) Table 4 Annual mean observed and mean estimated are 95% lower and upper confidence intervals ference from zero (P<0.05) in the paired t-test number of lactating females per assuming a normal distribution s. set in 100% sampled sets. Values in parentheses of differences. Bold type indicates significant dif- Year Northeastern stock Western-southern stock No. of sets Observed Estimated Difference (959S CI) No. of sets Observed Estimated Difference (95% CD 1973 116 0.55 0.61 -0.06 1-0.17.0.051 21 1.19 1.30 -0.11 (-0.63,0.421 1974 98 0.51 0.54 -0.03 1-0.13.0.07) 16 0.75 0.81 -0.061-0.36.0.24) 1975 99 0.57 0.48 0.09 (-0.05,0.22) 14 1.07 0.92 0.15 (-0.46.0.77) 1976 51 0.28 0.35 -0.08 1-0.18.0.02) 90 0.500 0.502 -0.002 (-0.119.0.115) 1977 167 0.55 0.46 0.09 (0.01.0.15) 163 0.49 0.37 0.12 (0.03,0.21) 1978 82 0.37 0.40 -0.03 1-0.14,0.08) 93 0.50 0.52 -0.02 (-0.19,0.13) 1979 75 0.47 0.46 0.01 (-0.13,0.14) 61 0.64 0.47 0.17 (0.01,0.33) 1980 54 0.39 0.38 0.01 (-0.11.0.13) 34 0.50 0.44 0.06 1-0.09,0.20) 1981 41 0.53 0.74 -0.21 (-0.81,0.38) 38 0.66 0.64 0.02 1-0.16.0.19) 1982 36 0.62 1.18 -0.56 (-1.40,0.27) 33 0.30 0.44 -0.14 1-0.37,0.10) 1983 33 1.33 2.14 -0.8K-7.89.6.28) 6 0.17 0.57 -0.40 (-1.57,0.77) 1984 4 0.25 0.49 -0.24 1-0.67,0.18) 29 0.48 1.08 -0.60 (-0.96,-0.23) 1985 70 0.34 0.50 -0.16 1-0.36,0.061 17 0.35 0.50 -0.15 1-0.49,0.20) 1986 45 0.71 0.47 0.24 1-0.04,0.51) 28 0.61 0.42 0.19 1-0.01.0.38) 1987 121 0.43 0.46 -0.03 (-0.18,0.11) 30 0.27 0.46 -0.19 1-0.44.0.06) 1988 6 0.44 0.57 -0.13 (-0.59,0.35) — — — — 1989 24 0.96 1.03 -0.07 1-0.59,0.44) 15 0.93 0.96 -0.03 (-0.68,0.64) 1990 16 0.56 0.47 0.09 1-0.25,0.44) 9 0.67 0.93 -0.26 1-0.94,0.42) All 1108 0.50 0.53 -0.03 1-0.08,0.02) 697 0.545 0.546 -0.001 (-0.053,0.051) ertson, in press) estimated the probability that a calf of a given age class was still suckling. Given that body length has a near linear relationship with age in these young age classes (Perrin, 1976), this meant that for any chosen length of independence, each individual smaller than that cutoff value would only be counted fractionally, in effect correcting for the probability that an animal of a given age is not suckling. This procedure caused the method in this paper to tally fewer "calves" in each set than in the previous study. A secondary result of this effect was that the mean deficit per set estimated in the present study tended to be slightly higher than that presented by Archer etal. in 2001. We estimated the total number of missing calves as a function of the number of dolphins killed in each stock (Table 3). Prior to 1995, only a fraction of the purse-seine trips carried scientific observers. To estimate the number killed in each stock, kill rates from the observed trips were applied to unobserved trips, stratified by area and stock (IATTC, 2002; Joseph, 19942). Since 1995 it has been re- ported that all dolphin sets have been observed, and that the number of dolphins killed is therefore known without error (IATTC, 2002). The total calf deficit could also be estimated as a function of the number of sets by multiplying the total number of sets made on each stock by Ds (Fig. 4). In the only study to estimate the number of sets made on each stock annually. Archer et al.5 used a relatively simple proration scheme of unobserved sets derived from ratios of the number of sets made on each stock in observed sets. However, because Archer et al.5 did not stratify unobserved sets by area, bas- ing the total calf deficit on these estimates would produce a different result from that presented in Table 3. Because the estimates of the kill by stock included stratification by area, estimates of the total calf deficit calculated by multiplying the kill estimates by D,. are likely to be more accurate. It is important to realize that the deficit that we present is directly related to the kill observed in the sets that we used. In other words, if proration schemes for un- observed sets were the same for the number of sets made and the number of dolphins killed, estimates of the total calf deficit with either D^ or Dk would be equivalent. Wade et al.1 explored the effects of 50% and 100' < ad- ditional fisheries-related mortality on the assessment of the northeastern spotted dolphin stock. The assumption of additional mortality led to higher estimates of maximum Archer. F.. T. Gerrodette. and A. Jackson. 2002. Prelim- inary estimates of the annual number of sets, number of dolphins chased, and number of dolphins captured by stock in the tuna purse-seine fishery in the eastern tropical Pacific. 1971-2000. National Oceanographic and Atmospheric Admin- istration Administrative Report LJ-02-10. 26 p. Southwest Fisheries Science Center, 8604 La Jolla Shores Dr., La Jolla, CA 92037. Archer et al.: Estimates of the incidental kill of Stenella attenuata attenuata calves in the tuna purse-seine fishery 243 Table 5 Annua 1 mean number of dolphins killed sl22 cm (calves killed based on length) and estimated number of suckling calves (calves based 3n weaning model 1 per set in 100rt sampled sets (first line for each year!. Mean deficit per set using .22 cm as cutoff length (calf deficit based on length ) and calf deficit as estimated in this article (calf deficit based on weaning model) on second line for each year. Values in parentheses are 959! lower and upper confidence intervals assuming a normal dist •ibution of differences. Differences in bold indicate significant difference from zero (PsO.05) in the paired Mest. Northeastern stock Western-southern stock Calves killed Calves killed Calves killed Calves killed based on based on based on based on length weaning model length weaning model No. Calf deficit Calf deficit No. Calf deficit Calf deficit of based on based on Difference of based on based on Difference Year sets length weaning model (95% CD sets length weaning model (95% CD 1973 116 0.54 0.21 0.33 (0.18,0.50) 21 0.33 0.06 0.27 (0.01,0.55) 0.35 0.48 -0.13 (-0.26,-0.03) 1.00 1.25 -0.25 1-0.79.0.29) 1974 98 0.39 0.05 0.34 (0.20,0.47) 16 0.56 0.09 0.47 1-0.53.1.47) 0.36 0.50 -0.14 (-0.26,-0.03) 0.56 0.74 -0.181-0.61,0.25) 1975 99 0.57 0.15 0.42 (0.20,0.64) 14 0.29 0.11 0.18(-0.03.0.39) 0.46 0.40 0.04 1-0.05.0.161 0.93 0.83 0.10(-0.45,0.66i 1976 51 0.18 0.11 0.07 1-0.01,0.15) 90 0.13 0.07 0.06 (0.001,0.13) 0.28 0.31 -0.031-0.14.0.06) 0.49 0.47 0.02 1-0.10.0.15) 1977 167 0.10 0.03 0.07 (0.02,0.12) 163 0.17 0.06 0.11 (0.06,0.16) 0.51 0.45 0.06 (-0.01,0.14) 0.46 0.35 0.11(0.03,0.20) 1978 82 0.17 0.03 0.14(0.05,0.23) 93 0.18 0.05 0.13 (0.04,0.23) 0.35 0.39 -0.04 (-0.14.0.07) 0.43 0.50 -0.07 1-0.22.0.09) 1979 75 0.09 0.04 0.05 (-0.02,0.13) 61 0.31 0.13 0.18(0.04,0.32) 0.44 0.43 0.01 (-0.11,0.13) 0.51 0.37 0.14 1-0.03,0.31) 1980 54 0.16 0.03 0.13 (0.02,0.25) 34 0.00 0.01 -0.01 (-0.02,-0.003) 0.373 0.371 0.002-0.115,0.119) 0.50 0.44 0.061-0.08.0.21) 1981 41 0.105 0.110 -0.005 1-0.194,0.185) 38 0.05 0.04 0.01 (-0.04.0.07) 0.53 0.65 -0.121-0.57,0.31) 0.63 0.62 0.01 (-0.17,0.20) 1982 36 0.44 0.21 0.23 (-0.10.0.55) 33 0.06 0.02 0.04 1-0.04,0.12) 0.44 1.00 -0.56(-1.27,0.14i 0.27 0.42 -0.15 (-0.37,0.08) 1983 33 0.00 0.14 0.14 1-0.64.0.36) 6 0.17 0.04 0.13 1-0.31.0.57) 1.33 2.00 -0.67 (-7.25.5.91) 0.17 0.56 -0.39 1-1.56,0.76) 1984 4 0.00 0.02 -0.02 1-0.08,0.04) 29 0.14 0.04 0.10 1-0.01,0.21) 0.25 0.49 -0.24 1-0.67,0.18) 0.35 1.04 -0.69 (-1.13,-0.26) 1985 70 0.13 0.04 0.09(0.02,0.15) 17 0.06 0.04 0.02 1-0.06.0.10) 0.29 0.47 -0.181-0.39,0.03) 0.35 0.49 -0.14 (-0.48,0.21) 1986 45 0.13 0.04 0.09 (0.01,0.17) 28 0.04 0.03 0.01 (-0.04,0.06) 0.64 0.44 0.20 1-0.04.0.44) 0.57 0.39 0.181-0.02,0.38) 1987 121 0.14 0.02 0.12 (0.05,0.20) 30 0.23 0.08 0.15(0.02,0.30) 0.38 0.45 -0.07 1-0.22,0.07) 0.27 0.43 -0.16 (-0.41,0.09) 1988 6 0.11 0.33 0.12 0.50 -0.01 (-0.23.0.22) -0.17 1-0.62,0.28) — — — — 1989 24 0.22 0.13 0.09 (-0.11,0.29) 15 0.47 0.20 0.27 (0.05,0.49) 0.87 0.95 -0.08 (-0.60,0.43) 0.73 0.82 -0.09 1-0.66,0.48) 1990 16 0.31 0.21 0.10 (-0.18,0.38) 9 0.89 0.17 0.72 1-0.18,1.62) 0.56 0.41 0.15 (-0.20.0.51) 0.33 0.77 -0.44 (-1.15,0.27) All 1108 0.25 0.08 0.17 (0.13,0.20) 697 0.18 0.06 0.12 (0.09,0.16) 0.42 0.48 -0.06 (-0.10,-0.01) 0.49 0.51 -0.02 (-0.08,0.03) 244 Fishery Bulletin 102(2) growth rates and lower estimates of the current size of the population in relation to carrying capacity. Wade et al.1 did not model the calf deficit estimated in our present study, but the effect of 14/r additional mortality would probably be less than the 50f> additional mortality that was modeled. The 50^ mortality was spread over all age classes, and additional mortality due to missing calves should be assigned to the first two year classes only. The important question is whether the calf deficit in the kill represents the main effect of mother-calf separation by the fishing process. As outlined in Archer et al. (2001t, the mechanism by which suckling calves are separated from their mothers is unknown. If separation is simply a function of the number of lactating females killed, then the deficit presented here is an accurate representation of the number of "missing" calves. However, there is some evidence that separation can occur without the mother being killed. In the early days of the backdown procedure, purse-seine skippers reported that "Babies swim around the outside of the net pushing to get back in probably because their mothers are still inside" i Gehresp (. It is unclear whether these calves were sepa- rated prior to encirclement or were released early during backdown, prior to their mothers. Regardless, given that dolphins exhibit some of their fastest swimming during a set immediately upon release from the net tChivers and Scott' ), separated calves waiting immediately outside the net may risk separation if their mothers join the rest of the school rapidly swimming away from the net. If this, or any of the other scenarios regarding the manner in which perma- nent separation can occur without the mother being killed i Archer et al.. 2001 1. then the calf deficit underestimates the actual number of orphaned calves. Future research should focus on the mechanism of calf separation because a better understanding of this process is the only way we will be able to estimate the magnitude of the unobserved calf mortality and its subsequent effects on the population. Acknowledgments The authors wish to thank Michael Scott and Xick Vogel of the IATTC for providing data as well as Jay Bar- B Gehres. L. E. 1971. Letter of July 2 to Alan R. Longhurst. 2 p. Southwest Fisheries Science Center, 8604 La Jolla Shores Dr.. La Jolla. CA 92037. 7 Olivers. S. J., and M. D. Scott. 2002. Tagging and tracking of Stenella spp. during the 2001 Chase Encirclement S Studies cruise. National Oceanographic and Atmospheric Administration Administrative Report LJ-02-33. 21 p. South- west Fisheries Science Center, 8604 La Jolla Shores Dr.. La Jolla. CA 92037. low and Bill Perrin for helpful reviews and analytical suggestions. Literature cited Archer. F.. T. Gerrodette, A. Dizon. K. Abella and S. Southern. 2001. Unobserved kill of nursing dolphin calves in a tuna purse-seine fishery Mar Mamm. Sci. 17:540-554. Archer. F.. and K. M. Robertson. In press. Age and length at weaning and development of prey preferences of pantropical spotted dolphins. Stenella attenuata, from the eastern tropical Pacific Mar Mamm. Sci. Dizon. A. E.. W. F. Perrin. and P. A. Akin. 1994. Stocks of dolphins iStenella spp. and Delphmux delphis) in the eastern tropical Pacific: a phylogeographic classification. NOAA Tech. Rep. NMFS-119. 20 p. Gerrodette. T. 2002. Tuna-dolphin issue. In Encyclopedia of marine mammals (W. F. Perrin. B. Wursig. and J. G. M. Thewis- sen, eds.l. p. 1269-1273. Academic Press. San Diego. CA. Hall. M. A., L. A. Dayton, and K. I. Metuzals. 2000. Bycatch: problems and solutions. Mar. Poll. Bull. 41:204-19. IATTC i Inter-American Tropical Tuna Commission). 2002. Annual report of the Inter-American Tropical Tuna Commission 2000, 171 p. IATTC. La Jolla. CA. Gosliner, M. L. 1999. The tuna-dolphin controversy. In Conservation and management of marine mammals (J. R. Twiss Jr.. and R. R. Reeves, eds.l, p. 120-155. Smithsonian Institution Press. Washington and London. Myrick. Jr.. A. C, A. A. Hohn. J.. Barlow, and P. A. Sloan. 1986. Reproductive biology of female spotted dolphins. Stenella attenuata, from the eastern tropical Pacific. Fish. Bull. 84:247-259. NRC t National Research Council I. 1992. Dolphins and the tuna industry. 176 p. National Academy Press. Washington. D.C. Perrin. W. F. 1970. Color patterns of the eastern Pacific spotted por- poise Stenella graffmani LbnnbeglCetacea, DelphinidaeL Zoologica NY) 54:135-149. Perrin. W, F.. J. M. Coe, and J. R. Zweifel. 1976. Growth and reproduction of the spotted porpoise. Stenella attenuata, in the offshore eastern tropical Pa- cific. Fish. Bull. 74:229-269. Smith, T. 1983. Changes in size of three dolphin [Stenella spp.1 populations in the eastern tropical Pacific. Fish. Bull. 81:1-13. Wade, P. R. 1995. Revised estimates of incidental kill of dolphins I Del- phinidae1 by the purse-seine tuna fishery in the eastern tropical Pacific. 1959-1972. Fish. Bull. 93:345-354. 245 Abstract— Psednos rossi new species (Teleostei: Liparidaei is described from two specimens collected in the North Atlantic Ocean off Cape Hatteras, North Carolina, at depths of 500- 674 m. Psednos rossi belongs to the P. christinae group, which includes six other species and is characterized by 46-47 vertebrae and the absence of a coronal pore. Psednos rossi dif- fers from those six species by having characters unique within the genus: straight spine, body not humpbacked at the occiput, and a very large mouth with a vertical oral cleft. Other distin- guishing characters include a notched pectoral fin with 15-16 rays, eye 17-19% SL, and color in life orange- rose. With P. rossi, the genus Psednos as currently known includes 26 de- scribed and five undescribed species of small meso- or bathypelagic liparids from the Atlantic. Pacific, and Indian Oceans. A remarkable new species of Psednos (Teleostei: Liparidae) from the western North Atlantic Ocean Natalia V. Chernova Zoological Institute Russian Academy of Sciences Unlversitetskaya nab- 1 St. Petersburg 199034. Russia David L. Stein NOAA/NMFS Systematlcs Laboratory Smithsonian Institution P.O. Box 37012 National Museum of Natural History, MRC-0153 Washington, DC. 20013-7012 E-mail address (for D L. Stem, contact author): david.stenvanoaa gov Manuscript approved for publication 7 January 2004 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Scientific Publications Office. Fish. Bull. 102:245-250 (2004). The liparid genus Psednos Barnard 1927 is a group of meso- and bathype- lagic snailfishes distinguished from the genus Paraliparis by having the infra- orbital canal of the cephalosensory system interrupted behind the eye and usually having a pronounced dorsal curvature of the spine, producing a "humpbacked" body. Psednos are small, easily damaged, and often misidenti- fied as juvenile Paraliparis. Until 1978, the genus was known only from two specimens of a single species (Psednos micrurus Barnard 1927) collected off Cape Point, South Africa. Two addi- tional specimens were collected in the southern Indian Ocean and reported by Stein (1978). No further specimens or species were described until Andria- shev (1992) described another new species. Since then, active searches for material from collections around the world have yielded many specimens from the Atlantic. Pacific, and Indian Oceans. To date, 25 species have been described (Andriashev, 1992, 199.3; Chernova. 2001; Stein et al.. 2001; Chernova and Stein, 2002) and an additional five are undescribed (one in Stein et al., 2001, three in Chernova and Stein, 2002, all in poor condition; and another that is currently being described by Stein). In this article, we describe an especially noteworthy spe- cies of the genus from two specimens collected from the North Atlantic off Cape Hatteras, North Carolina. Materials and methods All characters available for both speci- mens were studied. Characters and terms used were described by Andria- shev (1992), Chernova (2001), Stein et al. (2001), and Chernova and Stein (2002 1. Counts were made from a radio- graph of the holotype and from each specimen where possible; vertebral counts include the urostyle. The first caudal vertebra is that with the haemal spine supporting the first anal-fin ray. The posterior tip of the lower jaw in Psednos forms a distinct and promi- nent ventrally directed angle, the ret- roarticular process (Chernova, 2001). Counts and proportions are given as a percentage of standard length ( SL) and head length (HL). Nonstandard mea- surements are the following: distance from mandible to anus (from ante- rior tip of mandible to center of anus); distance from anus to anal-fin origin (from center of anus to anal-fin origin); interorbital width (measured between upper margins of eyes); postocular head length (distance from posterior margin of eye to tip of opercular flap). 246 Fishery Bulletin 102(2) Figure 1 Psednos rossi n.sp., paratype, USNM 372727. Adult, 51.8 mm SL, 57.2 mm TL. Sta. CH-01-047, off Cape Hatteras. Scale 5 mm. Infraorbital pore 6 not shown owing to damage. We selected the smaller specimen to serve as the holo- type owing to its better condition (skin, pores, shape of head) and the availability of more characters. Unfortu- nately, it is distorted and does not look natural; therefore the undistorted larger (adult) specimen, the paratype, is illustrated. It is also more useful to have a drawing of an adult for comparison with other Psednos specimens. In these small fishes, precise counts of number of tooth rows are possible only in disarticulated cleared and stained specimens; thus, we provide approximate counts. Similarly, the drawing of the gill arch of the paratype was made without dissection by viewing through an opening in the branchiostegal membrane. Although Andriashev (1986) and Andriashev and Stein (1998) demonstrated the importance of the pectoral girdle in distinguishing among species and in explain- ing liparid relationships, we did not dissect, clear, and stain a pectoral girdle from these specimens owing to the high probability of damaging them and destroying other characters (Chernova, 2001; Chernova and Stein, 2002). The new species is so easily distinguished from congeners that it is not necessary for a diagnosis of the species to look at additional characters that the pectoral girdle can provide. Future specimens should be used to study these characters. The holotype and paratype are permanently deposited in the Division of Fishes, Smithsonian Institution, Na- tional Museum of Natural History (USNM collection). Results Psednos rossi, n.sp. Holotype USNM 372726, juvenile, 37.2 mm SL. TL?, Sta. EL-00-033, off Cape Hatteras (The Point), 35°30.036'N, 74°46.497'W, 500-674 m over about 900 m depth, 23 July 2000, Tucker trawl. Good condition but distorted. Paratype USNM 372727, adult (sex not identified), 51.8 mm SL, 57.2 mm TL, Sta. CH-01-047, off Cape Hatteras (The Point), 35°28.93'N, 74°45.93'W, 628-658 m over 1090-704 m depth, 24 Aug. 2001, Tucker trawl. Throat slightly damaged, head slightly compressed, skin on head partly missing. Diagnosis Vertebrae 47, dorsal-fin rays 42-44, coronal pore absent. Mouth vertical, symphysis of upper jaw above level of eye. Body not humpbacked, vertebral column not curved behind cranium. Gill cavity enlarged. Anus on vertical behind head. Pectoral fin notched, rays 8+2+5-6. Eye 17-19% HL. Description Counts and proportions are given in Table 1. Head large, about one-third SL, its depth less than, and its width equal to or a little greater than, its length (Fig. 1). Head depth slightly greater than its width. Mouth very large, distinctly superior. Jaws almost vertical, at angle of about 90° to horizontal. Symphysis of upper jaw above level of eye. Ascending process of premaxilla horizontal, its distal end almost above center of eye. Posterior tip of lower jaw- exactly below symphysis of upper jaw. Posterior (lower) end of mouth cleft well below level of lower margin of eye. When mouth closed, ventral surface of lower jaw forms entire frontal surface of head. Lower jaw included. Sym- physeal process present at lower jaw symphysis, projecting forward prominently; retroarticular processes of lower jaw large, acute, directed anteroventrally (Fig. 2 A I. Teeth large, sharp, spear-shaped, strongly curved inward (Fig. 2B), in (smaller) holotype in approximately 22 and 24 (32 and 35) rows on upper and lower jaw; 5 (8-9) teeth in first full row near symphyses of both jaws. Snout short, 1.5 ( 1.0) times eye diameter. Olfactory rosette (7 lobes) and nostril above anterior third of eye. Eyes not large, close to upper Chernova and Stein: A new species of Psednos from the western North Atlantic Ocean 247 Table 1 Counts and proportions for the holotype and paratype of Psednos rossi new species. Proportions are in % of standard length (SL) followed by % head length (HL, in parentheses). Vertebrae Dorsal-fin rays Anal-fin rays Pectoral-fin rays Caudal-fin rays Gill rakers Head length Head width Head depth Body depth Body depth at anal-fin origin Predorsal-fin length Preanal-fin length Mandible to anus Anus to anal fin origin Upper pectoral-fin lobe length Pectoral-fin notch ray length Lower pectoral-fin lobe length Eye diameter Snout length Interorbital width Postocular head length Upper jaw length Lower jaw length Gill opening length Opercle length USNM 372726 Holotype 37.2 mm SL 47 44 35 16 [L] 15 [R] 6 32.3 22.0(68.1) 23.7(73.4) 21.5(66.6) 13.4(41.5) 29.6(91.6) 47.8(148.0) 34.9(108.0) 23.7(73.4) 13.4(41.5) 8.1(25.1) 9.4(29.1) 5.4(16.7) 8.1(25.0) 13.4(42.0) 18.8(58.0) 16.1(49.8) 16.1(49.8) 5.4(16.7) 13.4(41.5) USNM 372727 Paratype 51.8 mm SL 42 33 15 [L, R] 6 10 29.9 13.5(45.2) 17.4(58.2) 25.1(83.9) 17.0(56.8) 26.6(89.0) 48.3(161.5) 36.7(122.7) 21.2(70.9) 13.5(45.2) 5.8(19.4) 7.7(25.8) 11.2(37.4) 19.3(64.5) 12.5(41.8) 13.5(45.2) 5.4(18.1) 12.5(41.8) contour of head. Interorbital space flat, 2.5 (1.9) times eye diameter. Gill opening short, 1.0 (0.9) times eye diameter, at 45° angle, entirely above pectoral-fin base and slightly anterior to it (distance between ventral end of gill opening and base of upper pectoral ray about equal to length of gill opening). Opercular flap small, acute. Opercle very long, directed ventrally and posteriorly, its tip below level of pos- terior end of lower jaw. Interopercle of similar length, vis- ible externally, its anterior tip projecting anteriorly from ventral contour of head (Fig. 1). Long opercle, interopercle and elongated branchiostegal rays support membranes of enlarged branchial cavity that appears externally as a black posterior part of head. Branchial cavity length slightly more than half head length. Branchiostegal rays (4+2) long and distinctly visible externally. Gill rakers modified, closely but irregularly set, mostly alternating (especially on gill arch one), often paired on the outer and inner sides of each gill arch (central part of arches two and three); plates flattened, triangular, similar in shape to those in P. pallidus or Psednos sp.l of Chernova and Stein (2002, Figs. 9 and 13). spinule-bearing surface directed internally, flat and longitudinally oval. Spinules closely set, usually in two longitudinal rows, each of five to eight spinules, often with a few additional spinules in between (Fig. 2C). Sensory pores difficult to see because of thin transpar- ent skin (damaged in paratype). Nasal pores 2, the poste- rior on a vertical through center of eye. Paired nasal bones (through which the nasal canals run) long, tubular, and visible externally. Coronal pore absent. Lacrimal bones (bearing anterior portion of infraorbital canal) large, vis- ible externally, slightly prominent anteriorly. Infraorbital canal (better preserved in holotype) interrupted behind eye, infraorbital pores 6 (5+1), posteriormost above poste- rior margin of eye (Fig. 2A). In paratype, skin behind eye missing. Preoperculomandibular pores 6 (3 on lower jaw + 3 on preopercular area). Two temporal pores present: tx a short distance behind posterior margin of eye, and tsb, the suprabranchial pore, above and in front of gill opening (Fig. 2A). 248 Fishery Bulletin 102(2) Pectoral fin notched, of 16 (15) rays. Upper lobe of 8 (8) rays, the 2 (2) notch rays more widely spaced and placed exactly at middle of fin base. In holotype, left lower pec- toral lobe with 6, on right 5, rays. In paratype, 5 rays on each side. Bases of lower-lobe rays stronger and thicker than those of upper-lobe rays. Level of uppermost pectoral ray below horizontal through lower end of upper jaw. Base of pectoral fin close to vertical, lowest ray almost directly below uppermost. Upper-lobe rays not reaching anal fin origin, lower-lobe rays not reaching vertical through ends of upper lobe rays. In holotype, length of notch rays 1.7 times in upper pectoral-fin lobe length, lower pectoral-fin lobe 1.4 times in it. Body not humpbacked, dorsal contour of back almost straight; spine horizontal, its anterior end not dorsally "'Vt B Figure 2 Details of anatomy of Psednos rossi. (A) Cephalic pores and prominent features of head. Portions of sensory canals passing through bones are stippled. N = nostril and olfac- tory rosette; io = infraorbital pores, n = nasal pores, t = temporal pores; S = symphyseal knob; R = retroarticular process. (B) Teeth of paratype: (leftl frontal view; (right) lateral view. Tooth length about 0.25 mm. (C) First gill arch of paratype, USNM 372727. right side; view from inside of gill cavity. Raker height about 0.3 mm. curved (Fig. 3). Neural spines of vertebrae 1-4 neither longer nor broader than those posterior, unlike other spe- cies (see Fig. 5 in Chernova, 2001). Maximum body depth 4.2 (4.0) times in standard length and 1.6(1.5) times depth at anal-fin origin. In holotype, occiput slightly swollen (Fig. 3); in paratype, dorsal outline of head and back in front of dorsal fin origin almost perfectly flat (Fig. 1), pos- sibly an age-related difference. Abdominal part of body long, preanal length almost half of standard length. Inter- neural of first dorsal-fin ray between neural spines 3 and 4. Dorsal and anal fins moderately deep, maximum depth of erect dorsal fin in paratype 8.9 times in SL, 2.7 times in head length (damaged in holotype). Dorsal and anal fins overlapping about one-third of caudal-fin length. Anus on vertical behind head, slightly behind base of uppermost pectoral ray. Skin transparent. Gelatinous subcutaneous tissue weakly developed. In holotype (smaller specimen) body not as deep and jaws longer than in the paratype (larger specimen). Differences in head width and interor- bital width are great because head of paratype was slightly compressed during fixation. Other proportions similar to those of holotype. Body color in alcohol pale; under magnification, slightly dusky blotches of dots present caudally in paratype and absent in holotype. Head musculature pale. Black perito- neum visible through body wall. Mouth and gill cavities, gill arches, tongue, and both jaws black; gill rakers pale. Musculature of pectoral girdle appears pale on lateral surface of belly. Color in life orange-rose. Distribution Western North Atlantic off Cape Hatteras, mesopelagic at depths of 500-674 m. Etymology The patronym "rossi" after Steve W. Ross, who initially notified us of the captures and furnished the specimens to us for examination. Comparative notes Psednos rossi seems to belong to the P. christinae group (see Chernova, 2001; Chernova and Stein, 2002), includ- ing P. andriashevi, P. barnardi, P. christinae. P. dentatus, P. groenlandicus, and P. harteli. Species of this group are characterized by vertebrae 46-47, dorsal-fin rays 38-42, anal-fin rays 33-35, and coronal pore absent (versus the P. micrurus group having vertebrae 40-44. dorsal-fin rays 34-38, anal-fin rays 28-33, and coronal pore present) (Chernova, 2001). Psednos rossi distinctly differs from the other species of the christinae group in at least having occiput not swollen (vs. greatly swollen), not humpbacked because the vertebral column is straight (vs. humpbacked owing to the greatly curved anterior part of the spine), mouth vertical with jaws at 90° to horizontal, symphysis of upper jaw above level of eye (vs. a more or less oblique mouth at an angle of 30-45° and the upper jaw. symphysis on a horizontal with the lower half of the eve); and anus Chernova and Stem: A new species of Psednos from the western North Atlantic Ocean 249 Figure 3 Radiograph of Psednos rossi n.sp., holotype. USNM 372726. Juvenile, 37.2 mm SL. Sta. EL-00-033, off Cape Hatteras. behind the head (vs. anus below the posterior third of the head). The very oblique, almost vertical mouth occurs often in species of the P. micrurus group, five of which have the mouth at 75-85° to the horizontal (P. anoderkes, P. cathetostomus, P. microps, P. mirabilis, P. sargassicus). However, they all differ as described above. Discussion The physical features of Psednos rossi are unique in the genus. The straight vertebral column and body are outside the previous diagnosis of the genus, because all previously known species are humpbacked owing to the curved spinal column. Nevertheless, P. rossi clearly belongs in Psednos rather than Paraliparis because it has the other generic characters of Psednos (Chernova, 2001); particularly, its sensory canal system and pores are of Psednos type, having an interrupted infraorbital canal behind the eye. We suggest that its remarkable body shape is an extreme transformation of the usual Psednos body shape and is associated with the change of the mouth from oblique and of normal size to vertical and very large. In this process the anterior movement of the bony elements of the jaws greatly enlarges the branchial cavity. The morphology of Psednos rossi invites speculation about its ecology. The very large superior mouth with verti- cal jaws, eyes located close to the dorsal contour of the head and oriented to look forward and up, and straight body sug- gest adaptation to feeding on detritus and animals (such as copepods) above it in the water column. These adaptations, similar to those of hatchetfishes (family Sternoptychidae), are highly advantageous for a mesopelagic mode of life. Sudden opening of the very large vertical lower jaw could produce a strong orobranchial suction, simultaneously bringing food into the mouth and thus saving energy for this fish, which is probably a poor swimmer. Work over the last several years has made it clear that Psednos species exist at mesopelagic depths in the North Atlantic, Indian, North Pacific, and South Pacific Oceans. We confidently expect discovery of additional species from meso- and bathypelagic waters. Acknowledgments We wish to thank S. W. Ross, K. J. Sulak, and J. V. Gartner Jr. for collecting the specimens, bringing them to our attention, and loaning them to us for description. Collections were supported by the U.S. Geological Survey, State of North Carolina, North Carolina Coastal Reserve Program, and the Duke/UNCW Oceanographic Consor- tium. The figures are drawn by the senior author, who was supported by the Russian Science Foundation Grants 02-04-48669 and 00-15-07794. Literature cited Andriashev, A. P. 1986. Review of the snailfish genus Paraliparis (Scorpaeni- formes: Liparididae) of the Southern Ocean, 204 p. The- ses Zoologicae 7, Koeltz Scientific Books, Koenigstein 1992. Morphological evidence for the validity of the anti- tropical genus Psednos Barnard ( Scorpaeniformes, Lipari- didae) with a description of a new species from the eastern North Atlantic. UO, Tokyo 41:1-18. 1993. The validity of the genus Psednos Barnard (Scor- paeniformes, Liparidae) and its antitropical distribution area. Vopr. Ikhtiol. 33(11:5-15 [in Russian] J. Ichthyol. 33 (5):81-98. [English translation.] Andriashev, A. P., and D. L. Stein. 1998. Review of the snailfish genus Careproctus (Lipari- dae, Scorpaeniformes) in Antarctic and adjacent waters. Contr. Sci. Nat. Hist Mus. Los Angeles Cty. 470:1-63. 250 Fishery Bulletin 102(2) Chernova. N. V. 2001. A review of the genus Psednos (Pisces, Liparidae) with description often new species from the North Atlantic and southwestern Indian Ocean. Bull. Mus. Comp. Zool. 155:477-507. Chernova, N. V., and D. L. Stein. 2002. Ten new species of Psednos (Pisces, Scorpaeni- formes: Liparidae) from the Pacific and North Atlantic Oceans. Copeia 2002 (3):755-778. Stein, D. L. 1978. The genus Psednos a junior synonym of Paraliparis, with a redescription of Paraliparis mierurus (Barnardi (Scorpaeniformes: Liparidae). Matsya 4:5-10. Stein, D. L., N. V. Chernova, and A. P. Andriashev. 2001. Snailfishes (Pisces: Liparidae) of Australia, includ- ing descriptions of 30 new species. Rec. Austr. Mus. 53:341-406. 251 Abstract— Age and growth of sailfish (Jstiophorus platypterus) in waters off eastern Taiwan were examined from counts of growth rings on cross sections of the fourth spine of the first dorsal fin. Length and weight data and the dorsal fin spines were collected monthly at the fishing port of Shinkang (southeast of Taiwanl from July 1998 to August 1999. In total. 1166 dorsal fins were collected, of which 1135 (97r£> (699 males and 436 females) were aged suc- cessfully. Trends in the monthly mean marginal increment ratio indicated that growth rings are formed once a year. Two methods were used to back- calculate the length of presumed ages, and growth was described by using the standard von Bertalanffy growth function and the Richards function. The most reasonable and conserva- tive description of growth assumes that length-at-age follows the Rich- ards function and that the relationship between spine radius and lower jaw fork length ( LJFL I follows a power function. Growth differed significantly between the sexes; females grew faster and reached larger sizes than did males. The maximum sizes in our sample were 232 cm LJFL for female and 221 cm LJFL for male. Age and growth of sailfish Ustiophorus platypterus) in waters off eastern Taiwan Wei-Chuan Chiang Chi-Lu Sun Su-Zan Yeh Institute of Oceanography National Taiwan University No 1, Sec. 4, Roosevelt Road Taipei, Taiwan 106 E-mail address (for C L. Sun, contact author): chilufiintu edu.tw Wei-Cheng Su Taiwan Fisheries Research Institute No. 199, Ho-lh Road Keelung, Taiwan 202 Manuscript approved for publication 22 December 2003 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Scientific Publications Office. Fish. Bull. 102(2): 251-263 (2004). The sailfish (Istiophorus platypterus) is distributed widely in the tropical and temperate waters of the world's oceans. According to data from longline catches, sailfish are usually distributed between 30°S and 50°N in the Pacific Ocean, and highest densities are found in the warm Kuroshio Current and its subsidiary currents. This species has a tendency to be found close to the coast and near islands (Nakamura, 1985). During the 1990s the annual landings of sailfish off Taiwan ranged between 600 and 2000 metric tons, of which approximately 54% came from waters off Taitung (eastern Taiwan). Sailfish are seasonally abundant from April to October (peak abundance from May to July) and contribute substan- tially to the economic importance of the eastern coast of Taiwan where this species is taken primarily by drift gill nets, although they are also caught by set nets, harpoons, and as incidental bycatch in inshore longline fisheries. Age and growth of sailfish caught in recreational fisheries in the Atlan- tic Ocean have been studied by using various methods, including length- frequency analysis (de Sylva, 1957). analysis of release-recapture data (Far- ber1), and inferences from observed marks on hard parts, such as spines (Jolley, 1974, 1977; Hedgepeth and Jolley, 1983) and otoliths (Radtke and Dean, 1981; Radtke, 1983; Prince et al., 1986). In contrast, very few attempts have been made to age sailfish in the Pacific Ocean. Koto and Kodama ( 1962 ) estimated the growth of sailfish caught with longlines from 1952 to 1955 in the East China Sea using length-frequency analysis, and Alvarado-Castillo and Fe- lix-Uraga (1996, 1998) used the fourth spine of the first dorsal fin to estimate age and growth of sailfish caught from 1989 to 1991 in the recreational fishery off Mexico. However, western Pacific sailfish have not been aged with calci- fied structures in any previous study. The aging of fishes, and consequently the determination of their growth and mortality rates, is an integral compo- nent of modern fisheries science (Paul. 1992). Mortality and growth rates pro- vide quantitative information on fish stocks and are needed for stock assess- ment methods such as yield-per-recruit and cohort analysis (Powers. 1983). The objectives of this study were to estimate age and growth of sailfish by counting growth rings on cross sections of the fourth spine of the first dorsal fin and to determine which of the Richards function and the standard von Berta- lanffy growth function best represents growth of sailfish in waters off eastern 1 Farber, M.I. 1981. Analysis of Atlantic billfish tagging data: 1954-1980 Unpubl. manuscr. ICCAT workshop on billfish, June 1981. Southeast Fisheries Center Miami Laboratory. National Marine Fish- eries Service, NOAA, 75 Virginia Beach Drive, Miami, FL 33149. 252 Fishery Bulletin 102(2) 120 125 130 135 140 E Figure 1 Fishing grounds of the gillnet (cross lines) and longline (oblique lines) fish- ing boats based at Shinkang fishing port. Taiwan. This information could be used to determine the age composition of the catch and to assess the status of sailfish in these waters by using yield-per-recruit or se- quential population analysis techniques. Materials and methods Materials Data on total length (TL), eye fork length (EFL), lower jaw fork length (LJFL) (in cm), round weight (RW) (in kg) and the first dorsal fins of male and female sailfish were collected monthly at the fishing port of Shinkang (Fig. 1) from July 1998 to August 1999. In total, 304 TLs, 1166 LJFLs, 1166 RWs, and 1166 dorsal fins were collected. The dorsal fins were kept in cold storage before being boiled to remove surrounding tissue and to separate the fourth spines. Three cross sections (thickness 0.75 mm) were taken successively along the length of each spine with a low-speed "ISOMET" saw (model no. 11-1280) and diamond wafering blades, at a location equivalent to 1/2 of the maximum width of the condyle base measured above the line of maximum condyle width (Fig. 2A) (Ehrhardt et al., 1996; Sun et al., 2001, 2002). The sections were immersed in 95'/J ethanol for several minutes for cleaning, placed on disposable paper to air dry, and then stored in a labeled plastic case for later reading. Spine sections were examined with a binocular dissecting microscope (model: Leica-MZ6) under transmitted light at zoom magnifica- tions of 10-20x depending on the sizes of the sections. The most visible one of these three sections was read twice, approximately one month apart. If the two ring counts differed, the section was read again, and if the third ring count differed from the previous two ring counts, the spine was considered unreadable and discarded. The precision of reading was evaluated by using average percent error (APE) (Beamish and Fournier, 1981; Campana, 2001) and coefficient of variation (CV) (Campana, 2001) statistics. Images of the cross sections were captured by using the Image-Pro Image analysis software package (Media Cy- bernetics, Silver Spring MD, 1997) in combination with a dissecting microscope equipped with a charged coupled de- vice (CCD) camera (model: Toshiba IK-630) and a Pentium II computer equipped with a 640x480 pixel frame grab card and a high-resolution (800x600 pixel) monitor. The distance from the center of the spine section to the outer edge of each growth ring was measured in microns with the Image-Pro software package after calibration against an optical micrometer. The center of the spine Chiang et al.: Age and growth of Istiophorus platypterus in waters off eastern Taiwan 253 SPINE SHAFT VASCULARIZED CORE SECTION AREA 0.75 MM CONDYLE BASE CROSS SECTION Figure 2 Schematic diagram of the fourth dorsal spine of sailfish [I. platypterus) and the location of the cross section (A), and a cross section showing the measurements taken for age determination of sailfish (B). W= maximum width of condyle base, R = radius of spine, r, = radius of ring i, d = diameter of spine, dl = diameter of ring i. The vascularized core and growth rings (1, 2, 3, 4, 5) are also shown. section was estimated according to the methods of Cayre and Diouf (1983) (Fig. 2B). The distances (d,l were then converted into radii (r,0 by using the equation (Megalofo- nou, 2000; Sun et al., 2001): r; = d: - W/2), where ri d radius of the ring i; distance from the outside edge of ring i to the opposite edge of the cross section; and d = diameter of the spine. False growth rings were defined according to criteria of Berkeley and Houde (1983), Tserpes and Tsimenides ( 1995 ), and Ehrhardt et al. ( 1996 ). core of the spine. The number of early but missing growth rings was therefore estimated by the replacement method applied to Pacific blue marlin (Makaira nigricatis)by Hill et al. (1989). This method involved first compiling ring radii statistics from younger specimens that had at least the first or second ring visible. Radii of the first four visible rings from samples that had missing early rings were then com- pared with the radii for these younger specimens. When the radii of at least two successive rings of the first four visible rings each fitted well within one standard deviation from the mean radii of each of two or more rings from the data compiled from the younger specimens, the number of missing rings was computed as the difference between the ring counts for the matched radii compiled from younger specimens and those for the specimen of interest. Accounting for missing early rings The first several growth rings of the larger specimens may be obscured because of the large size of the vascularized Validation The marginal increment ratio (MIR), which was used to validate the rings as annuli, was estimated for each 254 Fishery Bulletin 102(2) specimen by using the following equation (Hayashi, 1976. Prince et al., 1988; Sun et al., 2002): MIR = {R-rn)l{rn-rn_1), where R = spine radius; and rn and rnl = radius of rings n and re— 1. The mean MIR and its standard error were computed for each month by sex for all ages combined, and also for the ages 1-5 and 6-11 for males and 1-5 and 6-12 for females. Growth estimation Growth for males and females was estimated by back-cal- culation of lengths at presumed ages. Two methods were used. Method 1 was based on the assumption that the rela- tionship between spine radius (R) and LJFL (L) is linear, i.e., L=a1+61i? (Berkeley and Houde, 1983; Sun etal, 2002), whereas method 2 was based on the assumption that this relationship is a power function, i.e., L=a0Rh- (Ehrhardt, 1992; Sun et al., 2002). The parameters of the relationships were estimated by maximum likelihood, assuming log-nor- mally distributed errors. Akaike's information criterion (AIC, Akaike, 1969) was used to select which of the linear and power functions best represented the data: AIC = -21nL + 2p, where InL = logarithm of likelihood function evaluated at the maximum likelihood estimates for the model parameters, and p = number of model parameters. The equations used to back-calculate the lengths at presumed ages were where L, = the mean LJFL at age t; Lx = the asymptotic length; o the hypothetical age at length zero; M/? o aj s N cd UJ CO 1 / to co N n n o _ — CO •* CN -— — rH c~ cN 00 CN - rt to ra - CO [^ 31 N ^] _ C£ ^h rH rj CD ^_ *-< ao co o ai as Q [^ X CO N CO CN CN CN CN CM CN I I 00 CO t> 00 00 I— 1 ai £ CO iH m CO o Tp CN c~ CM CN £ CM 00 o in CO CO 01 O o 00 CN CN CM CO CO CO CO CO CO CM CO o o 1 - £ ao CO CM CM 0- £ ■* t> 00 "CP CM O 1 1 CO o CN CO CO CO CO CO CO CO o o CM O O CO Tp CO 1~i CO CM o o o Tp O r-i d Q C CO in m oo T C CO ■^ -* ■* m m o o i> 3 5 in m 00 CD CO ■* m CN ■* ■<* •<* Tf o o [ - r*- »— i 00 CO CO CN ^p ao rp LO CO CM CO in CN m CO t- 3 3 CC o 00 IO CM CM o ao CN CO CN Tp Tp CO TP ■* TP o o C£ CO ^H [> ^H y-4 a cd © ^j oi co co co t^ ^ co co CO CO CO CO CO CO o ^ "^ t— --^ ^^ i-t CO CO i-t i^- ^h X O CO O O "-' rt CD UC rr ^-t CO CO CO CO CO CO CO 00 CO ■* o ^p CO a> CN o o CO o CO ^p 00 in ao CO ao CN CN CN CN CN CN ^ - CD W ' h~ CO ■>* iH CO CM o o o o CO ao m o CO o CM O 01 3 3 in t— CO ^H CN 3 in 00 in tp 00 ao CN in CM in CO in H H 1^ CO CO I I I I I I I I I I I I I s _i ^HCNco^incot^ooaio CO o o CO o o c o ■* CO CD ^ H ^ CO TP ^ I> ao r^ ^ 1 1 ^ ^* CO CM o CO 1 CM CO 0 CO CO CO CO CM CO o o co co ao CO CM CO cm d d ^p in CN CO CN O o CM o o TP o i-i d 3 S Chiang et al.: Age and growth of Ist/ophorus p/atypterus in waters off eastern Taiwan 259 09 - Male All ages combined 0.8 - 0.7 - 06 - f 43 117 158 3 18 Ti^A T /"I ^^-^r \ 48 8 12 S\ T^ 32 05 - I\ 17/ 0.4 - o | 09 - I 08 - E § 07- c 75 0,6 - c CD £ 05 - 5 04 - i T 50 81 Ages 1-5 /1^\I^^I^ 73 ' A 1/ ^ T^^^^k 10 r ^v T 3 / \J 6 T "\ 7 / P\ T A i — f r\ 7 / 09 -i 08 ■ 07 ■ 06 ■ i 1 , \ Ages 6-11 67 f ; '7 28 T I l_ — f^^^^T \ 38 26 T 05 ■ i — T\ 10 y\ 0.4 - JFMAMJJASOND Month Figure 5 Monthly means of marginal increment ratio for male sailfish (/. platy- pterus) in the waters off eastern Taiwan for all ages combined and for age classes 1-5 and 6-11, respectively. Vertical bars are ±1 SE; numbers above the vertical bars are sample sizes. Table 3 Mean back-calculated lower jaw fork lengths at age for sailfish in the waters off eastern Taiwan. Back-calculated length (cm) Method 1 Method 2 Age (yr) Method 1 Method 2 Age lyr) Male Female Male Female Male Female Male Female 1 108.53 113.41 99.90 103.51 7 181.11 185.36 181.86 186.09 2 125.70 130.79 121.79 126.32 8 188.99 192.82 189.84 193.67 3 138.82 143.90 137.27 141.96 9 194.98 200.60 196.59 201.47 4 150.80 156.02 150.56 155.54 10 200.78 207.85 201.74 208.81 5 161.78 166.22 162.12 166.38 11 208.05 213.29 209.14 214.66 6 171.63 176.60 172.18 177.12 12 217.15 219.05 260 Fishery Bulletin 102(2) 0.9 I Female All ages combined 0.8 ■ 0.7 - 0.6 • 0.5 • 2 J. 2 2 20 52 93 116 88 25 1\ m \ 19 NL 10 /i 04 - 03 - 1 — i — i — i — i — i 0.9 08 0.7 - 0.6 05 - 0.4 - 0.3 09 08 07 06 05 0.4 03 02 40 Ages 1-5 - 1 r~ —l 1 1 1 Ages 6-12 — i 1 1 1 1- JFMAMJJASOND Month Figure 6 Monthly means of marginal increment ratio for the female sailfish (/. platypterus) in the waters off eastern Taiwan for all ages combined and for age classes 1-5 and 6-12, respectively. Vertical bars are ±1 SE; num- bers above the vertical bars are sample sizes. (1996), and Sun et al. (2002) favored method 2 because they believed it to be more biologically realistic. When the back-calculated lengths-at-age are generated with this method the Richards function provides a statistically superior fit to the length-at-age data. Therefore, the pa- rameter estimates for the Richards function with method 2 listed in Table 4 are recommended as the most appropriate for calculating the age composition of sailfish in the waters to the east of Taiwan. It is perhaps worth noting that the tu values estimated for the Richards function with method 2 are much closer to zero than those estimated for the Richards function with method 1. Comparison with previous studies Figure 9 compares the age-length relationships of this paper with those for Atlantic (de Sylva, 1957; Hedgepeth and Jolley, 1983; Farber1) and Pacific sailfish (Koto and Kodama, 1962; Alvarado-Castillo and Felix-Uraga. 1998). De Sylva ( 1957 ) and Koto and Kodama ( 1962 1 used length- frequency analysis and concluded that sailfish are a very fast growing and short-lived species. However, they likely underestimated age and overestimated growth rate when their results are compared with those of other more recent studies. The maximum ages found in this study (11 years for males and 12 years for females) are close to the maximum longevity of at least 13 years proposed by Prince et al. (1986) based on tagging data. Farber1 analyzed Atlantic billfish tagging data and suggested that the asymptotic size was essentially reached by age 3 (Hedgepeth and Jol- ley, 1983). whereas the present study found a more gradual increase in length with age. in common with the results of Hedgepeth and Jolley (1983). Chiang et al.: Age and growth of Istiophorus platypterus in waters off eastern Taiwan 261 250 -I Male 200 - p aij*||»|Pe§>o' ylliliP^0 * 150 ■ 0o >^P^ _^l°J - 100 ■ ° n = 699 o -- LJFL =64.825 + 30.471 R r2 = 0.704 -J 50 ■ ll -J — LJFL = 79.833 R06'2 r2 = 0.720 length o i i i i i i 1 25°- Female S TO, S 200 - o iJ0^^ 150 - <0^ 100 - o 0 n =436 o -- LJFL =70.31 2 + 30.093 R r = 0.731 50 - — LJFL =83.461 R0596 r2 = 0.750 0 12 3 4 5 6 Spine radius (R, mm) Figure 7 Relationship between lower jaw fork length and spine radius for male and female sailfish (/. platypterus) in the waters off eastern Taiwan. Table 4 Parameter estimates and standard errors (in parenthesis) for the standai •d von Berta anffy growth function and the Richards function for sailfish in the waters off eastern Taiwan. Standard von Bertalan ffy growth function Richards function Method 1 Method 2 Method 1 Method 2 Parameter Male Female Male Female Male Female Male Female L, 252.6 261.4 240.4 250.3 271.8 280.4 294.0 343.8 (3.652) (3.397) (3.794) (4.278) (22.713) (19.882) (29.607) (47.921) k 0.115 0.110 0.145 0.138 (0.005) (0.004) (0.008) (0.008) t0 -3.916 -4.207 -2.781 -2.990 -2.473 -2.608 -0.704 -0.468 (0.143) (0.147) (0.154) (0.186) (0.931) (0.896) (0.279) (0.186) A' 0.051 0.049 0.023 0.011 (0.034) (0.030) (0.013) (0.007) m -0.551 -0.578 -1.288 -1.639 (0.472) (0.436) (0.308) (0.243) 262 Fishery Bulletin 102(2) 250 H 200 150- 100- 50- Male Standard VB - method 1 Standard VB - method 2 Richards function - method 1 Richards function - method 2 zsu- Female o 200- i L-4^**^^ 150- 100- 4/ Standard VB- method 1 - - - - Standard VB - method 2 50- ■' **/ / Richards function • method 1 f | Richards function - method 2 n - 1 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 -5-4-3-2-1 Age (year) 9 10 11 12 Figure 8 Observed and back-calculated length-at-age and standard von Bertalanffy and Richards function model-predicted growth curves for male and female sailfish <■!. platypterus) in the waters off eastern Taiwan. 300 -i 250 200 150 100 - 50 - -de Sylva (1957) -sexes combined' - Koto and Kodama (1 962) - sexes combined' - Farber (1981) - sexes combined* - Hedgepeth and Jolley (1 983) - male" - Hedgepeth and Jolley (1 983) - female' - Alvarado-C and Felix-U. (1998) - sexes combined - Present study - male - Present study - female 6 7 8 Age (year) 10 12 13 Figure 9 A comparison of the growth curves for sailfish (/. platypterus) esti by different authors. I ■ Data from Table 1 of Hedgepeth and Jolley mated 1983.1 use in stock assessments of the sailfish popu- lation in the western Pacific Ocean. Acknowledgments The authors express sincere gratitude to Andre Punt, School of Aquatic and Fishery Sciences. University of Washington, for his valuable comments and comprehensive edit- ing of the manuscript. This study was in part supported financially by the "Fisheries Agency, Council of Agriculture, Taiwan," through grant 91AS-2.5.1-FK7) to Chi-Lu Sun. Literature cited Even though the aging method used in the present study is the same as that of Hedgepeth and Jolley (1983) and Alvarado-Castillo and Felix-Uraga (1998), there are nev- ertheless differences in the estimated length-at-age. 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Radtke, R. L. 1983. Istiophorid otoliths: extraction, morphology, and pos- sible use as ageing structures. NOAA Tech. Rep. NMFS 8:123-129. Radtke, R. L., and J. M. Dean. 1981. Morphological features of the otoliths of the sailfish, Istiophorus platypterus, useful in age determination. Fish. Bull. 79:360-367. Richards, F. J. 1959. A flexible growth function for empirical use. J. Exp. Bot. 10:290-300. Sun, C. L., C. L. Huang, and S. Z. Yeh. 2001. Age and growth of the bigeye tuna, Thunnus obesus, in the western Pacific Ocean. Fish. Bull. 99:502-509. Sun, C. L., S. P. Wang, and S. Z. Yeh. 2002. Age and growth of swordfish (Xiphias gladius L.,) in the waters around Taiwan determined from anal-fin rays. Fish. Bull. 100:822-835. Tserpes, G., and N. Tsimenides. 1995. Determination of age and growth of swordfish, Xiphias gladius L., 1758, in the eastern Mediterranean using anal-fin spines. Fish. Bull. 93:549-602. von Bertalanffy, L. 1938. A quantitative theory of organic growth (Inquiries on growth laws. II). Hum. Biol. 10:181-213. Williams, F. 1970. The sport fishery for sailfish at Malindi, Kenya, 1958-1968, with some biological notes. Bull. Mar. Sci. 20(4):830-852. 264 Abstract— A density prediction model for juvenile brown shrimp (Farfan- tepenaeus aztecus) was developed by using three bottom types, five salinity zones, and four seasons to quantify pat- terns of habitat use in Galveston Bay, Texas. Sixteen years of quantitative density data were used. Bottom types were vegetated marsh edge, submerged aquatic vegetation, and shallow non- vegetated bottom. Multiple regression was used to develop density estimates, and the resultant formula was then coupled with a geographical informa- tion system (GIS) to provide a spatial mosaic (map) of predicted habitat use. Results indicated that juvenile brown shrimp (<100 mm) selected vegetated habitats in salinities of 15-25 ppt and that seagrasses were selected over marsh edge where they co-occurred. Our results provide a spatially resolved estimate of high-density areas that will help designate essential fish habitat (EFH) in Galveston Bay. In addition, using this modeling technique, we were able to provide an estimate of the over- all population of juvenile brown shrimp (<100 mm) in shallow water habitats within the bay of approximately 1.3 billion. Furthermore, the geographic range of the model was assessed by plotting observed (actual) versus expected (model) brown shrimp densi- ties in three other Texas bays. Similar habitat-use patterns were observed in all three bays — each having a coef- ficient of determination >0.50. These results indicate that this model may have a broader geographic application and is a plausible approach in refining current EFH designations for all Gulf of Mexico estuaries with similar geo- morphological and hydrological char- acteristics. A habitat-use model to determine essential fish habitat for juvenile brown shrimp (Farfantepenaeus aztecus) in Galveston Bay, Texas Randall D. Clark John D. Christensen Mark E. Monaco Biogeography Program Center for Coastal Monitoring and Assessment National Center for Coastal Ocean Science National Ocean Service. NOAA Silver Spring, Maryland 20910 E-mail address (For R. D Clark) Randy Clarkfflnoaa gov Philip A. Caldwell Geoffrey A. Matthews Thomas J. Minello Fishery Ecology Branch Galveston Southeast Fisheries Science Center Laboratory National Marine Fisheries Service, NOAA Galveston, Texas 77550 Manuscript approved for publication 22 December 2003 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Scientific Publications Office. Fish. Bull. 102:264-277 (200 1 1 Shallow estuarine habitats, whose com- plexity promotes survival and growth, are used by many young fish and macro- invertebrate species (Boesch and Turner, 1984). A complete understand- ing of how these habitats sustain spe- cies productivity is unknown and has become a focal point of federal fishery management programs. The National Marine Fisheries Service (NMFS) has developed guidelines to identify essential fish habitat (EFH) for all federally managed species based on four levels of available information that encompass the ecological linkages between habitats and fishery produc- tion. Examination of habitat-use pat- terns (habitat-related densities) are needed to determine which habitats are likely to be most essential. These patterns are measurable and can be reasonable indicators of habitat value. Relative habitat values have been esti- mated by comparing animal densities under the assumption that high densi- ties reflect greater habitat quality and preferred habitat (Pearcy and Myers, 1974; USFWS, 1981; Zimmerman and Minello, 1984; Sogard and Able, 1991; Baltzetal., 1993). Considerable bottom-type variation exists in northern Gulf of Mexico estu- aries, including intertidal marsh, sub- merged aquatic vegetation, oyster reef, mangroves, tidal mudflats, and sub- tidal bay bottom. Within each of these habitats, environmental and structural gradients may affect the functional role or importance of these habitats for particular species. To understand these relationships, fisheries indepen- dent monitoring (FIM) data are needed to determine species-habitat affini- ties that provide evidence that not all habitats are of equal importance for the maintenance of a population (Monaco et al., 1998; Minello 1999; Beck et al., 2001). Habitat affinities may change with spatial and temporal fluctuations of environmental variables, such as sa- linity and temperature (Copeland and Bechtel, 1974; Baltz et al., 1998). In this study we developed predictive models that estimate brown shrimp {Farfantepenaeus aztecus, formerly Penaeus aztecus [see Perez-Farfante and Kensley, 1997]) habitat-use pat- terns and interactions as a function of density-independent processes in Galveston Bay, Texas. Previous com- Clark et al.: A habitat-use model for juvenile Farfantepenaeus aztecus In Galveston Bay 265 29 5° N 29° N North A ■i- %&*% y Trinity Bay C^l Galveston ^ Bay "^*^- East <_k ■:■ • i. j? v. ■ ' .- '■ ... r' v . \. ) 7^ ' i j& m vf- *-\Christmas Kilometers i 1 Bay 0 10 95 W Figure 1 Map of Galveston Bay, Texas. 94.5° W parisons of brown shrimp densities among different bot- tom types in Louisiana and Texas estuaries have been conducted within limited temporal and spatial scales (Peterson and Turner, 1994; Zimmerman et al., 1984; Zim- merman et al., 1990b; Rozas and Minello, 1998; Minello, 1999). Our work expands upon these studies by developing a multivariate bottom-type use and environmental model incorporated into a geographic information system (GISi that provides a spatial assessment of habitat use. In ad- dition, the model is designed to be transferable to other northern Gulf of Mexico estuaries and thus would allow fishery managers to identify the relative importance of habitat types for population maintenance and recruitment into the fishery. Materials and methods Geographic setting The Galveston Bay complex (Fig. 1) encompasses approxi- mately 2020 km2 and is one of the largest estuaries in the northern Gulf of Mexico (NOAA, 1989). Comprising several major embayments, including Trinity, Galveston, East, and West bays, the complex contains many smaller interconnecting subbays, rivers, streams, tidal creeks, wetlands, reefs, and tidal flats around its periphery. The bay bottom is mostly flat and shallow (mean depth is approximately 2 m) and has slightly elevated oyster reefs, elevated dredge material areas, river channels, and deeper dredged navigation channels. Data collection Sixteen years (1982-97) of brown shrimp density data were analyzed to quantify areas of potential EFH. A total of 46,080 brown shrimp were captured during this time period with a mean total length of 27.5 mm (Fig. 2). Data from published studies by Czapla (1991), Minello et al. ( 1991), Minello and Zimmerman ( 1992), Minello and Webb ( 1997 ), Rozas and Minello ( 1998 ), Zimmerman et al. (1984, 1989, 1990a, 1990b), Zimmerman and Minello (1984), and various unpublished sources from the Galveston Laboratory of the National Marine Fisheries Service were combined to comprise a comprehensive density database of associated bottom-type and environmental data that would support model development and GIS analyses. All samples were collected by using a drop trap sampler, described in Zimmerman et al. (1984), which employs large cylinders ( 1.0 or 2.6 m2 area) released from a boom affixed to a boat to entrap organisms. This quantitative technique samples fishes and macro-invertebrates in highly structured shal- low-water habitats such as salt marshes, seagrass beds, and oyster reefs where the efficiency of conventional trawl and bag-seine gear is diminished. 266 Fishery Bulletin 102(2) Habitat mapping The underlying spatial framework for incorporating model predictions into the GIS consisted of six maps: four salinity periods, one bathymetric map, and one map defining bottom-type distribution. All GIS maps were developed in Universal Transverse Mercator projection, UTM, datum-1983, zone-15, using ArcView 3.1 (Redlands, CA) software. Each map consisted of 10 x 10 m grid cells where each cell contained pertinent salinity, depth, or bottom-type information. Salinity maps were developed from depth-aver- aged salinity models by using historical Galveston Bay data collected during 1979-90 (Orlando et al., 1993). Four salinity periods were identified to rep- resent typical salinity conditions under average sea- sonal freshwater inflow: low (March-June), increas- ing (July), high (August-October), and decreasing (November-February). Five isohalines were developed to display spatial salinity distribution (Christensen et al.1): 0-0.5, 0.51-5, 5.1-15, 15.1-25, and >25 parts per thou- sand (ppt) (Fig. 3). Bottom types from the drop sample database were di- vided into three categories: Marsh edge (ME) Submerged aquatic vegetation (SAV) Shallow non- vegetated bottom (SNB) intertidal marsh within 5 meters of open water habitat. This category consisted primarily of saltmarsh cord grass (Spartina alterniflora), and smaller proportions of salt meadow- grass {Spartina patens), black needle- rush {Juncus roemerianus), salt grass (Distichlis spicata), bullrushes iScir- pus spp.), and cattails (Typha spp.); consisted primarily of shoalgrass {Halodule wrightii), wigeongrass iRuppia maritima), and a sporadic distribution of wild celery (Vallisneria americana); generally restricted to waters less than 1 meter deep, including creeks, ponds, shoreline, and open bay habitat. Density data for other bottom types were limited and were not used in the analysis. Wetland maps, used in the creation of the bottom type map in the GIS, were obtained from the U.S. Fish and Wild- life Service's national wetland inventory (NWI). The NWI maps were obtained as vector files, created by digitizing boundaries between wetland types from 1989 aerial photo- graphs and classified by using the classification scheme of Cowardin et al. ( 1979). Regularly flooded emergent vegeta- tion and submerged aquatic vegetation distributions from Christensen, J. D., T. A. Battista, M. E. Monaco, and C. J. Klein. 1997. Habitat suitability modeling and GIS technol- ogy to support habitat management: Pensacola Bay, Florida Case Study, 58 p. NOAA/NOS Strategic Environmental Assessments Division, Silver Spring, MD. 40 50 60 70 Total length (mm) Figure 2 Total-length frequency distribution for juvenile brown shrimp captured in drop traps within Galveston Bay (1982-971. the NWI maps of Galveston Bay were chosen to represent ME and SAV, respectively, from the drop sample database. Nonvegetated open water areas with depths greater than 1 m were eliminated throughout the bay to reflect depth range from the drop sample database. This elimination was done by plotting approximately 400,000 depth sound- ings obtained from the National Geophysical Data Center (NGDC ), and a bathymetric grid map was developed in 1-m contours with ArcView 3.1 (6 nearest neighbors, power=2). The nonvegetated open water map from NWI was overlaid with the bathymetric map and only those areas within the 1-m contour were extracted and added to the bottom-type map (Fig. 4). Two maps were used to plot (map) seasonal model predictions, bottom type, and the respective salinity period. The salinity maps did not completely correspond temporally with seasons defined by cluster analysis of in situ temperature recordings from the density database. Salinity periods were chosen to correlate with temporal seasons based on maximum monthly overlap to develop the seasonal prediction maps: low salinity (spring); increas- ing salinity (summer); high salinity (fall); and decreasing salinity (winter). The total area of Galveston Bay (2020 km2) was deter- mined by combining the total areas for regularly flooded emergent vegetation, irregularly flooded emergent vegeta- tion, SAV, and open water classifications from NWI data. The bottom-type map reflects the study area and totaled 565.6 km2 after excluding all areas >1 m in depth and with irregularly flooded emergent vegetation: SNB = 476.2 km2. ME = 84.9 km2, and SAV = 4.5 km2. Initially, NWI's SAV classification totaled 5.7 km2, but the final SAV coverage was reduced to 4.5 km2 based on SAV mapping by White etal. (1993). Regression modeling ANOVA and Tukey-Kramer multiple means comparisons were used to determine if mean density varied significantly by bottom type, salinity zone, and season. Multiple regres- sion with significant predictors was used to predict mean log density. The model was then applied to the GIS maps Clark et al.: A habitat-use model for juvenile Farfantepenaeus aztecus in Galveston Bay 267 Low salinity (April-June) Increasing salinity (July) &m High salinity (August-October) Decreasing salinity (November-March) Salinity Zone (ppt) □ 0-0.5 CZZI 0 51-5 CD 51"15 CZ] 15 1-25 H > 25 Figure 3 Galveston Bay seasonal salinity distribution maps. to spatially display model predictions in each 10 x 10 m cell. The resulting values for each cell (predicted mean log density) were converted to numbers/m2 and reclassified into 5 percentiles based on their resultant distribution: 0-20%, 21-40%, 41-60%, 61-80%, and 81-100%. All statistical analyses were conducted with JMP statistical software (SAS Institute, Cary, NO. Due to difficulties in creating continuous salinity and temperature contour maps in GIS, these variables were classified as follows: salinity was classified by one of the five isohaline zones described previously and analyzed as such to determine its influence on brown shrimp distri- bution; and water temperature was classified by season determined by cluster analysis and analyzed to examine possible temporal effects of brown shrimp distribution. Spatial patterns were evaluated by comparing the pre- dicted mean log density values with the observed mean log density values from Galveston Bay drop samples. Addi- tionally, the model's predictive performance was assessed by comparing the predicted mean log density values with observed mean log density values from samples collected in Matagorda, Aransas, and San Antonio bays using the same collection method. With this approach, the assump- tion was made that brown shrimp modeled in Galveston Bay respond similarly to the range of biotic and abiotic factors in the other bay systems. Drop sample data collected during July-September 1984 (/i=128), and April- June 1985 (re=144) from West Bay (ME, SNB) and Christmas Bay (ME, SAV, and SNB) were used to examine bottom-type preference or selectivity. Tukey- Kramer multiple comparisons test was used to compare log density patterns in areas where ME and SAV occurred together and in areas where SAV was not present. Results Brown shrimp model ANOVA and Tukey-Kramer pair-wise comparisons showed significant differences in brown shrimp log density between the three bottom types, five salinity zones, and four seasons (Fig. 5). Multiple regression models were run with these discreet variables (Mahon and Smith, 1989; 268 Fishery Bulletin 102(2) "T .^ Nonvegetated bottom (SNB) Marsh edge (ME) Submerged aquatic vegetation (SAV) Figure 4 Spatial distribution of Galveston Bay bottom types used in the multivariate regression model. Results of the least squares multiple * = significant at P < 0.05. regression model for Table 1 predicting seasonal brown shrimp density in Galveston Bay, Texas. Model fit r2 Mean Observations in) Mean square error 0.73 0.47 47 0.20 Source ANOVA df Sum of squares Mean square F ratio Prob > F Model Error Total 17 29 46 5.74 1.61 6.90 0.33 0.04 8.43 <0.0001* Source Effects df Sum of squares F ratio Prob > F Season Bottom type Salinity zone Bottom typex Salinity zone 3 2 4 8 1.85 0.61 3.15 0.86 15.43 7.57 19.68 2.69 <0.0001* 0.0023* <0.0001* 0.0242* Krumgalz et al., 1992; Garrison, 1999) and we tested for possible interactions between the variables. Only the interaction between bottom type and salinity zone yielded statistically significant results. ANOVA results for the model including the bottom-type and salinity-zone interac- tion term (Table 1) and variable coefficients (Table 2 (fitted Clark et al.: A habitat-use model for juvenile Farfantepenaeus aztecus in Galveston Bay 269 Table 2 Variable coefficients (log +1) derived from brown shrimp multivariate regression model. ME aquatic vegetation; SNB = shallow nonvegetated bottom. marsh edge; SAV = submerged y-intercept Bottom type Season 0.335 0.113 (ME) 0.043 (SAV) -0.156 (SNB) 0.239 (spring) 0.165 (summer) -0.045 (fall) -0.359 (winter) Salinity zone Bottom type x salinity zone -0.525 -0.147 0.079 0.286 0.307 (0-0.5) (0.5-5) (5-15) (15-25) (>25) -0.104 -0.055 0.159 0.273 -0.396 0.123 -0.030 0.049 -0.018 -0.119 0.288 -0.168 -0.018 0.114 -0.096 (ME/0-0.5) (SAV/0-0.5) (SNB/0-0.5 (ME/0.5-5) (SAV/0.5-5) (SNB/0.5-5) (ME/5-15) (SAV/5-15) (SNB/5-15) (ME/15-25) (SAV/15-25) (SNB/15-25) (ME/>25) (SAV/>25) (SNB/>25) the data well (r2=0.73, n=47). Overall, density predictions were highest in the spring, declined through summer and fall, and reached the lowest values during winter (Fig. 6). SNB density predictions were highest in the >25 ppt salin- ity zone and declined as salinity declined in the estuary. ME density predictions exhibited similar density predic- tion trends; however, a smaller peak was observed in the 0.5-5 ppt salinity zone. This result may be an artifact of two fall samples that exhibited high density within this salinity zone. Density predictions within SAV were near zero in the lower two salinity zones, peaked in the 15-25 ppt salinity zone, and slightly decreased in the >25 ppt salinity zone. Model prediction maps For all seasons, highest density predictions corresponded with ME and SAV bottom types within the region of the bay with highest salinity — Christmas and West bays (Fig. 7). Density predictions decreased within all bottom types as salinity declined in the middle and upper regions of the bay. Spring density predictions were the highest; maxi- mum values were predicted within ME (6.14/m2) and SAV (14.49/m2) located in Christmas and West bays (Fig. 7). Density predictions steadily declined through the middle bay and declined to 1/m2 or less within SAV and SNB in the upper region of the bay (Trinity Bay) where salinities were less than 5 ppt. Density predictions during summer, fall, and winter were lower than those observed during the spring but exhibited similar spatial trends — higher pre- dictions within the high salinity vegetated bottom types, and decreasing with decreasing salinity. Model performance Spatial patterns were assessed by plotting predicted mean density values from the model and observed mean density A 1" K 08 * 0 6 0.4 ■ * 0.2 ■ /•2=0.16 P<0.0001 SAV ME SNB Bottom type B 08 * >. 06 I 0.4- * 3 0.2 r2=0.06 $ P<0.0001 0-0.5 0-5.5 5-15 15-25 >25 Salinity zone c '■ 0 8 - 0.6 - * * 04 - * 02 ■ r2=0.13 P<0.0001 spring summer fall winter Season Figure 5 Analysis of variance and Tukey-Kramer pair-wise comparisons of brown shrimp density between (A) bottom type, (B) salinity zone and, (C) season. Mean densities are represented by solid diamonds and lines determine standard error. SAV = submerged aquatic vegetation; ME = marsh edge; SNB = shallow non- vegetated bottom. 270 Fishery Bulletin 102(2) SNB 1.2 1 0.8 0.6 0.4 0.2 0 li ifll spring summer fall winter Salinity zone |a 0-0.5 D 0.5-5 □ 5-15 G15-25B> 25 | Figure 6 Seasonal density predictions for brown shrimp (F. aztecus) by bottom type and salinity zone. ME = marsh edge; SAV = submerged aquatic vegetation; SNB = shallow nonvegetated bottom. values from drop sample data collected in Galveston Bay. Regression analysis from this plot exhibited a strong posi- tive relationship (r2=0.83, P<0.0001) between predicted and observed density data (Fig. 8). This analysis was per- formed to verify how the model represented the observed density data. Model performance and transferability were assessed by regressing predicted mean density values from the Galveston Bay model on observed mean density values from drop sample data collected in Matagorda, San Antonio, and Aransas bays (Fig. 9). Regression analy- sis produced a positive relationship for the entire drop sample data from these bays combined (r2=0.56) and in- dividually: Matagorda — r2=0.54; San Antonio — r2=0.57; and Aransas — r2 = 0.56. In Aransas and San Antonio bays, brown shrimp densities were greatest during the spring within the SAV bottom type and within salinities >15 ppt. In Matagorda Bay, brown shrimp densities were greatest in the spring within ME bottom types in waters >15 ppt. No SAV samples were taken in this estuarine system. Use of bottom types Results from spring (1985) and fall (1984) drop samples within Christmas and West Bay (in lower Galveston Bay) bottom types revealed significantly greater brown shrimp densities in Christmas Bay SAV than adjacent ME and SNB (P<0.0001). Brown shrimp densities in West Bay ME were not significantly different from Christmas Bay SAV but were significantly greater than densities within adjacent SNB and Christmas Bay ME and SNB bottom types (Fig. 10). The model results were also used to roughly estimate an overall population of approximately 1.3 billion juve- nile brown shrimp in Galveston Bay during the spring season, by multiplying predicted densities by bottom-type area (Table 3). Total area of bottom types in Galveston Bay were as follows: 4.5 km2 (SAV); 84.9 km2 of marsh edge (ME); and 1627.2 km2 of nonvegetated bottom (29% [476.2 km2] of the latter area was considered SNB). On the basis of predicted densities in different salinity regimes, we estimated that there would be 51.0 million shrimp Clark et al.: A habitat-use model for juvenile Farfantepenaeus aztecus in Galveston Bay 271 spring summer winter Predicted density (#/m2 ) m 0-0.12 0.13-1.51 1.52-2.72 2.73-5.46 ■I 5.47-14.85 Figure 7 Seasonal spatial distribution maps of predicted densities for brown shrimp (F. aztecus). in SAV and 858.7 million shrimp in SNB. We used marsh edge densities to estimate 473.5 million shrimp in regu- larly flooded vegetation or about 55,700 shrimp per hectare of this habitat type. Discussion Various factors are considered important in defining nursery areas for juvenile estuarine-dependent organ- isms; however, the specific contributions of these factors are poorly understood (Beck et al., 2001). Specific combi- nations of physiochemical conditions and cyclic primary production that are related to food availability, growth, and sanctuary from predation often define optimal envi- ronments (Miller and Dunn, 1980). Barry et al. (1999) considered prey availability to be a necessary component defining the nursery function of estuarine habitats. Shrimp and blue crab production has been correlated with the availability of wetland habitat in estuaries (Turner, 1977; Zimmerman et al., 2000). In the present study, brown shrimp were most abundant in the lower bay where vegetated habitats were most abundant. Zimmerman et al. ( 1990b) reported that benthic infauna are most abundant in vegetated habitats within lower Galveston Bay and are nutritionally important for penaeids (Zein-Eldin and Renaud, 1986; McTigue and Zimmerman, 1991, 1998). In addition, field and laboratory experiments have shown that brown shrimp growth is positively correlated with the abundance of marsh epiphytes and phytoplankton (Gleason and Zimmerman, 1984). Most estuarine nekton are adaptable to the highly dynamic environmental conditions exhibited within es- tuaries (Gifford, 1962; Tagatz, 1971; Zimmerman et al., 1990b). These organisms are commonly found in a wide range of salinities and temperatures and are most affected by sudden changes in these environmental conditions 272 Fishery Bulletin 102(2) Table 3 Estimated area (km2) of each bottom type and salinity zone combination sampled dur ing spring (March- -May), and estimated brown shrimp population based on spring density predictions from the model. ME = marsh edge; SAV = submerged aquatic vetetation; SNB = shallow nonve getated bottom. Salinity zone Bottom type area Density estimate Population estimate Shrimp/ha. Bottom type (ppt) (km2) (number/m2) (millions) (thousands) ME 0-0.5 1.4 0.14 0.2 1428 0.5-5 1.6 5.50 8.8 55,000 5-15 22.4 4.44 99.4 44,375 15-25 59.5 6.14 365.3 61,394 >25 0 8.46 0 0 Total 84.9 473.5 55,771 SAV 0-0.5 1.0 0.09 0.09 9000 0.5-5 0.03 0.18 0.005 1667 5-15 0.02 4.56 0.09 45,000 15-25 3.5 14.52 50.8 145,142 >25 0 9.91 0 0 Total 4.5 51.0 114,680 SNB 0-0.5 29.6 0 0 0 0.5-5 54.2 1.01 54.7 10,092 5-15 183.6 1.61 295.6 16,100 15-25 203.3 2.41 489.9 24,097 >25 5.5 3.37 18.5 33,636 Total 476.2 858.7 18,032 Total 565.6 1383.2 24,455 (Christensen et al., 1997). In laboratory experiments, Zein-Eldin and Aldrich (1965) concluded that higher sa- linities are more favorable for brown shrimp. Salinities of ME + SAV x SNB 0.2 0.4 0.6 0.( Observed log density Figure 8 Relationship between predicted and observed densities of brown shrimp (F. aztecus) in Aransas, Matagorda and San Antonio bays and predicted densities from the Galveston Bay model. ME = marsh edge; SAV = submerged aquatic vegetation; SNB = shallow nonvegetated bottom. 20 ppt or greater were considered optimum in data from Louisiana (Barrett and Gillespie, 1973). In the present study, brown shrimp were captured throughout Galveston Bay, but highest densities were observed in the lower bay where salinities were greater than 15 ppt. This spatial trend was further strengthened by greater abundance of vegetated bottom types in the lower portions of the bay, where nearly half of the total marsh edge and 90% of sea- grass beds are located (Fig. 4). These bottom types are regularly inundated and provide stable substrate for brown shrimp prey (epiphytic algae and infauna), whereas seasonal oligohaline marsh and SAV habitats in the upper bay may not promote favorable condi- tions for prey organisms (Zimmerman et al., 1990b). Therefore, salinity effects and the greater availability of vegetated habitats in the lower bay may work in a complementary manner to provide nursery areas for brown shrimp in Galveston Bay. Previous attempts to examine spatial patterns of abundance and to determine linkages between organisms and habitat included the development of habitat suitability index (HSI) models. Early methods were derived by the U.S. Fish and Wildlife Service (USFWS) for freshwater species, where the HSI was defined as a numerical index that represented the capacity of a given habitat to support a selected spe- cies. The scale of HSI values (0-1.0) reflects a linear relationship between suitability and carrying capacity Clark et al.: A habitat-use model for juvenile Farfantepenaeus aztecus in Galveston Bay 273 1.2 1.0 0.8 0.6 0.4 02 0.0 -0.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 + V + jr ■ >^ ■ + ■ + S ■ y* * - ■ ■ ■ ./ • X * x . •/*& s + x is « _ ■>♦ All Bays ^ ♦ n = 63 r2 = 0.56 + + yT X /^ ' x S ♦ X Aransas Bay n = 9 r2 = 0.56 x ■ _, X x •\^ x x^^^' X - X > Matagorda Bay n = 25 r2 = 0.54 I I I i i i + + s *s* * x ^r X v^ San Antonio Bay ^ + n = 29 r2 = 0.57 i i I i i i 0 0.2 0.4 0.6 0.8 1.0 1.2 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Observed log density ME + SAV x SNB Figure 9 Relationships between observed densities of brown shrimp iF. aztecus) in Aransas, Matagorda, and San Antonio Bays and predicted densities from the Galveston Bay model. Relationships for all bays combined are shown in the upper left graph. For each relationship, the r2 is shown for the least squares regression, and the number of observations (n ) and the total number of samples in parentheses. ME = marsh edge; SAV = submerged aquatic vegetation; SNB = shallow nonvegetated bottom. (USFWS, 1981). Recently, Christensen et al.1 and Brown et al., 2000, developed suitability indices, based on lit- erature reviews and expert opinion, and raster-based GIS models that produce a spatial view of relative suitability. The Florida Fish and Wildlife Conservation Commis- sion-Marine Research Institute (FMRI) and the National Ocean Service's Center for Coastal Monitoring and As- sessment (NOS/CCMA) collaborated to develop a suite of quantitative HSI modeling approaches, using fisheries- independent monitoring catch-per-unit-of-effort (CPUE) data (Rubec et al., 1998, 1999, 2001). These studies used an unweighted geometric mean formula as part of the HSI models to assess overall suitability. This approach assigns equal weight to all factors by using scaled suitability indi- ces as inputs to the model. The regression approach used in this study more appropriately weights density according to the factors in the model and allows a more robust tech- nique to elucidate spatial patterns of habitat use by using actual CPUE data. In addition, the method described in our study can support more complex analyses, such as interaction effects or trophic relationships (or both). Our ANOVA (Table 1) revealed that season, bottom type, salinity, and the interaction between salinity and bottom type are significant factors that influence the distribution of juvenile brown shrimp in Galveston Bay. The addition of the interaction effect to the model increases the coefficient of determination from 0.63 to 0.73. Without this term in the model, predicted values for brown shrimp density are overestimated compared to the observed density data. Seagrass beds in salinities greater than 15 ppt supported significantly greater densities of brown shrimp than did marsh edge. However, in locations with salinities less than 15 ppt, brown shrimp densities were not significantly different between the two bottom types. These results in- dicate significantly lower use among all the bottom types analyzed in the fresher portion of the estuary. It is likely that salinity and a combination of other environmental factors directly or indirectly (or directly and indirectly) affect abundance on bottom types and habitat quality in this region. The results indicate that SAV supports greater brown shrimp density than do ME and SNB; however, SAV accounts for less than 1% of the total bottom type within 274 Fishery Bulletin 102(2) April-June 1985 1.5 - * 1.0 - i 0.5 - i r2=0.46 * * P<0.0001 July-September 1984 1.5 - i i 1.0 - $ 0.5 - i r2=o.59 i 0 - P<0.0001 i I i i West Bay ME West Bay SNB Xmas Bay ME Xmas Bay SAV Xmas Bay SNB Figure 10 Brown shrimp (F. aztecus) observed log density and standard deviation for bottom types in Christmas Bay and West Bay. ME = marsh edge: SAV = submerged aquatic vegetation; SNB = shallow nonvegetated bottom. Galveston Bay. Our data suggest that brown shrimp select SAV over ME when these habitats co-occur (Christmas Bay) and select ME when grassbeds are absent (West Bay) ( Fig. 10 ). Habitat submergence time may explain high SAV use in Christmas Bay (Rozas and Minello, 1998). Subtidal grassbeds may provide more continuous refuge and food supply at both low and high tides than the marsh surface, which can be accessed only during high tides. Additionally, brown shrimp were significantly smaller in SAV (,v = 17 mm) than in ME (5=25 mm) (t-test, P<0.001>. which may imply ontogenetic changes in habitat or trophic require- ments (Conrow et al., 1990; Thomas et al., 1990; Rozas and Minello, 1999). Differences in the use of bottom types may correspond with the population's size distribution at the time of sampling. Additional research is needed to reveal ontogenetic habitat shifts and relationships among shal- low estuarine bottom types (Mclvor and Rozas, 1996). Assessment of the model performance was based on FWS HSI theory where there is a positive relationship between HSI value and the carrying capacity of the avail- able habitat. In the present study, the relationship equates high brown shrimp densities with optimal habitat condi- tions that promote high carrying capacity. Therefore, low densities would reflect a low suitability or a low capacity to support the population. Comparisons of predicted den- sity with that of observed values from Galveston Bay, and other Texas bays (Figs. 7 and 8) agree with FWS theory by exhibiting a strong relationship between density and suitable habitat as determined from the model. Model per- formance and transferability were examined by applying Clark et al.: A habitat-use model for juvenile Farfantepenaeus aztecus in Galveston Bay 275 the Galveston Bay model (with interaction term) to brown shrimp density data from Aransas, Matagorda, and San Antonio bays. The results indicated similar habitat-use patterns in Aransas and San Antonio bays; there were higher densities in high-salinity seagrass beds and a de- clining density as salinity decreased in these bay systems. No SAV samples were taken in Matagorda Bay; however, the model performed well in predicting greater brown shrimp density in higher-salinity marsh-edge habitats. Our analysis suggests that although the empirical model is complex, it is general enough to be applicable across a broader range of habitat types. The model results may, however, have some geographic limitations. For instance, the model may not perform well within the Laguna Madre in south Texas, where freshwater inflow is diminished and hypersaline conditions exist. This conclusion is consistent with Rubec et al. (1999), who used similar methods to demonstrate that HSI models are applicable across estuar- ies in central Florida. Our results are promising in view of previous efforts where predictions of nekton abundance with empirical models have proven difficult. Currently, estuarine EFH for most federally managed species in the Gulf of Mexico exists as mapped estimates of relative abundance from NOS's estuarine living marine resources (ELMR) database (GMFMC, 1998; Nelson and Monaco, 2000). The entire Galveston Bay complex was considered EFH for brown shrimp based on ELMR relative abundance data. Our model, generated by using brown shrimp density data, provides a more spatially resolved delineation of EFH (in waters <1 m depth) for brown shrimp <100 mm. The analyses described in the present study focused on bottom types in waters less than 1 m which comprise about 25% of the available habitat in Galveston Bay. Trawl CPUE data from Texas Parks and Wildlife De- partment (TPWD) were analyzed to compare abundance and distribution patterns in waters >1 m. These trawls (3.8-cm stretched mesh) do not capture small size classes (<50 mm TL) of brown shrimp efficiently; thus the trawl analysis provides information only on larger size classes (mean=89 mm). However, few individuals in smaller size classes of shrimp (<50 mm TL) are likely to inhabit deeper bay waters; density estimates of small nekton, including shrimp, decline rapidly with depth (Mock, 1966; Baltz et al., 1993; Rozas, 1993; Rozas and Zimmerman, 2000). In addition, these CPUE values are likely underestimates of brown shrimp density; catch efficiency for shrimp in trawls can be roughly estimated at 20f/f (Zimmerman et al., 1984; Rozas and Minello, 1997). Despite these problems, shrimp abundance estimates in water >1 m appear low; abun- dance estimates from TPWD trawl data in deep open-bay waters were almost two orders of magnitude lower than densities in shallow water habitats. Brown shrimp population estimates from the present study (Table 3) were highest in the lower bay (224,568 per ha.). Seagrass beds accounted for more than 607i of the es- timate ( 145,142 per ha.) and marsh edge and nonvegetated bottom types combined were estimated at approximately 79,000 per ha. As noted earlier, the NWI regularly flooded emergent vegetation classification is not all marsh edge but is a complex of SNB, marsh edge, and inner marsh with different shrimp densities associated with each of these microhabitat types. Minello and Rozas (in press) modeled small-scale density patterns on the marsh surface in a 437-ha. salt marsh of lower Galveston Bay and applied these data to a GIS analysis of marsh landscape patterns. In this highly fragmented marsh complex that was 37% SNB and 63% marsh vegetation, they estimated brown shrimp populations at 37,000 per ha. We could not estimate brown shrimp populations in irregularly flooded emergent vegetation, although the areal coverage of this habitat type was large. Compared with the regularly flooded wetlands, overall densities of brown shrimp in these irregularly flooded systems should be relatively low because of higher marsh surface elevations (Rozas and Reed, 1993; Minello et al., 1994; Minello and Webb, 1997) and restricted tidal access (Rozas and Minello, 1999). We also were unable to assess the contribution of oyster reef as habitat for brown shrimp. Coen et al. ( 1999), however, reported brown shrimp on oyster reefs, and Powell ( 1993 ) estimated that there was 108 km2 of this habitat in Galveston Bay. Our modeling results provide evidence that estuarine habitat types are discriminately used by brown shrimp. The success of transferring our empirical model from Galveston Bay to adjacent bay systems in Texas suggests that the model has a broad application and can possibly be used to simulate patterns of habitat use in systems that lack sufficient density data. Continuing collections of density data in Gulf estuaries are necessary to make additional interestuary comparisons and to determine whether these habitat-use patterns differ throughout the distributional range of brown shrimp. The use of other habitat types also needs to be examined. For example, other available habitat types from Galveston Bay, such as oyster reef and inner marsh, and from other Gulf estuar- ies, such as mangrove, calcium carbonate rock formations, and sponge communities, may be important habitats for this federally managed species. Acknowledgments Funding and support for this work was provided by the Southeast Region of NOAA's National Marine Fisheries Service, The Southeast Fisheries Science Center, and the Biogeography Program of the National Ocean Service. We would like to thank Pete Sheridan, Lawrence Rozas, Ken Heck, and Roger Zimmerman for providing access to pub- lished and unpublished data sets. John Boyd helped with construction of the nekton density database. Literature cited Baltz, D. M., J. W. Fleeger, C.F. Rakocinski, and J. N. McCall. 1998. Food, density, and microhabitat: factors affecting growth and recruitment potential of juvenile saltmarsh fishes. Environ. Biol. Fish. 53:89-103. Baltz, D. M., C. Rakocinski, and J. W. Fleeger. 1993. Microhabitat use by marsh-edge fishes in a Louisiana estuary. Environ. Biolog. Fish. 36:109-126. 276 Fishery Bulletin 102(2) Barret, B. B., and M. C. Gillespie. 1973. Primary factors which influence commercial shrimp production in coastal Louisiana. La. 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Minello, and G. Zamora. 1984. Selection of vegetated habitat by Penaeus aztecus in a Galveston Bay salt marsh. Fish. Bull. 82:325-336. 278 Abstract— Queen conch (Strombus gigas) stocks in the Florida Keys once supported commercial and recreational fisheries, but overharvesting has decimated this once abundant snail. Despite a ban on harvesting this spe- cies since 1985, the local conch popu- lation has not recovered. In addition, previous work has reported that conch located in nearshore Keys waters are incapable of spawning because of poor gonadal condition, although reproduc- tion does occur offshore. Queen conch in other areas undergo ontogenetic migrations from shallow, nearshore sites to offshore habitats, but conch in the Florida Keys are prevented from doing so by Hawk Channel. The pres- ent study was initiated to determine the potential of translocating non- spawning nearshore conch to offshore sites in order to augment the spawning stock. We translocated adult conch from two nearshore sites to two off- shore sites. Histological examinations at the initiation of this study confirmed that nearshore conch were incapable of reproduction, whereas offshore conch had normal gonads and thus were able to reproduce. The gonads of nearshore females were in worse condition than those of nearshore males. However, the gonadal condition of the translocated nearshore conch improved, and these animals began spawning after three months offshore. This finding suggests that some component of the nearshore environment (e.g.. pollutants, tem- perature extremes, poor food or habitat quality) disrupts reproduction in conch, but that removal of nearshore ani- mals to suitable offshore habitat can restore reproductive viability. These results indicate that translocations are preferable to releasing hatchery- reared juveniles because they are more cost-effective, result in a more rapid increase in reproductive output, and maintain the genetic integrity of the wild stock. Therefore, translocating nearshore conch to offshore spawn- ing aggregations may be the key to expediting the recovery of queen conch stocks in the Florida Keys. Translocation as a strategy to rehabilitate the queen conch (Strombus gigas) population in the Florida Keys Gabriel A. Delgado Claudine T. Bartels Robert A. Glazer Florida Fish and Wildlife Conservation Commission Florida Marine Research Institute 2796 Overseas Highway, Suite 119 Marathon, Florida 33050 E-mail address (for G. A Delgado) gabneLdelgado;g>fwc. state fl us Nancy J. Brown-Peterson Department of Coastal Sciences College of Science and Technology The University of Southern Mississippi P.O. Box 7000 Ocean Springs, Mississippi 39566 Kevin J. McCarthy National Marine Fisheries Service, NOAA 75 Virginia Beach Drive Miami, Florida 33149 Manuscript approved for publication 24 November 2003 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Scientific Publications Office. Fish. Bull. 102:278-288(20041. The queen conch (Strombus gigas) is a large marine gastropod harvested intensively throughout the Caribbean for its meat and shell. In the Florida Keys, conch once supported commercial and recreational fisheries, but overhar- vesting severely depleted the popula- tion. The harvesting of conch has been banned in Florida since 1985, but the population has not recovered to levels that can support exploitation (Glazer and Berg. 1994; Berg and Glazer, 1995; Glazer and Delgado, 2003). Intensive fishing may invoke depensatory mecha- nisms as densities are reduced, limit- ing the ability of conch to locate mates and increasing the chance of recruit- ment failure (Appeldoorn, 1995). This seems to be the case in Florida because the lack of recovery has been attrib- uted to diminished recruitment due in part to small spawning aggregations (Stoner et al., 1997; Stoner and Ray- Culp, 2000). Queen conch occur in the various oceanside habitats of the Florida Keys archipelago with the exception of Hawk Channel (Glazer and Berg, 1994). This naturally occurring deep-water channel runs parallel to the Florida Keys, be- tween the island chain and the offshore reef tract. The substrate on the bottom of Hawk Channel is predominantly soft sediment, which is poor conch habitat; consequently, Hawk Channel serves as a barrier to migration and isolates nearshore from offshore conch aggre- gations (Glazer and Berg, 1994). We have been monitoring queen conch stocks throughout the Florida Keys since 1987, and despite extensive sur- veys, we have never observed reproduc- tive activity among conch in nearshore aggregations (Glazer and Berg, 1994). Conversely, reproductive behavior has been commonly observed among conch in offshore aggregations (Glazer and Berg, 1994). Moreover, a preliminary histological examination of conch from these two regions indicated that the gonads of offshore conch were capable of undergoing gametogenesis, whereas the gonads of nearshore conch were nonfunctional (Glazer and Quintero, Delgado et al.: Translocation of Strombus gigas as a strategy to rehabilitate the Florida Keys conch population 279 DK Key West 4jSt-^*' PS Atlantic Ocean I I Hawk Channel Reef Tract 25 00' N 24 30' N 8130'W 81 00' W 80 30' W Figure 1 Queen conch lStroi7ibus gigas) translocation sites in the Florida Keys (adapted from McCarthy et al. 2002). The nearshore region is the stretch of water on the landward side of Hawk Channel; the offshore region is the stretch of water on the other side of the channel, contiguous with the Atlantic Ocean. Nearshore conch were translo- cated from Tinglers Island (TI) to Alligator Reef (AR) and from Duck Key (DK) to Pelican Shoal (PS). 1998; McCarthy et al., 2002). In a metapopulation context, the nearshore region in the Florida Keys can be considered a "blackhole sink" for larval recruitment because conch that settle there do not spawn and thus do not contribute to the reproductive output of the stock (se?isu Morgan and Botsford, 2001). In 1990, the Florida Fish and Wildlife Conservation Commission's (FWC) Florida Marine Research Institute constructed an experimental hatchery to test the feasi- bility of rehabilitating queen conch stocks in the Florida Keys by releasing hatchery-reared juveniles. A series of experiments to determine the best size of juveniles, time of release, and area to release hatchery-reared juvenile conch were conducted, and a cost-benefit analysis was performed. Unfortunately, the high mortality of conch after release, coupled with high production costs, caused us to examine alternate strategies (Glazer and Delgado, 2003). Translocation is defined as the intentional introduction or reintroduction of animals in an attempt to establish, reestablish, or augment a population in order to aid in the recovery of a native species whose numbers have been re- duced by overharvesting or habitat loss (or both) (Griffith et al., 1989). This method of population recovery has been used to facilitate the recovery of numerous species of birds and mammals (Griffith et al., 1989) and several aquatic species, including cutthroat trout (Harig et al., 2000) and corals (Edwards and Clark, 1999; Rinkevich, 1995; van Treeck and Schuhmacher, 1997). Nest translocations have also proven effective in efforts to recover sea turtles (Garcia et al., 1996). The present study was initiated to determine the po- tential of translocating nonspawning nearshore conch to offshore sites as a method to augment spawning aggrega- tions and as an aid in the recovery of the queen conch population in the Florida Keys. However, this strategy will be beneficial only if the translocated conch regain their reproductive capacity. To test this approach, we translo- cated adult conch from the nearshore region into existing offshore breeding aggregations and examined changes in reproductive behavior (i.e., mating and spawning) and gonadal development. Materials and methods Translocations and reproductive behavior During March 1999, we translocated adult conch from nearshore aggregations to aggregations offshore. Near- shore aggregations were located at Tinglers Island (24°41'N, 8r05'W; water depth 75%. For statistical analyses, this index was reduced to two categories: <50% and >50%. Statistical analyses We evaluated differences in reproductive behavior (mating and spawning) between resident nearshore and translo- cated nearshore conch for each season by using Fisher's exact test because it is not sensitive to small sample sizes (Zar, 1996). We also examined differences in gonadal condition (i.e., gonadal maturity and the percentage of gametogenic tissue) between resident nearshore and resident offshore conch for each season by using Fisher's exact test. Males and females were analyzed separately. In order to assess the effectiveness of the translocations to the offshore region, we used Fisher's exact test to compare the gonadal condition of translocated nearshore conch with the gonadal condition of resident nearshore conch in summer and in fall. Again, the sexes were analyzed separately. All tests were run on SPSS 9.0 (SPSS Inc., Chicago, ID for Windows. Results were considered sig- nificant if P<0.05. Results Reproductive behavior: mating Approximately 84% of the tagged resident nearshore conch. 69% of the tagged translocated nearshore conch, and 88% of the tagged resident offshore conch were observed at least once during monitoring. Resident nearshore conch Table 3 Percentage of mating (the number of males and females mating divided by the total number of conch observed during that season) and spawning (the number of females spawning divided by the total number of females observed during that season I in nearshore conch and offshore conch by season I adapted from McCarthy et al.. 2002 ). Numbers in parentheses represent the number of observations; P represents the probabilities from Fisher's exact test of differences in reproductive behavior between resi- dent nearshore and translocated nearshore conch. The asterisk (*) indicates that the test was statistically sig- nificant. N/A indicates that statistical analyses were not conducted because no mating or spawning was observed among either resident nearshore or translocated near- shore animals. Offshore conch Nearshore conch Resident Resident Translocated P Mating Spring Summer Fall Spawning Spring Summer Fall 5.3(95) 0.0 1 37 i 2.4(4671 0.0(1061 0.9(2321 0.0(20) 46.2(39) 0.0(6) 16.8(191) 0.0(34) 5.2(97) 0.0(9) 0.0(19) N/A 0.0(81) N/A 0.0(51) N/A 0.0(10) N/A 12.2(41) 0.041* 18.5(27) 0.214 and translocated nearshore conch were not observed mating during any of the field surveys; conversely, resi- dent offshore conch were observed mating throughout the study (Table 3). The mating frequency of resident offshore conch was highest during the spring ( 5.3% ) and decreased during subsequent seasons to 0.9% in the fall (Table 3). All observed mating occurred between resident offshore animals. Reproductive behavior: spawning Neither resident nearshore females nor translocated near- shore females were observed spawning during the spring (Table 3). However, by summer, translocated nearshore females had attained the capacity to spawn and had a significantly higher spawning frequency than resident nearshore females (12.2% vs. 0.0% , respectively) (Table 3). During the fall, spawning frequency of translocated nearshore females peaked at 18.5%, whereas resident nearshore females had still not exhibited any spawn- ing behavior (Table 3). However, this difference was not statistically significant because of the small number of resident nearshore conch observed (Table 3). Looking at individual performance instead of spawning frequency, seven (or about 14%) of the approximately 50 nearshore females translocated offshore were observed spawning at least once during the study period. 282 Fishery Bulletin 102(2) " " TV.- w . '■ "0 o k O-' VJ, ■■ B i • - &* < • >: ^ it ' ■ / . •■ S§i s , ; . u E i:; 4 ..<-_.■> v* Figure 2 Photomicrographs of the gonadal condition of resident nearshore, resident offshore, and translocated nearshore queen conch (Strombus gigas). (A) Resident nearshore female during spring, no tissue and <25% gametogenic tissue. (B) Resident offshore female during spring, ripe and >75% gametogenic tissue. (C) Translocated nearshore female during summer, late development and 25-50% gametogenic tissue. (D) Resident nearshore male during spring, early development and <25% gametogenic tissue. (E) Resident offshore male during spring, ripe and >75% gametogenic tissue. (F) Translocated nearshore male during summer, ripe and 25-5095 gametogenic tissue. Resident offshore females were observed spawning throughout the study (Table 3 1. Their spawning frequency peaked during the spring at 46.2% and decreased during subsequent seasons to 5.2% in the fall (Table 3). Histology: females Histological examinations revealed that the gonadal con- dition of resident nearshore and resident offshore female Delgado et al.: Translocation of Strombus gigas as a strategy to rehabilitate the Florida Keys conch population 283 Females 100 80 • 60 40 20 100 80 60 40 20 • 100 80 60 40 20 0 Spring n i Summer i i n . n .1 i Fall n n Jllii A« 6^ J Males Spring ill ■ 1 Summer Ml 1 Li Fall n 1 <8 6B s" i c5- 9 d? ■& resident nearshore resident offshore Figure 3 Gonadal maturity of resident nearshore and resident offshore queen conch (Strom- bus gigas) by sex and season. The dotted line separates the categories that were combined for statistical analyses. conch were markedly different at the beginning of the study (Fig. 2. A and B). There were significant differences in gonadal maturity between resident offshore and resi- dent nearshore female conch during the spring, summer, and fall (Table 4). During the spring, the gonads of most resident offshore females were categorized as being in late development; by summer most were ripe and by fall most were either spent, in atresia, or regressed (Fig. 3). In contrast, the gonads of most resident nearshore females contained no germ cells during the spring (Fig. 3). By summer, the gonads of some resident nearshore females were found to be in the early stages of development, but most females were still incapable of spawning, and by fall, all the resident nearshore females sampled were incapable of spawning (Fig. 3). There were also significant differ- ences in the percentage of gametogenic tissue between resident offshore and resident nearshore females during the spring, summer, and fall (Table 4). The gonads of most resident offshore females contained >75% gametogenic tissue throughout the study period, whereas those of most resident nearshore females had <25% (Fig. 4). The gonadal condition of translocated nearshore females (Fig. 2C) improved when compared with the gonadal condition of resident nearshore females (Fig. 2A). There were significant differences in gonadal maturity between Table 4 Probabilities from Fisher's exact test of differences in gonadal maturity and the percentage of gametogenic tissue between resident nearshore and resident offshore conch by sex and season. n r ?presents the total number of observations. Asterisks ( I indicate that the test was statistically significant. Females Males n P H P Gonadal maturity Spring 35 <0.001* 32 0.004* Summer 33 <0.001* 32 0.002* Fall 35 0.006* 21 <0.001* % gametogenic tissue Spring 35 <0.001* 32 <0.001* Summer 33 <0.001* 32 <0.001* Fall 35 0.002* 21 <0.001* translocated nearshore and resident nearshore females during both the summer and fall (Table 5). There was 284 Fishery Bulletin 102(2) Females Males a 60 40 ■ 20 ■ 0 80 i- 60 CD ~ 40 | 20 D- 0 80 60 40 20 0 Spnng I J_i Q_m In rfi J1 &' ,5? & & Spnng mmer It rail IL n resident nearshore resident offshore Figure 4 The percentage of gametogenic tissue of resident nearshore and resident offshore queen conch iStrombus gigas) by sex and season. The dotted line separates the cat- egories that were combined for statistical analyses. Nearshore females Nearshore males 100 80 60 40 20 100 80 • 60 ■ 40 20 0 An L n n L Summer J nl I Fall [ ] n ■ ?*/ & 8 75' i of the gonad, whereas most resi- dent nearshore females still had <259c gametogenic tissue (Fig. 6). Histology: males There were marked differences in gonadal condition of resident near- shore and resident offshore male conch (Fig. 2, D and E). There were significant differences in gonadal maturity between resident offshore and resident nearshore male conch during the spring, summer, and fall (Table 4). During the spring and summer, the gonads of most resi- dent offshore males were catego- rized as ripe; by fall most were spent (Fig. 3). In contrast, at least half of the resident nearshore males were not capable of spawning during the spring and summer, although some were in the early stages of tes- ticular development and some were even ripe (Fig. 3). However, all the sampled resident nearshore males were incapable of spawning by fall and none were identified as spent (Fig. 3). Histological examinations also revealed significant differ- ences in the percentage of game- togenic tissue between resident offshore and resident nearshore males during the spring, summer. Delgado et al.: Translocation of Strombus gigas as a strategy to rehabilitate the Florida Keys conch population 285 Nearshore females Nearshore males Figure 6 The percentage of gametogenic tissue of resident nearshore and translocated near- shore queen conch {Strombus gigas) by sex and season. The dotted line separates the categories that were combined for statistical analyses. and fall (Table 4). Most resident offshore males had >75% gametogenic tissue throughout the study period, whereas most resident nearshore males had <25% (Fig. 4). The gonadal condition of translocated nearshore males (Fig. 2F) improved in relation to the gonadal condition of resident nearshore males (Fig. 2D). There were significant differences in gonadal maturity between translocated nearshore and resident nearshore males during both the summer and fall (Table 5). Almost 80% of the translocated nearshore males were ripe during the summer, whereas about half of the resident nearshore males were incapable of reproducing (Fig. 5). By fall, most translocated near- shore males were still capable of reproduction, whereas none of the resident nearshore males were (Fig. 5). There were also significant differences in the percentage of gametogenic tissue between resident nearshore and translocated nearshore males during the summer and fall (Table 5). During the summer, the gonads of most of the resident nearshore males contained <25% game- togenic tissue, whereas translocated nearshore males were divided equally among the four gametogenic tissue categories (Fig. 6). During the fall, the gonads of most of the resident nearshore males still had <25% gametogenic tissue; however, most translocated nearshore males had developed >50'/r of the gonad (Fig. 6). Discussion In the nearshore region of the Florida Keys, adult queen conch had severe deficiencies in reproductive behavior and gonadal development. Histological examinations of resi- Table 5 Probabilities from Fisher's exact test of differences in gonadal maturity and the percentage of gametogenic tissue between resident nearshore and translocated near- shore conch by sex and season, n represents the total number of observations. Asterisks (*) indicate that the test was statistically significant. Females Males Gonadal maturity Summer Fall 7o gametogenic tissue Summer Fall 26 0.019* 23 <0.001* 26 0.130 23 0.038* 24 10 0.045* 0.033* 24 0.014* 10 0.033* dent nearshore conch revealed that most were incapable of reproducing, whereas resident offshore conch exhibited a normal reproductive cycle (as described by Egan, 1985, and Stoner et al., 1992). Furthermore, our results suggest that female conch may be more sensitive to the nega- tive effects of nearshore conditions than male conch. For example, during the spring and summer, some resident nearshore males were ripe (although their reproductive output would have been severely reduced because of a low percentage of gametogenic tissue), whereas the gonads of 286 Fishery Bulletin 102(2) most resident nearshore females contained no germ cells. The latter condition may have been due to the fact that egg production is more costly bioenergetically than sperm production (Ricklefs, 1990). Mating and spawning do not occur among resident near- shore conch presumably because of their retarded gonadal development; however, the translocation of nearshore conch to the offshore region mitigated the deleterious ef- fects that the nearshore environment had on their gonadal development. The reproductive tissues of translocated nearshore conch began to develop during the summer after the conch had spent about three months offshore. Most translocated female conch were in the early stages of gonadal development, whereas most translocated male conch were ripe. We believe this difference in gonadal de- velopment is due to the fact that the starting gonadal con- dition of nearshore females was worse than the starting condition of male conch. By fall, after six months offshore, most translocated females had become ripe. In addition, the percentage of gametogenic tissue in the gonads of both sexes increased through the summer and fall. In conjunction with the improvement in gonadal condi- tion, nearshore females translocated to the offshore region were observed spawning during the summer and fall; however, no mating was observed among nearshore conch translocated offshore. Resident offshore conch also had low mating frequencies (<6"7r ). Similarly low mating frequen- cies have been reported in the Virgin Islands (Randall, 1964) and the Bahamas (Stoner et al., 1992). We suspect that the lack of observations of nearshore conch mating in the offshore region may have been an artifact of the low probability of encountering that activity due to the small number of nearshore conch translocated offshore. Never- theless, we believe mating must have occurred because translocated nearshore conch were observed spawning. However, it is unknown if queen conch are capable of lay- ing unfertilized egg masses. The beginning of reproductive activity in queen conch is linked to the start of spring, when water temperatures begin rising (Randall, 1964; Stoner et al, 1992; Weil and Laughlin, 1984). This same seasonal pattern was observed in our study with resident offshore conch. They exhibited the highest mating and spawning frequencies during the spring and reproductive behavior decreased during the ensuing seasons. However, compared with the spawn- ing pattern of resident offshore conch, peak spawning in translocated nearshore conch was delayed; peak spawning occurred during the fall. Nevertheless, there was evidence to suggest that the timing of reproductive behavior of both resident offshore and translocated nearshore conch might eventually become similar. Our results indicated that it takes at least three months after translocation for the negative effects of the nearshore environment to be mitigated and for gonadal maturation to occur. The out- of-phase spawning may have been prevented if the trans- locations had occurred earlier in the year (e.g., January, instead of March). Identifying the causative factor or factors that inhibit the reproductive viability of nearshore queen conch re- quires further study. However, the juxtaposition of the nearshore conch aggregations with human population cen- ters suggests that anthropogenic changes to the nearshore region may be partially responsible. Decreased reproduc- tive output caused by anthropogenic contaminants has been observed in several marine invertebrates, including dogwinkles iNucella lapillus) (Bryan et al., 1987; Gibbs and Bryan, 1986), scallops (Gould etal., 1988), sea urchins (Krause, 1994; Thompson et al., 1989), and shrimps and crabs (Wilson-Ormond et al., 1994). For example, chronic exposure to tributyltin has been shown to sterilize females of several species of mollusks (Matthiessen and Gibbs, 19981, and sublethal levels of copper greatly inhibited gamete production and maturation in scallops (Gould et al., 1988). There have also been numerous reports impli- cating eutrophication in nearshore habitat degradation in the Florida Keys (Lapointe et al., 1990; Lapointe and Clark, 1992; Szmant and Forrester, 1996); however, very little is known about the effects of increased nutrient levels at the organismal level. The retarded gonadal condition in nearshore queen conch may also be due to environmental factors such as suboptimal habitat, poor food quality, or temperature extremes associated with shallow water. Research on bivalves has shown that habitat, diet, and food quality directly affect gamete production (Le Pennec et al.. 1998: Madrones-Ladja et al., 2002). As they increase in age and size, queen conch undergo ontogenetic migrations from shallow, nearshore sites to deeper-water habitats (Ran- dall, 1964; Sandt and Stoner, 1993; Stoner, 1989; Weil and Laughlin, 1984). It has been hypothesized that as conch grow larger and require more food, they migrate to take advantage of the augmented food supply in more produc- tive offshore habitats (Sandt and Stoner. 1993; Stoner. 1989). However, nearshore queen conch in the Florida Keys are prevented from migrating offshore by Hawk Channel (Glazer and Berg, 1994). Therefore, translocat- ing nearshore conch offshore would, in effect, link these isolated environments. The implications of this study are of particular impor- tance to the FWC-Florida Marine Research Institute's ongoing queen conch stock restoration program. Trans- locating naturally recruiting nearshore conch to offshore areas would be more cost effective than hatchery produc- tion of juvenile conch, especially because production costs are eliminated and survival of translocated conch is likely to be much greater than that of hatchery outplants (see Stoner, 1997. for a review of juvenile mortality in stock enhancement efforts). Translocations would also have a more immediate effect on reproductive output than would the release of hatchery-reared conch. A translocation pro- gram would focus on moving large juveniles and adults offshore, whereas a hatchery program must release small juveniles (to minimize production costs) that would then have to survive to maturity. Consequently, translocations would quickly alleviate the depensatory mechanisms de- scribed by Appeldoorn (1995) that can affect the recovery of queen conch stocks. Finally, translocations provide the added benefit of maintaining the genetic diversity of the population. Hatchery-reared conch are typically derived from a few egg masses and there is a concurrent loss in Delgado et al.: Translocation of Strombus gigas as a strategy to rehabilitate the Florida Keys conch population 287 rare alleles (Allendorf and Ryman, 1987). However, the use of wild conch to enhance the spawning aggregations eliminates this problem. Queen conch appear to be a prime candidate for reha- bilitation by translocation because they meet the criteria associated with successful translocations reported by Griffith et al. (1989). These factors include release within the historical range of the species or into areas of in- creased habitat quality (or both). Additionally, herbivorous animals stand a greater chance of translocation success than do carnivores or omnivores. Lastly, wild animals translocate more successfully than captive-bred animals. According to these parameters, queen conch are ideally suited for translocations. However, before a full-scale translocation program can be implemented, there are some theoretical considerations that must be addressed. For example, Stoner and Ray- Culp (2000) reported that conch reproductive behavior reached an asymptotic level near 200 conch/ha.; therefore, it would seem advantageous to enhance reproductive ag- gregations to that density. However, without high habitat quality, translocations have low success rates regardless of how many animals are released (Griffith et al., 1989). First, we must ascertain if offshore habitats can support the added number of conch or if the translocated or na- tive animals (or both) will simply disperse after release because of density-dependent factors (e.g., intraspecific competition for limited resources). Conch grazing has been shown to significantly reduce the biomass of seagrass mac- rodetritus and epiphytes (Stoner, 1989). In addition, the effects of removing nearshore conch from the nearshore environment need to be investigated. Additionally, if increased recruitment is the ultimate goal of the translocation program, larvae must survive and be retained within the Florida Keys. At this point, it is unknown whether larvae produced from translocated nearshore conch are viable or as viable as the larvae pro- duced by native offshore conch. Furthermore, the relative contribution of local and upstream sources to recruitment is unknown. Stoner et al. (1996, 1997) suggested that most of the queen conch larvae entering the Florida Keys come from upstream sources. If this is indeed the case, then local translocations will not be as effective as an international or regional management strategy. However, mechanisms for larval retention in the Florida Keys have been described by Lee and Williams (1999), who suggested that the pe- riodic formation of gyres in the lower Keys may facilitate the retention and recruitment of locally produced larvae. If larvae are retained within the Florida Keys system, any increase in local larval production will increase larval sup- ply and may increase recruitment. Therefore, translocation sites should be located in the lower Keys in order to ensure maximum larval retention and recruitment. The present study has shown that translocation may be a viable method for rehabilitating queen conch populations in the Florida Keys. We have demonstrated that nearshore conch that were translocated offshore regained some of their reproductive capacity and abilities. Therefore, mov- ing conch from nearshore larval sinks to offshore larval sources may be the key to expediting the recovery of queen conch stocks. Further research (e.g., larval retention studies, studies on the effect of water quality on larval survival, carrying capacity studies) and monitoring will determine the efficacy of this restoration strategy. Acknowledgments John Hunt, William Sharp, James Colvocoresses, Allan Stoner, and one anonymous reviewer provided insightful comments on the manuscript. Judy Leiby and Jim Quinn provided editorial comments. We thank Mary Enstrom and Sherry Dawson of The Nature Conservancy (TNC) as well as the numerous TNC volunteers who participated in the field surveys. Meaghan Darcy and other staff members at the Florida Marine Research Institute assisted in the field and in sample processing. 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Prentice Hall, Upper Saddle River, NJ. 289 Abstract— This study examines gene- tic variation at five microsatellite loci and at the vesicle membrane protein locus, pantophysin, of Atlantic cod {Gadus morhua) from Browns Bank, Georges Bank, and Nantucket Shoals. The Nantucket Shoals sample rep- resents the first time cod south of Georges Bank have been genetically evaluated. Heterogeneity of allelic dis- tribution was not observed (P>0.05) between two temporally separated Georges Bank samples indicating potential genetic stability of Georges Bank cod. When Bonferroni correc- tions («=0.05, P<0.017) were applied to pairwise measures of population differentiation and estimates of FST, significance was observed between Nantucket Shoals and Georges Bank cod and also between Nantucket Shoals and Browns Bank cod. However, nei- ther significant differentiation nor sig- nificant estimates ofFST were observed between Georges Bank and the Browns Bank cod. Our research suggests that the cod spawning on Nantucket Shoals are genetically differentiated from cod spawning on Browns Bank and Georges Bank. Managers may wish to consider Nantucket Shoals cod a sepa- rate stock for assessment and manage- ment purposes in the future. Genetic differentiation among Atlantic cod (.Gadus morhua) from Browns Bank, Georges Bank, and Nantucket Shoals Christopher Lage Department of Biological Sciences Murray Hall University of Maine Orono, Maine 04469 Kristen Kuhn Irv Kornfield School of Marine Sciences Murray Hall University of Maine Orono, Maine 04469 E-mail address (for I Kornfield, contact author): irvk@maine.edu Manuscript approved for publication 5 November 2003 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Scientific Publications Office. Fish. Bull. 102:289-297 (2004). The Atlantic cod (Gadus morhua) is a migratory gadid found on both sides of the North Atlantic. In the Northwest Atlantic, cod are distributed nearly continuously along the continental shelf from Greenland to North Caro- lina, spawning in relatively discrete, temporally stable areas, and differ- ent regions are regarded as different management units defined primarily by latitude and bathymetry ( Ruzzante et al, 1998). Atlantic cod historically supported economically important fisheries in the Northwest Atlantic (Halliday and Pinhorn, 1996). In U.S. waters, cod are assessed and managed as two stocks: 1) Gulf of Maine and 2) Georges Bank and southward (includ- ing Nantucket Shoals). Growth rates differ between the two stocks; growth is slower in the Gulf of Maine compared to growth in Georges Bank (Pentilla et al., 1989 ); each stock is exploited by the same gear type and may show similar biological responses towards such gear selection. Although both stocks sup- port important commercial and recre- ational fisheries, each is overexploited and remains at a low biomass level (Mayo and O'Brien, 1998; O'Brien and Munroe, 2001; Mayo et al„ 2002). Over- exploitation may result in significant life-history changes such as a decline in time to reproductive maturity which has been observed in Georges Bank cod (O'Brien, 1998); such changes maybe a compensatory response to overfishing but may also be influenced by shifts in underlying genetic control (Policansky. 1993). Commercial fisheries are conduct- ed year round, using primarily otter trawls and gill nets. The Canadian fishery on Georges Bank is managed under an individual quota system. United States cod fisheries are man- aged under the New England Fishery Management Council's Northeast Mul- tispecies Fishery Management Plan (FMP)1 as implemented by the U.S. Federal Register, 50 CFR Part 648 (U.S. Federal Register, 2003). Under this FMP, cod are included in a com- plex of 15 groundfish species managed by time and area closures, trip limits, gear restrictions, minimum size limits, days-at-sea restrictions, and a permit moratorium. The FMP's goal is to re- duce fishing mortality to levels that will allow stocks within the complex to initially rebuild above minimum biomass thresholds, and, ultimately, to remain at or near target levels. When ecological and evolutionary processes are responsible for stock structuring, it is necessary to incorpo- 1 New England Fishery Management Coun- cil. 2003. Northeast Multispecies Fishery Management Plan. NEFMC. 50 Water St., Mill 2, Newburyport. MA, 01950 290 Fishery Bulletin 102(2) rate them into strategies designed to manage exploited species (Avise, 1998). High dispersive capabilities of many marine fish often correlate with low levels of population di- vergence over vast areas (Ward et al., 1994; Graves, 1998) and may be particularly true for species characterized by high fecundity, large population size, and potentially long- distance egg and larval dispersal. Although marine fish predominantly have high dispersal rates and low levels of population structuring, migratory species with continuous distributions may develop and maintain stock structure if they show fidelity to natal spawning sites or limited egg and larval dispersal. Fidelity to natal grounds has been shown in Greenland-Iceland cod (Frank, 1992) and Georges Bank haddock (Polacheck et al., 1992). Genetic divergence between areas originates when populations are formed or through the restriction of gene flow. Cod in some regions are known to migrate long distances, whereas in other regions they are nearly stationary (Lear and Green, 1984). Tagging studies in the Gulf of Maine show little exchange between the region east of Browns Bank and Georges Bank, and the inner Gulf of Maine (Hunt et al., 1999); however exchange has been reported among Bay of Fundy, southern Nova Scotia, Browns Bank, and Georges Bank populations (Klein-MacPhee, 2002). Such exchange among cod from different management areas may be im- portant for stock assessments and management practices. Determining underlying genetic structure of spawning stocks is paramount to the conservation and management of overexploited species. In the last 30 years the use of molecular-based stud- ies in fisheries science has become common (Shaklee and Bentzen, 1998). In cod, a number of studies have used allozymes (Moller, 1968; Jamieson, 1975; Cross and Payne, 1978; Dahle and Jorstad, 1993), but their use and sensitivity are limited because of weak statistical power resulting from low levels of polymorphism and because of processes of balancing selection (Mork et al., 1985; Pogson et al, 1995). Mitochondrial DNA (mtDNA) char- acterization among Northwest Atlantic cod indicates that there is limited, albeit significant, population structuring throughout most the species' range (Smith et al., 1989; Carr and Marshall, 1991; Pepin and Carr, 1993; Carr et al., 1995; Arnason and Palsson, 1996). Genetic divergence at the vesicle membrane protein locus, pantophysin (Panl), originally called GM798 and identified as synaptophysin {SypT) (Fevolden and Pogson, 1997), has been reported among populations of cod from the Northwest Atlantic (Pogson, 2001; Pogson et al., 2001), Norway and the Arctic (Fevolden and Pogson, 1997), and Iceland (Jonsdottir et al., 1999, 2002). High levels of variation have been re- ported at nuclear RFLP loci (Pogson et al., 1995; Pogson et al., 2001), and especially at microsatellite loci (Bentzen et al., 1996; Ruzzante et al., 1996a, 1996b, 1997, 1998; Beacham et al., 1999; Miller et al., 2000; Ruzzante et al., 2000, 2001). By using microsatellites, significant genetic structuring has been detected among cod populations on major continental shelves and on neighboring banks that are separated by deep channels and have gyre-like circula- tion patterns hypothesized to act as retention mechanisms for eggs and larvae (Ruzzante et al., 1998). Although both Browns and Georges Bank maintain persistent gyre-like circulation patterns that may act to retain eggs and lar- vae, they are separated by the Fundian Channel (>260 m ) which may pose a barrier to juvenile and adult migration (Klein-MacPhee, 2002). Evaluation of Northwest Atlantic haddock by using microsatellites showed similarly signifi- cant stock structuring from Newfoundland to Nantucket Shoals (Lage et al., 2001). Current assessment and man- agement of cod in U.S. waters combine Georges Bank with the regions to its south including Nantucket Shoals. This study investigates genetic stock structure among cod from this region and provides additional insight for scientists and managers. Materials and methods Sampling Samples of adult cod were collected through the U.S. National Marine Fisheries Service and the Canadian Department of Fisheries and Oceans groundfish surveys between 1994 and 2000. Adult cod were obtained from each of the following spawning grounds (Fig. 1): Browns Bank (July 1994, n = 30), Georges Bank (March 1994, n = 48; March 1999. >*=96; In = 144), and Nantucket Shoals (March 2000. n = 97). Blood or tissue (or both) was obtained from individual fish and preserved in 95% ethanol for subsequent DNA extraction. DNA extraction, amplification, and visualization DNA was extracted by using either a Qiamp DNA Mini Kit ( Qiagen Inc., Valencia, CA) or by following a published protocol designed for nucleated blood cells ( Ruzzante et al., 1998). Five microsatellite loci — Grnol, Gmol32 (Brooker et al., 1994), Gmo8, Gmol9, Gmo34 (Miller et al, 2000). and the pantophysin locus. Panl (Fevolden and Pogson, 1997; Pogson, 2001) — were used to evaluate genetic diver- sity. Polymerase chain reactions (PCR) of all loci were per- formed in an Eppendorf Mastercycler Gradient thermal cycler. Final concentrations of reagents in a 25 uL PCR cocktail were as follows: -10 ng of genomic DNA. lxPCR buffer pH 9.5 110 mM KC1, 20 mM Tris-HCl pH 8.3, 10 mM (NH4)2SOJ, 1.5 mM MgCl2, 200 /iM each dNTP, 0.15 nM forward primer. 0.15 ,«M reverse primer (unlabeled for the Panl locus and 5-labeled with a TET, FAM, or HEX ABI dye for all microsatellite loci), and 0.75 units of Taq DNA polymerase. PCR conditions were as follows: initial 5 min at 95°C. 30 cycles of denaturing at 95°C for 1 min, anneal- ing at 50°C (Gmo8, Gmol9, and Gmo34>. 55°C (Pan I), and 57°C (Gmol and Gmol32) for 1 min 30 s, and extending at 72°C for 1 min 30 s with a final extension of 72°C for 10 min. Gmol9 and G/?2o34, as well as Gmol and Gmol32, were multiplexed in two 25 «L PCR reactions. Flourescent microsatellite PCR products were visualized on an ABI377 automated DNA sequencer (Perkin-Elmer Corporation. Foster City, CA) and were analyzed by using GeneScan (vers. 2.1) and Genotyper (vers. 2.1) software programs (Perkin-Elmer Corporation, Foster City, CA). Panl PCR Lage et al.: Genetic structuring of Gadus morhua 291 44 « 42° - 40 - West longitude Figure 1 Map of Northwest Atlantic sampling regions for Atlantic cod [Gadus morhua). Dashed lines indicate the 100-m isobath. products were digested with the restriction endonuclease Dral for at least 2 hours at 37°C and visualized on 2% agarose gels to determine presence of PanlA or PanlB (or both) allelic variants. Genetic analyses Samples were tested for conformation to Hardy- Weinberg equilibrium (HWE) expectations by the Markov chain method (Guo and Thomson, 1992) by resampling 2000 iterations per batch for 200 batches with GENEPOP vers. 3. Id (CEFE/CNRS, Montpelier, France; available at http://www.cefe.cnrs-mop.fr/) (Raymond and Rous- set, 1995); the null hypothesis tested was random union of gametes within a population. All loci were tested for genotypic disequilibrium across the entire data set, as well as for individual populations by using Markov chain resa- mpling with 2000 iterations per batch for 200 batches in GENEPOP vers. 3. Id; the null hypothesis tested was that the genotypes at one locus are independent from genotypes at the other locus. Tests of allelic and genotypic differentiation among and between population samples were conducted by using FSTAT 2.9.1 (UNIL, Lausanne, Switzerland; available at http://www.unil.ch/izea/softwares/fstat.html) (Goudet, 1995); the null hypothesis tested was homogeneous distri- butions across samples. Because alleles can be considered as independent when samples conform to HWE, it is valid to permute alleles among samples to test for population differentiation. On the other hand, when HWE is rejected within samples, alleles within an individual cannot be considered independent, and thus permuting genotypes among samples is the only valid permutation scheme. In both cases, contingency tables were generated and classi- fied by using the log-likelihood statistic G (Goudet et al, 1996). Estimates of among- and between-sample FST's were generated according to Weir and Cocherham (1984) with FSTAT vers. 2.9.1 and GENETIX vers. 4.04 (available 292 Fishery Bulletin 102(2) Table 1 Genetic variation in sampled populations of Atlantic cod (Gadus morhua) and P-value (in parentheses I for among sample popula- tion differentiation, n = observed number of alleles; H0 = observed heterozygosity; FST = the among-sample P-valueJ; bp = base pairs; * = significant deviation from HWE («=0.05. P<0.0083); t = P <0.05; ? P= sO.OOl Locus In Allelic range Browns Bank Georges Bank Nantucket Shoals Fst Differentiation H0 n Ho n Ho n Panl 2 PcmlA/PanlB 0.0385 2 0.0397 2 0.0222 2 -0.0052(0.767) 0.7820 Gmol 5 96-110 bp 0.1000 4 0.1319 5 0.1505 5 0.0019(0.307) 0.3210 Gmo8 23 118-201 bp 0.8929 17 0.8370 19 0.8444* 20 0.0001(0.437) 0.5200 Gmol9 26 120-237 bp 0.8846 17 0.8148* 25 0.7975* 23 -0.0021(0.857) 0.8320 Gmo34 11 82-120 bp 0.7778 5 0.5683 11 0.6630 7 -0.0033(0.797) 0.8400 Gmol32 21 105-155 bp 0.8333 13 0.8214 17 0.7727 16 0.0255(0.000)? 0.0010? All loci 88 — — — — — — — 0.0047(0.001)? 0.0240t at http://www.univ-montp2.fr/~genetix/genetix/genetix. htm) (Belkhir et al.2). Significance of FST estimates was determined with 2000 randomizations. Tests of population differentiation and estimations of FST were calculated at each locus individually and at all loci combined. To correct for simultaneous comparisons, standard Bonferroni cor- rections were applied by using a global significance level of 0.05 (Rice, 1989). Results Genetic variation Observed numbers of alleles, allelic ranges, heterozygosi- ties, and deviations from HWE are presented in Table 1. All tests of genotypic linkage disequilibrium were non- significant at the global and population levels. When Bonferroni corrections for multiple tests were applied to tests of HWE (a=0.05, P<0.0083), the pooled Georges Bank sample deviated significantly at Gwol9, and the Nantucket Shoals sample deviated at Gmo8 and at Gmol9. Interestingly, these two loci have the greatest variation based on number of alleles and heterozygosity. In each case, the cause of deviation was due to an excess of homo- zygotes. Population samples that generally conform to expectations of random mating but show a lack of concor- dance to HWE at one or more loci may be due to a number of processes including null alleles, genetic drift, admix- ture, selection, and insufficient sampling (e.g., Ruzzante, 1998). Possible explanations of homozygote excess include sample admixture ( Wahlund effect) or drift; however these explanations are unlikely because one would expect to see similar results at all loci. More likely explanations are the - Belkhir K., P. Borsa, L. Chikhi, N. Raufaste, and F. Bon- homme. 2002. GENETIX 4.04, logiciel sous Windows TM pour la genetique des populations. Laboratoire Genome, Populations, Interactions, CNRS UMR 5000, Universite de Montpellier II, Montpellier i France). presence of null alleles or selection. Deviations of HWE at Gmo8 and Gmol9 were not observed in all population samples, indicating that null alleles were not present at a global-level but may be present at the population-level for these two loci. Subsequently, any significant population structuring observed at these loci should be viewed with caution (see below). Population structure Tests of population structure are shown in Table 2. Al- though Gmo8 and Gmol9 showed significant deviations from HWE, they did not support any significant population structuring even when tests of population differentiation were performed without assuming conformation to HWE (i.e., permuting among genotypes rather than alleles). Heterogeneity of allelic distribution was not observed (P>0.05) between the 1994 and 1999 Georges Bank samples at each locus individually and at all loci combined, thus indicating potential genetic stability of Georges Bank cod. These samples were subsequently pooled to form a single Georges Bank population sample to facilitate statistical analyses by allowing for better estimations of allele frequencies and by reducing the number of pairwise tests. Tests of population differentiation among samples showed significant divergence at Gmol32 (P<0.01) and at all loci combined (P<0.05). When Bonferroni correc- tions were applied to pairwise measures of divergence («=0.05, P<0.017), significance was observed between Nantucket Shoals and Georges Bank at Gwol32 and at all loci combined, and also between Nantucket Shoals and Browns Bank at Gwol32. No significant differentiation was observed between individual or pooled Georges Bank samples and the Browns Bank sample. Significant among-population FST values were esti- mated at Gmol32 (0.0255, P<0.001) and at all loci com- bined (0.0047, P<0.01). When Bonferroni corrections were applied, significant pairwise-population FST values were estimated between Nantucket Shoals and Browns Bank at Gwol32 (0.0624, P<0.001) and at all loci combined Lage et al.: Genetic structuring of Gadus morhua 293 Table 2 Genetic structuring in sampled populations of Atlantic cod ^Gadus morhua): Above diagonal are P values for pairwise differen- tiation. Below diagona are pairwise FST values; upper va ues are for all loci combined; lower values are for Cm 3132. *=P<0.0167 1 1< = 0.05 for three comparisons); ** = P sO.001. Browns Bank Georges Bank Nantucket Shoals Browns Bank — 0.5440 0.1120 0.2970 0.0020* Georges Bank 0.0012 0.0124 0.0030* 0.0010** Nantucket Shoals 0.0114* 0.0624** 0.0045* 0.0226** — (0.0114, P<0.017), and between Nantucket Shoals and Georges Bank at Gmol32 (0.0226, P<0.001) and at all loci combined (0.0045, P<0.0i7). Estimates of FST values between Browns Bank and Georges Bank samples were all nonsignificant. No significant genetic structuring was observed in any comparison when Gmol32 was excluded from the analysis. Discussion Georges Bank, a large, shallow offshore bank located along the southern edge of the Gulf of Maine off the U.S. and Canadian coasts (Fig. II, supports a large fish biomass. High primary productivity and tightly bound system energetics on the bank result in relatively stable levels of overall biomass and total fish production, although major shifts in species composition routinely occur (Fogarty and Murawski, 1998). The largest spawning aggregation of cod on Georges Bank is found on the Northeast Peak, a gravel region that is an important habitat for the early demersal phase of cod, and may represent a limiting resource for this stock (Lough and Bolz 1989; Langton et al.. 1996). The bank maintains its own circulation pattern in a slow clockwise gyre which may act as a transportation and retention mechanism for planktonic eggs and larvae (Smith and Morse, 1984; Lough and Bolz, 1989). There may be exchange of biota among regions by episodic fluxes of shelf water carrying eggs and larvae away from the Sco- tian Shelf and Browns Bank onto Georges Bank (Cohen et al., 1991; Townsend and Pettigrew, 1996; Bisagni and Smith, 1998). Once on Georges Bank, planktonic eggs and larvae may, depending on depth, be entrained and transported to gravel settlement sites along the western edge of Georges Bank (Smith and Morse 1984; Lough and Bolz, 1989; Werner et al., 1993). However, wind-driven advection may cause egg and larval loss from the North- east Peak and southern flank of Georges Bank (Lough et al., 1989). Cod spawned in the Gulf of Maine usually drift southeasterly towards Georges Bank because of the counterclockwise Gulf of Maine gyre, but the extent of egg and larval exchange between these regions is unknown (Serchuketal., 1994). Cod have been found from the surface to depths greater than 450 meters; however few cod proximate to the Gulf of Maine occur deeper than 180 meters (Klein-MacPhee, 2002). Browns Bank and Georges Bank are bathymetri- cally separated by the relatively deep (>260 meters) Fun- dian Channel which may act as a barrier to adult migra- tion, whereas Georges Bank and Nantucket Shoals are separated by the relatively shallow (<100 meters) Great South Channel. Although the latter channel is probably not a significant barrier to adult migration, it is an area of strong recirculation towards Georges Bank and could limit egg and larval dispersal. Tagging studies show little ex- change of adults between the inner Gulf of Maine and the region east of Browns Bank and Georges Bank (Hunt et al., 1999), but limited exchange has been reported among the Bay of Fundy, southern Nova Scotia, Browns Bank, and Georges Bank (Klein-MacPhee, 2002). The likelihood of determining correct population struc- ture increases when population differentiation is sta- ble over time (Waples, 1998). Results from this study are concordant with observations of temporal stability of mic- rosatellite variation observed in Atlantic cod ( Ruzzante et al., 1996a, 1997, 2001). Tests of population differentiation and subdivision cannot reject the maintenance of genetic homogeneity among Georges Bank cod from 1994 to 1999 and thus may indicate some degree of temporal genetic stability among adult Georges Bank cod. Our results indicate that cod from Nantucket Shoals are genetically distinct from those from Browns Bank and Georges Bank, and cod from the two Banks are more genetically similar. The observed lack of heterogeneity be- tween Browns Bank and Georges Bank is consistent with gene flow — perhaps due to episodic larval transport and some level of limited adult exchange. Nantucket Shoals cod may be genetically distinct because of egg and larval isolation by entrainment in the Georges Bank gyre or be- cause of limited movement of adults between regions (or a combination of both). Eggs and larvae spawned on Nan- tucket Shoals most likely do not enter the Georges Bank gyre system; these early life history forms may be retained on the shoals or transported to the southwest by prevailing circulation (Fogarty and Murawski, 1998). Some North Atlantic cod stocks have shown substantial differences in 294 Fishery Bulletin 102(2) growth rate, reproductive capacity, and maturity sched- ules related to temperature (Brander, 1994). Cod within our study zone generally avoid water temperatures greater than 10°C, but Nantucket Shoals cod are abundant in temperatures as warm as 15°C (Klein-MacPhee, 2002). This differential thermal tolerance may support genetic structuring of Nantucket Shoals cod by selecting against individuals from other areas. Closely related gadid species such as cod and haddock may exhibit similar patterns of population genetic struc- turing associated with similar life histories, selective pressures, and ecological constraints. Our results are concordant with a previous study suggesting that had- dock from Browns Bank and Georges Bank are genetically similar and that haddock from Nantucket Shoals are dis- tinct (Lage et al., 2001). However, Ruzzante et al. (1998) observed significant genetic differentiation between cod from Browns Bank and Georges Bank. Our results do not agree with this previously observed heterogeneity between Browns Bank and Georges Bank and may be due to the examination of different loci, different sampling compari- sons, or small sample sizes used in both studies (or to a combination of these variables) (Ruzzante, 1998; Smouse and Chevillon, 1998). Among loci, the greatest genetic differentiation was observed at locus Gmol32. Indeed, observed statistical significance of population differentiation and FST depends entirely on Gmol32. Length variation at Gmol32 is a func- tion of mutations in the repetitive array and of an indel in a flanking region (Ruzzante et al., 1998) causing bimodal allele distributions in some populations. When compared to other microsatellite loci, Gwol32 has shown the greatest differentiation among other Northwest Atlantic cod popu- lations (Bentzen et al., 1996; Ruzzante et al, 1998, 2001) and among Northwest Atlantic haddock populations (Lage et al., 2001) by an order of magnitude. Other loci examined have not shown similarly strong measures of population structuring. Observed genetic structuring may be due to forces currently determining regional larval and adult distributions, including bathymetry and oceanographic patterns. However, because similar genetic structuring is not observed at all loci, another potential explanation is that structuring at Gmol32 is due to forces that acted during the formation of populations rather than to forces presently maintaining strong reproductive isolation. Once genetic structure was generated during the formation of these populations subsequent to the last ice age. biological and oceanographic forces may have maintained such struc- ture; other loci may show an absence of structure simply be- cause it may not have been present when populations were formed. Pogson et al. (2001) reported that the recent age of populations, rather than extensive gene flow, may be re- sponsible for weak population structure in Atlantic cod, and that interpreting limited genetic differences among popula- tions as reflecting high levels of ongoing gene flow should be made with caution. This suggests that the observed lack of heterogeneity between Browns Bank and Georges Bank may not be due to high levels of ongoing gene flow, but to similarities between recently generated populations main- tained by small but adequate levels of gene flow. Alternatively, significant structuring associated with Gmol32 in both cod and haddock may suggest that selec- tion is acting at this or at a linked locus. Although micro- satellites themselves may be generally considered neutral, there is, in theory, potential for physical linkage or drift- generated linkage disequilibrium between microsatellite and functional loci. There is however, recent evidence of selection acting directly on microsatellite loci in tilapia in high-salinity environments. Streelman and Kocher ( 2002 ) found a strong functional genotype-environment interac- tion and suggested that microsatellite repeats of varying length might induce promoter conformations that differ in their capacity to bind transcriptional regulators. A poten- tial selective mechanism to support the observed genetic structuring of Nantucket Shoals cod (and haddock) may be differential thermal tolerance, although this hypothesis remains untested. There is strong evidence for an unusual mix of balanc- ing and directional selection at the pantophysin (Pa«I ) lo- cus in cod but no evidence of stable geographically varying selection among North Atlantic populations ( Pogson, 2001; Pogson et al., 2001). In the present study, the Paul locus showed little variation and no significant genetic structur- ing (Table 1). The observed lack of geographic structuring at Panl provides no evidence for local adaptation. How- ever, our observations may be due to strong balancing selection among the geographically proximate populations examined or, if Panl is not under selection, insufficient variation to resolve genetic structure. Alternatively, this observed lack of genetic divergence at Panl could be due to similarities among recently generated populations of North Atlantic cod. Our research suggests that the cod spawning on Nan- tucket Shoals are genetically differentiated from cod spawning on Browns Bank and Georges Bank. Managers may wish to consider Nantucket Shoals cod as a separate stock for assessment and management purposes in light of current practices that combine Georges Bank with regions to the south as one management unit. Cod from within the Gulf of Maine can potentially migrate along the coast to Nantucket Shoals where there is little geographic barrier to adult movement. If this is true, the Nantucket Shoals sample that we analyzed may actually be representative of a mixed Gulf of Maine and Nantucket Shoals population. Additional analyses are needed to evaluate the hypothesis that Nantucket Shoals cod are genetically distinct from cod spawning within the Gulf of Maine. Further studies should address the issues of temporal stability and robust sampling and should incorporate cod samples from within the Gulf of Maine. Acknowledgments We thank three anonymous reviewers for their insightful comments. 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Oceanogr. 2:43-64. 298 Abstract -A major cause of the steep declines of American oyster ( Crassos- trea virginica) fisheries is the loss of oyster habitat through the use of dredges that have mined the reef substrata during a century of intense harvest. Experiments comparing the efficiency and habitat impacts of three alternative gears for harvesting oys- ters revealed differences among gear types that might be used to help im- prove the sustainability of commercial oyster fisheries. Hand harvesting by divers produced 25-32^ more oysters per unit of time of fishing than tradi- tional dredging and tonging. although the dive operation required two fish- ermen, rather than one. Per capita returns for dive operations may none- theless be competitive with returns for other gears even in the short term if one person culling on deck can serve two or three divers. Dredging reduced the height of reef habitat by 34rr . sig- nificantly more than the 23fr reduction caused by tonging, both of which were greater than the 6Q< reduction induced by diver hand-harvesting. Thus, con- servation of the essential habitat and sustainability of the subtidal oyster fishery can be enhanced by switch- ing to diver hand-harvesting. Man- agement schemes must intervene to drive the change in harvest methods because fishermen will face relatively high costs in making the switch and will not necessarily realize the long- term ecological benefits. Conserving oyster reef habitat by switching from dredging and tonging to diver-harvesting Hunter S. Lenihan Bren School ol Environmental Science and Management University of California, Santa Barbara Santa Barbara, California 93106-5131 E-mail address: tenihamfflbren ucsb edu Charles H. Peterson Institute of Marine Sciences University of North Carolina at Chapel Hill Morehead City, North Carolina 28557 Manuscript approved for publication 25 November 2003 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Scientific Publications office. Fish. Bull. 102:298-305(2004). Commercial fishing for demersal fish- es and benthic invertebrates, such as mollusks and crabs, is commonly under- taken with bottom-disturbing gear that can inflict damage to seafloor habitats (Dayton et al., 1995; Engel and Kvitek, 1995; Jennings and Kaiser, 1998; Wat- ling and Norse, 1998). Habitat damage from dredges and analogous gear, designed to excavate invertebrates that are partially or completely buried beneath the surface of the seafloor, is generally much more severe than the damage caused by bottom trawls ( Collie et al., 2000). Furthermore, impacts on and recovery from bottom-disturbing fishing gear vary with habitat type; generally smaller effects and more rapid rates of recovery are found for infauna in sedimentary habitats and the most severe and long-lasting damage in biogenic habitats that emerge from the seafloor (Peterson et al., 1987; Collie et al., 2000). Such bio- genic habitats include seagrass beds, fields of sponges and bryozoans. and invertebrate reefs. Biogenic reefs that provide important ecosystem services such as habitat for other organisms include not only tropical coral reefs but also temperate reefs constructed by oysters (Bahr and Lanier, 1981; Lenihan et al., 2001), polychaetes like Petaloproctus (Wilson, 1979; Reise, 1982), and vermetid gastropods (Saf- riel, 1975). The recovery of such emer- gent invertebrate reefs is a slow process because of the relative longevity of the organisms that provide structure for the reef after they die and because of the nature of reefs as accumulations of multiple generations of reef builders. One widespread temperate reef builder, the American oyster iCrassos- trea virginica, also known as the "east- ern oyster," Am. Fish. Soc), has been especially affected by bottom-disturb- ing fishing gear as the target of fisher- ies. More than one hundred years of dredging and tonging oysters in the Chesapeake Bay and Pamlico Sound have caused severe degradation of the oyster reef matrix (deAlteris, 1988; Hargis and Haven, 1988), such that reef area and elevation have been dra- matically reduced (Rothschild et al., 1994; Lenihan and Peterson, 1998). Reduction in reef height has a serious consequence for the oyster population because one function of naturally tall subtidal oyster reefs is to elevate the oysters up into the mixed surface layer of the estuary; this layer of mixed sur- face water allows them to avoid mass mortality from persistent exposure to seasonally anoxic and hypoxic bottom water (Lenihan and Peterson. 1998). Reef height and structure also control reef hydrodynamics (e.g., flow speed, turbulent mixing, and particle delivery and deposition), which influence oyster population dynamics and production (Lenihan, 1999). Consequently, har- vest-related reef destruction and degra- dation are considered major factors that have led to declines of American oys- Lenihan et al.: Conserving oyster reef habitat 299 ters in many estuaries located along the coasts of the At- lantic Ocean and Gulf of Mexico (Lukenbach et al., 1999). Loss of oysters and the biogenic habitat that they provide appears from archaeological and paleontological evidence to be a worldwide phenomenon in temperate estuaries (Jackson et al., 2001). Oyster loss hurts not only the oys- ter fishery but, more importantly, imperils the ecosystem services provided by the oysters. These include, especially, the provision of emergent habitat and reef-dependent prey resources for many fish and crustacean populations of com- mercial and recreational importance (Peterson et al.. 2000; Lenihan et al, 2001; Peterson et al, 2003), the filtration of estuarine waters (Newell, 1988), and the promotion of estuarine biodiversity by provision of hard-bottom habitat in fields of mobile sediments (Wells, 1961). Because of the importance of restoring and sustaining oysters and their reefs to serve both the oyster fishery and the ecosystem, we designed a field test of the habitat im- pacts of three oyster harvesting methods: dredging, tong- ing, and hand extraction by divers (diver-harvesting). Our study is a gear comparison, in which we assess not only the traditional response variable of quantitative harvest per unit of effort with each gear but also the degree of reef habitat damage induced by the extraction of the oysters (analogous to Peterson et al., 1983). We additionally ex- amine the quality of the oysters harvested as a function of gear type. The results indicate that diver-harvesting is a more environmentally sound way of harvesting oysters than traditional methods with dredges and tongs and may be more compatible with conserving oyster reef habitat. Methods Study site Gear comparisons were conducted on subtidal oyster reefs in the Neuse River estuary. North Carolina (35°00'20"N, 76°33'50"W). Environmental conditions of this estuary are well described elsewhere (Paerl et al., 1998; Lenihan, 1999). Briefly, the estuary is mesohaline, an optimal habitat for the American oyster, and was once an impor- tant oyster fishery ground (Lenihan and Peterson, 1998). The estuary contains remnants of many large, natural subtidal oyster reefs that have been intensely mined by oyster harvesting gear for over a century. Dredging is the most common fishing practice. Mining of the reef matrix has combined with sediment loading and eutrophication- associated hypoxia (Paerl et al., 1998) to degrade the oyster reef habitats and greatly reduce oyster populations (Lenihan and Peterson, 1998). In harvested areas, reefs that were 2-3 m tall in quantitative surveys in the late 1800s (n = 8 reefs) were all <1 m tall in our survey con- ducted in 1994 — a modification of habitat caused entirely by the removal of oysters and shells during harvesting with dredges and tongs (Lenihan and Peterson, 1998). To help maintain oyster harvests, the North Carolina Division of Marine Fisheries (NCDMF) restores oyster reefs throughout many locations in the estuary by creat- ing piles of oyster shell, or marl, on the seafloor. These restored oyster reefs are also targeted by oyster fishermen using dredges and, less often, using manual oyster tongs (Marshall1). Experimental oyster reefs Gear comparisons were conducted in March 1996 on 16 subtidal oyster reefs that had previously been created in July 1993 as part of a reef restoration experiment (Leni- han and Peterson, 1998; Lenihan, 1999) in collaboration with NCDMF. The experimentally restored reefs (referred to as "experimental reefs" in this gear-comparison study) were piles of oyster shells 1 m tall, 6-7 m in diameter (28.3-38.5 m2 in area), and generally hemispherical in shape. Natural subtidal reefs located elsewhere in the estuary are typically larger, rectangular biogenic struc- tures, ranging from 8-13 m wide and 20-30 m long. Experimental reefs were constructed in 3-4 m of water on a firm and sandy bottom, and were separated by at least 50 m. From the time of their construction until use in our experiments, the restored oyster reefs remained research sanctuaries, protected from commercial and recreational shellfishing. As oysters settle and undergo metamorphosis on the shells of other (live and dead) oysters, to which they are attracted by chemical cues (Tamburri et al., 1992), they help cement together and add to the shell matrix of the reef over years. Prior to our harvest treatments, the ex- perimentally restored reefs were colonized by at least three generations of oysters, many of which grew to adult size (range of oyster sizes on experimental reefs at the start of our experiment: 2-11 cm in shell height). Consequently, the shell matrices of the reefs had become somewhat cohe- sive, although probably less so than natural oyster reefs. In February 1996, before initiation of experimental harvests, there was no significant difference in the mean density of adult (>1 cm in shell height) oysters (mean ±SD 179.1 ±18.4/m2) among the four sets of four experimental reefs randomly selected to receive the four harvesting treatments (one-way ANOVA; F3 12=0.29; mean square error=285.06; P=0.83). Experimental reefs in the Neuse River usually had slightly higher oyster densities nearer their base and larger oysters near the crest (see Lenihan, 1999). Experimental harvests We compared three types of oyster-harvesting techniques: dredging, hand-tonging, and diver-harvesting. In March 1996, each of 16 reefs was either dredged, tonged, diver- harvested, or left unharvested as a control (four replicates of each treatment). Experimental dredging and hand-tong- ing were conducted in the manner applied by commercial oyster fishermen. The dredge, 25 kg in weight and 1 m in width, was pulled behind a powerboat operated by NCDMF personnel with commercial oyster-dredging experience. Hand-tonging was also conducted by a professional oyster Marshall, M. 1999. Personal commun. North Carolina Division of Marine Fisheries, 3431 Arendell St., Morehead City, NC 28557. 300 Fishery Bulletin 102(2) fisherman, R. A. Cummings. Oysters and shell material col- lected by dredges and tongs were separated aboard the boat on a culling board, using the common culling techniques (i.e., breaking apart oysters and shell with hammers, mal- lets, and chisels). As mandated by law, oyster shell and undersized oysters (<7 cm in height) were thrown overboard above the reef from which they had been collected. Hand collections of oysters were conducted by scuba divers (J. H. Grabowski and H. S. Lenihan). Unlike profes- sional oyster divers in Chesapeake Bay and other areas, who rake large quantities of shell and attached oysters into baskets that are pulled aboard ship to be culled, the divers in this trial adopted a different method designed to preserve reef habitat. Instead of collecting shell and oys- ters indiscriminately, the divers chose only those oysters that appeared alive and of market-size. Selected oysters were hand picked from the reef and placed in heavy plastic mesh baskets that, when full, were subsequently pulled aboard the boat with haul lines. To standardize fishing effort, each of the 12 harvested reefs was harvested for 2 hours, regardless of the num- ber of oysters collected. A 2-h harvest period for each 28.3-38.5 m2 reef was considered to be a thorough but not excessive level of harvesting by the professional fishermen. The numbers of oysters collected in the final three or four dredge hauls and oyster tongs were typically lower (by -10-20%) than the preceding dredge hauls and tongs. This reduction in the catch per unit of effort was great enough that a fisherman foraging optimally would normally cease harvesting at that time and move on to another reef. Similarly, after 2 hours of diver-harvesting, most of the clearly visible market-size oysters had been harvested. Quantifying reef structure Measurements of oyster reef height and diameter were conducted on all 16 experimental reefs both before and after application of the three fishing methods. In Febru- ary 1996, the preharvest height and radius of each oyster reef were measured by scuba divers using a custom-made "square angle," consisting of two pieces (2 m and 5 m long) of 3-cm wide steel angle-iron, each with an attached 1-m long carpenter's level. Both pieces of angle iron were marked at 1-cm intervals. The 5-m long (cross) piece was attached to the 2-m long (upright) piece by a roller-joint. The roller-joint allowed the cross piece to move up and down the upright piece, thus providing a measure of reef height, and to move horizontally in relation to the upright piece, thus providing a measurement of reef radius. The 2-m long piece also had a 0.75-m long piece of angle iron attached perpendicularly near its bottom so that it would not sink into the seafloor when placed upright. One diver held the 2-m long angle iron perpendicular to the seafloor at the edge of a reef, while the other diver placed the 5-m long angle iron parallel to the seafloor, so that one end rested on the highest point of a reef and the other end met the upright angle iron at the reefs edge. The height and radius of the reef were then measured by recording the height at which the cross piece met the upright piece, and the distance at which the upright piece met the cross-piece. For each reef, a mean diameter was calculated by measuring three separate radii (oriented at three compass bearings, all 120° apart), multiplying the radii by two to estimate diameters, and then averag- ing the three diameters. This averaging procedure was undertaken because the reefs were not perfectly circular. Measurements of reef height and radius were repeated in March, two-five days after experimental harvests were completed. Sampling oyster populations We sampled live and dead oysters on each treatment and control reef before (late February 1996) and immediately after (late March) experimental harvests to estimate the proportion of oysters incidentally killed but not har- vested by each harvesting treatment. Specifically, oyster data was collected within 30 hours of the application of the harvest treatment on each replicate reef. Densities of live and dead oysters were quantified by divers who hap- hazardly placed eight 0.5-m'2 weighted PVC quadrats on the reef surface at haphazard locations and recorded the number of live and dead oysters greater >1 cm in height. The density of dead oysters was measured by count- ing the number of oyster shells that were articulated and appeared relatively fresh (i.e., not black in color or decayed), or oysters with somatic tissue exposed because of cracked, broken, or punctured shells. Oysters with exposed somatic tissue almost certainly die because of predation by fishes and crabs in the Neuse River estuary (Lenihan, 1999; and see Lenihan and Micheli, 2000). Mean proportions of dead oysters were computed (dead oysters/dead+alive oysters), as well as mean densities of live and dead oysters on each reef. Catch per unit of effort The relative efficiency of each harvesting method was determined by comparing the numbers of bushels (1 bushel=36.4 L) of market-size oysters taken per hour of fishing. We quantified numbers of bushels for each har- vesting method aboard the boat by placing oysters of legal size in premeasured mesh baskets. After being counted, and upon termination of the harvest trial, many of the oysters were returned to other nearby reefs not involved in the experiment. Statistics One-way analysis of variance (ANOVA) was used to com- pare the following across harvest treatments and controls: 1) changes in mean reef height and diameter; 2) catch per unit of effort; 3) the proportion of oysters found dead on reefs before harvest; 4) the proportion of oysters found dead on reefs after harvest; and 5) the absolute difference in the proportion of oysters found dead before versus after harvesting ([after minus before]). Data from all treat- ment (dredging, tonging, and diver-harvesting; n = 4 for each treatment) and the control Ui = 4) reefs were used in Lenihan et al.: Conserving oyster reef habitat 301 Table 1 Results of one-way ANOVAs comparing differences in reef height (cm), reef diameter (cm), and catch per unit of effort (number of oysters collected per hour) among experimental reefs harvested by different methods (dredging, tonging, diver-harvesting, and controls), df = degrees of freedom; ms = mean square; F = F-value; P = P-value; ss = sum of squares. Partial r2= treatment ss/total ss. Reef height Reef diameter partial Source df ms partial 7'2 Catch per unit of effort partial Harvesting treatment 3 0.09 36.90 0.0001 0.90 0.07 15.79 Residual 12 0.003 0.005 Total 15 Total ss: 0.31 0.0002 0.80 0.27 3.21 0.08 17.84 0.0001 0.11 9.64 the ANOVA. Before ANOVA, homogeneity of variances was tested by using Cochran's method (a=0.05). All data passed this test. After ANOVA, post hoc differences among means were compared by using Student-Newman-Keuls (SNK) tests (a=0.05). Results Reef height and diameter Dredge harvesting on experimental reefs removed the largest amount of shell material from the reefs, based on the reduction of reef height (Fig. 1A) and on the qualitative assessment of increases in numbers of oyster shells found on the seafloor around the reefs. Hand-tonging removed an intermediate amount of reef materials, and diver-har- vesting removed far less shell matrix than either dredging or tonging. All harvesting methods reduced the height of restored oyster reefs (Fig. 1A), but dredging (34% of reef height) and tonging (23%) had greater impacts than did diver-harvesting (6%). ANOVA demonstrated significant differences among harvest treatments in mean change in reef height (Table 1); all harvest treatments induced a loss in reef height as compared with unharvested control reefs (SNK; P<0.05). Dredging reduced reef height more than any other treatment (SNK, P<0.05), and tonging reduced reef height more than diver-harvesting (SNK, P<0.05). The reduction in reef height caused by diver-harvesting was small (mean ±SD: 6 ±3 cm). However, diver-har- vesting nearly eliminated the veneer of live market-size oysters on reefs, which provides substantial structure on reef surfaces. Oyster harvesting either slightly increased or slightly decreased reef diameter, depending upon method ( Fig. IB). Reef material was apparently removed from edges of reefs by tonging. thereby reducing reef diameter. Shell was spread around the reefs by dredging, thereby increas- ing reef diameter after application of that harvesting method. The effects of oyster harvesting on reef diameter proved significant (Table 1). Tonging significantly re- duced reef size compared with controls and the other two harvesting treatments (SNK; P<0.05), whereas dredging DC I A A T B 1 C T D ] B A 1 B i T i C 1 1 1 D control diver-harvested tonged dredged Figure 1 Modification of reef size and structure caused by various harvesting techniques. (A) Mean (+SE) reduction in the height of experimentally restored oyster reefs caused by three types of oyster harvesting: hand-harvesting by divers, hand tonging. and dredging. Dredges are pulled behind power boats. Reefs were located in the Neuse River estuary, North Carolina. Letters represent results of SNK post hoc tests: dredged>tonged>diver-harvested>control at P<0.05. (B) Mean ( + SE) change in the diameter of experimental oyster reefs caused by different oyster- harvesting techniques. Letters represent results of SNK post hoc rests: dredged>diver-harvested>control>tonged atP<0.05. 302 Fishery Bulletin 102(2) Table 2 Results of one-way ANOVAs comparing differences in the proportion of oysters found dead ("mortality" i on reefs before and after harvesting by different methods (dredging, tonging, diver-harvesting, and controls), and the absolute difference (\after-before]) in the proportion of dead oyster found before versus after harvesting, df = degrees of freedom; ms = mean square; F = F-value; P = P-value; ss = sum of squares. Partial r2 = treatment ss/total ss. Before mortality After mortality partial Source df ms partial r2 Difference in mortality partial Harvesting treatment 3 0.001 0.49 0.69 0.11 0.02 Residual 12 0.001 0.002 Total 15 Total ss: 0.01 7.90 0.004 0.58 II us 0.01 0.08 7.56 0.004 0.57 0.04 increased reef diameter compared to the other treat- ments (SNK; P<0.05). The increase in diameter of diver-harvested reefs was also greater than that for controls (SNK; P<0.05). The substantial increase in shell material (with oysters of all sizes) spread out on the seafloor on dredged reefs indicates that the collec- tion efficiency of dredges is less than 100%. Catch per unit of effort Catch per unit of effort of oysters included the time required to collect oysters from the reef and the time needed to separate (i.e., cull) them from undersized oysters and shell material. Two of the harvesting methods, hand-tonging and oyster dredging, are one- man operations in which one fisherman can operate the harvesting gear, cull oysters, and drive the boat. Therefore, measurements of catch per unit of effort for dredging and tonging represent the numbers of bush- els of oysters one fisherman can collect per hour. In contrast, scuba diving is rarely attempted alone and it is usually necessary for someone else to tend the diver (e.g., helping him or her in and out of the water) and to haul oysters up to the boat when given a signal by the diver on the reef. Divers should preferably work as a team using the "buddy" system for safety reasons. Data for diver-collections are given in bushels per hour collected by one diver but hauled up to the boat and culled by a second person. There was a significant difference in the numbers of bushels collected per hour by the different harvesting techniques (Table 1). Diver-harvesting had a higher catch efficiency than all other treatments ( SNK; P<0.05; Fig. 2). Diver-harvesting was about 25% more time efficient than dredge harvesting and 32% more efficient than tonging. There was no statistically significant difference in effi- ciency between dredging and tonging (SNK; P>0.05). Incidental oyster mortality The proportion of oysters found dead on experimental reefs in February 1996 (-20%), prior to experimental 3.0 -I 1 bushel = 36.4 L - 2.5 - o 1 2.0- A T B harvested B T I I '■»■ sz CO ™ 0.5- C control diver-harvested tonged dredged Figure 2 Mean (+SE) number of bushels collected per hour on experi- mental reefs by different oyster-harvesting techniques. Letters represent results of SNK post hoc tests: diver- harvested>dredged and tonged>control at P<0.05. harvesting, was similar to that found on other nearby ex- perimental and natural reefs in the Neuse River estuary in preceding years (e.g., Lenihan and Peterson. 1998: Lenihan 1999). In February, the proportions of dead oys- ters did not differ among the four sets of reefs destined to be experimentally harvested (Table 2. Fig. 3A). In contrast, there was a large and statistically significant difference in the proportions of dead oysters on the reefs after harvesting (Table 2, Fig. 3A). The proportions of dead oysters on reefs that had been tonged and dredged were significantly greater than on diver-harvested and control reefs (SNK; P<0.05). Before-after-control-impact (BACI) comparison of the change in proportions of dead oysters from before to after harvesting ( [after— before] ), a direct estimate of incidental mortality caused by harvesting gear, showed a similar pattern to mortality inferred from in situ proportions of dead oysters in March after harvesting (Table 2, Fig. 3B). Lenihan et al.: Conserving oyster reef habitat 303 A significant treatment effect in the after period (Table 2) indicated that the change over time in proportion of dead oysters varied among harvest treatments. Tonging and dredging increased the fraction of dead among in situ oysters on reefs (SNK; P<0.05; Fig. 3B), but diver-harvesting did not. Immediately after harvesting, divers found that many oysters on tonged and dredged reefs had been broken open, severely cracked, or punctured. Discussion Our comparisons of gear revealed relatively unambiguous differences in their harvesting efficiency for oyster dredges, tongs, and hands of divers. Dredging and tonging had similar and statistically indistinguishable catch efficiencies, which seems reasonable given that both tech- niques are commonly employed in the same loca- tions and times in the oyster fishery. Presumably, fishermen choose between these two gears on the basis of personal preference, history, and skill, as well as on the basis of water depth, bottom type, and other factors that did not vary in our study. Diver-harvesting of oysters resulted in higher rates of harvest per hour, but this enhancement in catch efficiency required the presence of two people, one diver beneath the surface and another person on deck involved in hauling baskets of oysters onto the deck and culling out market- able oysters. Because the increase in efficiency was only 25-32%, this enhancement falls short of the 100% required to compensate each fisherman to the same degree that dredging and tonging pro- vide. Nevertheless, the immediate economics of diver-harvesting could prove competitive or even superior if the single deckhand could serve two or more divers, which seems likely from our experi- ence with the workload on deck, and if the oysters taken are priced more favorably because of larger size or less damage, which seems possible. A complete short-term economic comparison would need to include higher costs for fuel in dredging and costs of filling air tanks for diving, as well as depreciation of gear. This discussion of the basic efficiencies and eco- nomics of the methods of commercial oyster fish- ing is based upon short-term considerations only. That short-term time perspective is the cause of failures to achieve sustainability in fisheries quite generally (Ludwig et al., 1993; Botsford et al., 1997). We show that adoption of hand-harvesting by divers would result in substantially less fishery-induced reduction in reef height by a factor of four to six, implying greater preservation of the habitat and thus a more sustainable fishing practice. Our data on the changes in area covered by reefs as a function of harvest treatment revealed only small differences among treatments. The height of a reef 40 30 20- 10 control diver- longed dredged harveted Before control diver- tonged dredged harveted After 14 - B 12 - 10 - 8 - 6 - A - B T I T 2 - control diver-harvested tonged dredged Figure 3 Mortality of oysters caused by various harvesting techniques. (A) Mean ( + SE) % dead within oyster populations on experimental reefs before and after being harvested by three different harvest- ing techniques: dredging, tonging, and diver-harvesting. Control reefs were not harvested. Letters represent results of SNK post hoc tests: dredged, after>tonged, after>all other treatments at P<0.05. There was no difference among treatments before harvesting. (B) Mean ( + SE) absolute difference in the % dead oysters on experi- mental reefs before and after harvesting. Difference calculated by: \9o after-% before]. Letters represent results of SNK post hoe tests: dredged and tonged>diver-harvested and control at P<0.05. is a critical variable in sustaining the reef as an engine of oyster production because short reefs can be easily covered by sediment (Lenihan, 1999), can be abraded by sediment transport (Lenihan, 1999), and can fail to extend above hypoxic bottom waters (Lenihan and Peterson, 1998). Tall reefs (i.e., reefs not degraded by harvesting) produce faster flow speeds and more turbulence for oyster popula- tions, which in turn increase oyster growth rate, increase 304 Fishery Bulletin 102(2) physicalogical condition, reduce disease incidence and intensity, and decrease mortality (Lenihan, 1999). Con- sequently, assessment of economics of the oyster fishery over longer time frames would likely demonstrate higher returns from practicing diver-harvesting, assuming that this technique conserved reef structure. Diver-harvesting also killed fewer of the oysters that remained on the bot- tom, thereby sustaining future harvests better through reduced wastage and by retention of more live oysters that would produce more reef material. Although the relative advantage of diver-harvesting for conserving reef structure is evident, the absolute conser- vation of reef habitat under the various oyster harvesting methods is not clear from our study. Our data on impacts of diver-harvesting revealed slight declines in reef height, but whether these same declines would apply to an older reef, as opposed to a recently restored reef, is open to ques- tion. The level of cementation that binds the shells of the reef is not as great on recently restored reefs, making them more susceptible to degradation with physical disturbance. Our study measured only the immediate drop in reef eleva- tion after fishing at a level that removed a large fraction of legally marketable oysters. In a well-managed fishery, this drop in reef elevation would represent virtually an en- tire season's decline, after which substantial reef growth would occur through recruitment and growth of smaller oysters before a new harvesting season. Thus, a healthy oyster reef may well be able to compensate for the modest reduction in elevation caused by diver-harvesting. If so, oyster reef sanctuaries now being created throughout the Chesapeake Bay (Luckenbach et al., 1999) could conceiv- ably be opened to diver-harvesting (without implements) and still preserve the reef services to the ecosystem. This possibility deserves to be evaluated in order to minimize conflicts between the goals of restoring oyster reef habitat for conservation purposes and restoring oyster reefs for the restoration of lost fisheries. Application of the results of our gear comparisons to management of oyster fisheries will likely encounter some impediments. Although various artisanal fisheries worldwide have employed free diving as a fishing tech- nique and some modern fisheries, including the American oyster fishery, involve the use of scuba, diving is not a skill possessed by most oyster fishermen and probably is not a method under consideration for oyster fishing in general. In addition, the peak of oyster harvesting season on the Atlantic and Gulf coasts is usually during winter months (e.g., November-March) when water temperatures in estuaries are quite low (0-10°C). Such conditions require cold-water diving equipment (e.g., dry-suits), which will further increase the cost of this new harvesting tech- nique. Thus acceptance of diver-harvesting by the indus- try would require training in diving skills and safety, education and demonstration of the advantages of this gear, and perhaps even investment of public funds to de- fray costs of the transition from traditional dredges and tongs to scuba or hookah. Because the gains of switching to diver-harvesting accrue to the industry over the long term, while individual fishermen who switch may suffer economically in the short-term, gear choice represents a modified example of the tragedy of the commons (Ludwig et al., 1993). Only when armed with some form of owner- ship rights and an attendant long-term perspective would an individual oyster fisherman choose to switch to diver- harvesting. The precipitous declines of over 99r< in oyster landings in mid-Atlantic estuaries (Rothschild et al., 1994; Lenihan and Peterson, 1998) mean that oyster fishermen can hardly be expected to bear the costs of switching fish- ing methods. Therefore, government intervention would be required to convert subtidal oyster dredge and tong fisheries into diver-harvesting operations for two reasons; the need for compensation of start-up costs and the need to overcome the tragedy of the commons. Given the dire state of oyster fisheries today ( Rothschild et al., 1994 ), the habi- tat destruction in these declines (deAlteris, 1988; Hargis and Haven, 1998; Rothschild et al., 1994; Lenihan and Peterson, 1998). the broad ecosystem services provided by healthy oyster reefs (Jackson et al., 2001; Lenihan et al. 2001), and the very real potential for restoring oysters and their reefs (Luckenbach et al., 1999: Lenihan, 1999). a mandate to switch fishing methods for subtidal oyster fisheries could pay large dividends. Acknowledgments We thank Mike Marshall, Jeff French, and those many NCDMF people working on deck for initially creating experimental reefs to our specifications and for later applying the experimental dredge harvesting treatment. We thank Robert A. Cummings for applying the hand- tonging treatment, and Jonathan H. Grabowski for help- ing with diver-harvesting of reefs. This work was funded by the North Carolina General Assembly through the Cooperative Institute of Fisheries Oceanography (to C. H. Peterson), and NOAA-Chesapeake Bay Program Oyster Disease Program (to H. S. Lenihan, C. H. Peterson, and F. Micheli) Literature cited Bahr. L. M.. and W. P. Lanier. 1981. The ecology of intertidal oyster reefs of the South Atlantic coast: a community profile. U.S. Fish Wildl. Serv. Office of Biological Services FWS/OBS-81/15. Botsford, L. W., J. C. Castilla, and C. H. Peterson. 1997. The management of fisheries and marine ecosys- tems. Science 277:509-515. Collie, J. S., S. J. Hall, M. J. Kaiser, and I. R. Poiner. 2000. A quantitative analysis of fishing impacts on shelf- sea benthos. J. Anim. Ecol. 69:785-798. Dayton. P. K.. S. F. Thrush. M. T. Agardy. and R. J. Hoffman. 1995. Environmental effects of marine fishing. Aquat. Conserv. Mar. Fresh. Ecosyst. 5:205-232. deAlteris, J. T. 1988. The geomorphologic development of Wreck Shoal, a subtidal oyster reef of the James River, Virginia. Estu- aries 11:240-249. Engel, J., and R. G. Kvitek. 1995. Effects of otter trawling on a benthic community in Lenihan et al.: Conserving oyster reef habitat 305 Monterey Bay National Marine Sanctuary. Conserv. Biol. 12:1204-1214. Hargis, W. J., Jr., and D. S. Haven. 1988. The imperiled oyster industry of Virginia: a critical analysis with recommendations for restoration. Special report 290 in applied marine science and ocean engin- eering, 145 p. Virginia Sea Grant Marine Advisory Services, Virginia Institute of Marine Science, Gloucester Point, VA. Jackson. J. B. C, M. X. Kirby, W. H. Berger, K. A. Bjorndal. L. W. Botsford, B. J. Bourque, R. H. Bradbury. R- Cooke, J. Erlandson, J. A. Estes, T. R Hughes, S. Kidwell, C.B. Lange, H. S. Lenihan, J. M. Pandolphi, C. H. Peterson, R. S. Steneck, M. J. Tegner, and R. R. Warner. 2001. Historical overfishing and the recent collapse of coastal ecosystems. Science 293:629-638. Jennings, S., and M. J. Kaiser. 1998. The effects of fishing on marine ecosystems. Adv. Mar. Biol. 34:201-352. Lenihan, H. S. 1999. Physical-biological coupling on oyster reefs: how hab- itat structure influences individual performance. Ecol. Monogr. 69:251-276. Lenihan. H. S., and F. Micheli. 2000. Biological effects of shellfish harvesting on oyster reefs: resolving a fishery conflict by ecological experi- mentation. Fish. Bull. 98:86-95. Lenihan, H. S., and C. H. Peterson. 1998. How habitat degradation through fishery distur- bance enhances impacts of hypoxia on oyster reefs. Ecol. Appl. 8:128-140. Lenihan, H. S., C. H. Peterson, J. E. Byers, J. H. Grabowski, G. W. Thayer, and D. R. Colby. 2001. Cascading of habitat degradation: oyster reefs in- vaded by refugee fishes escaping stress. Ecol. Appl. 11:764-782. Luckenbach, M. A., R. Mann, and J. A. Wesson (eds.). 1999. Oyster reef restoration: a synopsis and synthesis of approaches, 366 p. Virginia Institute of Marine Sciences Press, Gloucester Point, VA. Ludwig, D., R. Hilborn, and C. Walters. 1993. Uncertainty, resource exploitation, and conservation: lessons from history. Science 260:17-18. Newell, R. I. E. 1988. Ecological changes in the Chesapeake Bay: Are they the result of overharvesting the American oyster, Cras- sostrea virginica? In Understanding the estuary: advances in Chesapeake Bay research (M. P. Lynch, E. C. Krome, eds.), p. 536-546. Chesapeake Bay Research Consor- tium, Publ 129 CBP/TRS 24/88, Gloucester Point, VA. Paerl, H. W., J. L. Pinckney, J. M. Fear, and B. L. Peierls. 1998. Ecosystem responses to internal and watershed organic matter loading: consequences for hypoxia in the eutrophying Neuse River Estuary, North Carolina, USA. Mar. Ecol. Prog. Ser. 166:17-25. Peterson, C. H., H. C. Summerson, and S. R. Fegley. 1983. The relative efficiency of tow clam rakes and their contrasting impacts on seagrass biomass. Fish. Bull. 81:429-434. 1987. Ecological consequences of mechanical harvesting of clams. Fish. Bull. 85:281-298. Peterson, C. H., H. C. Summerson, E. Thomson, H. S. Lenihan. J. H. Grabowski, L. Manning, F. Micheli, and G. Johnson. 2000. Synthesis of linkages between benthic and fish communities as a key to protecting essential fish habitat. Bull. Mar. Sci. 66:759-774. Peterson, C. H., J. H. Grabowski, and S. P. Powers. In press. Quantitative enhancement of fish production by oyster reef habitat: restoration valuation. Mar. Ecol. Prog. Ser. Reise, K. 1982. Long-term changes in the macrobenthic invertebrate fauna of the Wadden Sea: are polychaetes about to take over? Neth. J. Sea Res. 16:29-36. Rothschild, B. J., J. S. Ault, P. Goulletquer, and M. Heral. 1994. Decline of the Chesapeake Bay oyster population: a century of habitat destruction and overfishing. Mar. Ecol. Prog. Ser. 111:29-39. Safriel, U. N. 1975. The role of vermetid gastropods in the formation of Mediterranean and Atlantic reefs. Oecologia 20:85-101. Tamburri, M. N., R. K. Zimmer-Faust, and M. L. Tamplin. 1992. Natural sources and properties of chemical induc- ers mediating settlement of oyster larvae: a re-examin- ation. Biol. Bull. 183:327-337. Watling L., and E. A. Norse. 1998. Disturbance of the seabed by mobile fishing gear: a comparison to forest clearcutting. Cons. Biol. 12:1180- 1197. Wells, H. W. 1961. The fauna of oyster reefs with special reference to the salinity factor. Ecol. Monogr. 31:239-266. Wilson, W. H„ Jr. 1979. Community structure and species diversity of the sediment reefs constructed by Petaloproctus socialis (Poly- chaeta: Maldanidae). J. Mar. Res. 37:623-641. 306 Abstract— Loligo opalescens live less than a year and die after a short spawning period before all oocytes are expended. Potential fecundity iEP), the standing stock of all oocytes just before the onset of spawning, increased with dorsal mantle length (L), where EP = 29. 8L. For the average female squid (L of 129 mm), EP was 3844 oocytes. During the spawning period, no oogonia were produced: therefore the standing stock of oocytes declined as they were ovulated. This decline in oocytes was correlated with a decline in mantle condition and an increase in the size of the smallest oocyte in the ovary. Close agreement between the decline in estimated body weight and standing stock of oocytes during the spawning period indicated that maturation and spawning of eggs could largely, if not entirely, be supported by the conversion of energy reserves in tissue. Loligo opalescens, newly recruited to the spawning population, ovulated about 36^ of their potential fecundity during their first spawning day and fewer ova were released in subsequent days. Loligo opalescens do not spawn all of their oocytes; a small percentage of the spawning population may live long enough to spawn 78% of their potential fecundity. Loligo opalescens are taken in a spawning grounds fishery off Califor- nia, where nearly all of the catch are mature spawning adults. Thirty-three percent of the potential fecundity of L. opalescens was deposited before they were taken by the fishery (December 1998-99). This observation led to the development of a management strategy based on monitoring the escapement of eggs from the fishery. The strategy requires estimation of the fecundity realized by the average squid in the population which is a function of egg deposition and mortality rates. A model indicated that the daily total mortality rate on the spawning ground may be about 0.45 and that the average adult may live only 1.67 days after spawning begins. The rate at which eggs escape the fishery was modeled and the sen- sitivity of changing daily rates of fish- ing mortality, natural mortality, and egg deposition was examined. A rapid method for monitoring the fecundity of the L. opalescens catch was developed. Manuscript approved for publication 19 December 2003 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Scientific Publications Office. Fish. Bull. 102:306-327 (2004). Fecundity, egg deposition, and mortality of market squid (Loligo opalescens) Beverly J. Macewicz 1. Roe Hunter Nancy C. H. Lo Erin L. LaCasella Southwest Fisheries Science Center National Marine Fisheries Service, NOAA 8604 La Jolla Shores Drive La Jolla, California 92037-1508 E-mail address (for B J. Macewicz): Bev Macewiczi5noaa.gov Many loliginid squid populations depend entirely upon the reproduc- tive output of the preceding genera- tion because individuals live less than a year (Yang et al., 1986; Hatfield, 1991, 2000; Natsukari and Komine, 1992; Arkhipin, 1993; Arkhipin and Nekludova, 1993; Jackson, 1993, 1994; Jackson et al., 1993; Boyle et al., 1995; Jackson and Yeatman, 1996; Jackson et al., 1997; Moltschaniwskyj and Sem- mens, 2000; Semmens and Moltschani- wskyj, 2000). In California waters, Loligo opalescens (market squid, also known as the opalescent inshore squid [FAO]) live only 6-12 months (Butler et al., 1999) and die after spawning (McGowan, 1954; Fields, 1965). Thus, fecundity of L. opalescens is a criti- cal life history trait and, in addition, must be known in order to estimate the biomass with either egg deposition or larval production methods (Hunter and Lo, 1997). Loligo opalescens is one of the most valuable fishery resources in Cali- fornia waters and is monitored under the Coastal Pelagics Species Fishery Management Plan of the Pacific Fishery Management Council as market squid. Laptikhovsky (2000) pointed out that squid fecundity estimates would be biased if the females spawned ova prior to capture, if oocytes remained in the ovary after death, or if some of the standing stock of oocytes were lost because of atresia. Previous field work on squid fecundity has been limited to the traditional method of simply count- ing oocytes or ova (or both) of animals taken on the spawning grounds, and none of the biases identified by Lap- tikhovsky (2000) have been evaluated (Boyle and Ngoile, 1993; Coelho et al., 1994; Guerra and Rocha, 1994; Boyle et al„ 1995; Collins et al., 1995; Moltscha- niskyj, 1995; Lopes et al., 1997; Lap- tikhovsky, 2000). On the other hand, laboratory studies (Ikeda et al., 1993; Bower and Sakurai, 1996; Sauer et al., 1999; and Maxwell and Hanlon, 2000) have indicated that oocytes remain in the ovaries after spawning and death. Additionally, atresia was found to oc- cur in all stages of oocytes of Loligo vulgaris reynaudii (Melo and Sauer, 1998). Modern approaches to estimat- ing lifetime fecundity in fishes take the potential biases of past spawning history, residual fecundity, and atresia into account (Hay et al., 1987; Hunter et al., 1992; Macewicz and Hunter. 1994; Kjesbu et al., 1998). The initial objectives of the present study were to estimate the fecundity of L. opalescens by using a modern approach that con- siders such biases, and to provide a histological description of those aspects of ovarian structure upon which mod- ern fecundity analyses are based. As our work progressed, we realized that it might be practical to manage the market squid fishery by monitoring egg escapement based on fecundity mea- surements. Thus, we added two new objectives: to conduct a preliminary evaluation of the use of egg escapement as a tool for management of the market squid fishery; and to develop a method Macewicz et al.: Fecundity, egg deposition, and mortality of Loligo opalscens 307 0 7^ Santa Cruz 1. Female Market Squid, -v Loligo opalescens, V^^ Collection sites 34°N Santa Rosa 1 a\ Research Cruises ° Jan. 1998 R/V Jordan * Dec. 1998 R/V Mako Santa ^-♦O Catalina I. 33° ■^ 121°W 1 20° 119c 1 1 8: Figure 1 Collection locations for female Loligo opalescens during two joint research cruises during 1998 by California Department of Fish & Game (CDF&G) and National Marine Fisheries Service (NMFS) and for three immature females (triangles) collected during February 2000 (CDF&G). to monitor the fecundity of the catch that avoids the costly process of counting all oocytes and ova. In this study we consider four aspects of the fecundity of L. opalescens: potential fecundity, minimum residual fecundity, maximum fecundity, and the fecundity depos- ited by the average female in the population. Potential fecundity, or potential lifetime fecundity, is the standing stock of all oocytes in the ovary just before the onset of the first ovulation. Because L. opalescens are semelparous, the standing stock of all oocytes in the ovary just before first ovulation equals their potential lifetime fecundity. Clearly, once ovulation and spawning (deposition of ova in egg cap- sules on the sea floor) begin, the standing stock of oocytes can no longer be considered a measure of the potential fecundity of the female. Minimum residual fecundity is the minimum number of oocytes that might be expected to remain in the ovary at death. Because ovaries of dying L. opalescens contain oocytes (Knipe and Beeman, 1978), only a portion of the potential fecundity will be spawned in their lifetime. We use ancillary information on L. opal- escens (an index of mantle condition and extent of ovarian development) to project what the minimum residual may be. Maximum fecundity (potential fecundity less the mini- mum residual fecundity) is the maximum number of eggs a female might be expected to deposit in a lifetime. We also estimate the fraction of the potential fecundity deposited by the average female, a key vital rate we approximate by modeling the daily rates of total mortality and egg deposition. Lastly, the term "standing stock of oocytes" is used throughout this article to indicate the total number of oocytes at all stages in an ovary. Whether the standing stock of a particular female is to be considered a potential fecundity, a residual fecundity, or something in between, depends upon ancillary information (i.e., presence of post- ovulatory follicles in the ovary, ova in the oviduct, mantle condition, or the level of ovarian maturity). Materials and methods We collected Loligo opalescens during two southern Cali- fornia research cruises in 1998 (7-15 January and 3-10 December) (Fig. 1). Most specimens were taken at night by using trawls, jigging, or by removing them from com- mercial purse-seine catches at sea; some specimens were collected during the day by using bottom trawls. We mea- sured dorsal mantle length (mm), weighed the whole body (g), and classified the ovary and preserved it with viscera and oviduct attached in 10% neutral buffered formalin. To determine reproductive state we decided not to use the familiar ovary classification systems but rather tabulated gross anatomical characters and, later on, selected the most useful characteristics. See Table 1 for characters selected for scoring. Preserved ovaries and oviducts were reclassified in the laboratory and weighed (to nearest 0.001 g). A piece of the preserved ovary from each of the 135 female L. opalescens from January and the 117 females from December was sectioned and stained (hematoxylin and eosin). Analyses of the histological sections included identification of the oocytes in the various development stages (I-VI) as de- scribed by Knipe and Beeman (1978), and identification of atresia and postovulatory follicles (Fig. 2 ). We use the term "ova" to indicate an ovulated mature oocyte (stage VI). 308 Fishery Bulletin 102(2) Figure 2 Slide of the ovary (stained with hematoxylin and eosin) of a mature spawning female L. opaleseens. Bar = 1.0 mm. Table 1 Classification system for the gross anatomical characteristics of the reproductive system of female market squid (Loligo opaleseens). Female organs Character Grade Nidamental gland Accessory nidamental gland Oviduct Ovary Ovary length color number of large clear eggs number of large clear oocytes number of opaque or white oocytes millimeters 0=clear, l=whitish, 2 = pink. 3=peach. 4 =reddish-orange l=none. 2 = 1-20. 3=21-200, 4=>200 l=none, 2 = 1-20, 3=21-200. 4=>200 l=none, 2 = 1-20, 3=21-200, 4=>200 Postovulatory follicles were classified as either new, degen- erating, or very degenerative. We assigned the females to one of the following reproductive categories on the basis of the histology of their ovaries (numerical stages in Knipe and Beeman, 1978): Immature Ovary contains only unyolked oocytes; oocyte development ranged from stages I (oogonia) to IV (follicular invagination oocyte) and requires microscopic examination. Mature No postovulatory follicles are present. Ova- preovulatory ries contain oocytes with yolk (stage V, yolking begins about 1.1 mm in size); ovary usually contains unyolked oocytes. Mature Ovary contains postovulatory follicles spawning ( POFs) of any degree of degeneration (none to extensive); more than one degenerative POF class may be present. Oocyte develop- ment stages III— VI are often present but stages Ic-II are rare. (29t of the ovaries have late stage Ic oocytes and none have any of the earliest stages, la or lb.) In some histological sections of ovaries we saw 1-10 yolking oocytes (development stage V) with a broken follicle layer, and the yolk seemed to be oozing out between the other oocytes. Because this may have been an artifact of hand- ling, we did not use such females to estimate fecundity. Macewicz et at: Fecundity, egg deposition, and mortality of Lo/igo opalscens 309 We used the gravimetric method (Hunter et al., 1985, 1992) to estimate the standing stock of oocytes in 98 L. opalescens ovaries. The gravimetric method overes- timated the total number of oocytes of Loligo pealeii, but the difference between a count of all oocytes and a weight-based estimate was slight (Maxwell and Hanlon, 2000). We did not compare our estimates with a count of all oocytes in the ovary because we used a portion of the ovary for our histological examinations, and each value is the mean of the counts from two tissue samples (aver- age coefficient of variation between samples was 0.12). All oocytes in each tissue sample were macroscopically classified (Fig. 3) as either unyolked, yolked, mature, or atretic; they were then counted by class and all stages were summed. "Atretic" was defined as oocytes in the alpha stage of atresia (Hunter and Macewicz, 1985b), recognizing, however, that poor preservation can create oocytes of similar macroscopic appearance. The number of ova in the oviduct was also counted directly (usually when n was less than 300) or the mean number was estimated from two tissue samples by using the gravimetric method. To illustrate the form of the oocyte-size distribution in the ovary, we measured (to 0.01 mm) the major axis of all the oocytes in one tissue sample from each ovary of six females by using a digitizer linked by a video camera to a dissection microscope. In all other ovaries used for fecundity estimation, we measured only the smallest and largest oocyte in the sample. The length of the major axis of the smallest oocyte (D) was used as an index of the extent of ovarian maturity. D is a crude index of time elapsed during the spawning period — as long as oocyte maturation continues throughout the spawning period and no new oocytes are produced — both of which appear to be true for L. opalescens. To monitor body condition we cut a tissue sample disc from the mantle using a number 11 cork borer (area of 251.65 mm2) and removed the outer dermis and the in- ner membrane. The mantle sample discs were frozen and subsequently dried at 56°C to a constant weight. An index of mantle condition (C) was calculated as the weight of the dry mantle in milligrams divided by disc surface area and is expressed as mg/mm-. We evaluated the extent that body reserves might be used to support egg production by comparing dry weight of the eggs and capsules to prespawning female body dry weight. For these calculations we made the following mea- surements: 1) the mean dry weight of one squid egg was 0.00177 g, including a fraction of the egg capsule because the value is based on the dry weight of 34 egg capsules (1-2 days old) containing 2 to 403 eggs each (total of 7341 eggs, capsules collected from La Jolla Canyon 6 July and 11 September 2000); 2) the relationship of dorsal mantle length (L) and whole-body wet weight (Ww) for immature and mature preovulatory females of Wu. = 0.000051L2 8086, where Ww is in grams and L is in mm (Fig. 4); and 3) the mean wet weight to dry weight conversion factor of 0.24 (2SE = 0.001), based on the wet and dry weights of mantle tissue sampled from 214 mature females. The latter con- version factor was constant regardless of mantle condition index; apparently, in L. opalescens, starvation does not unyolked j?m M .early yolking ~ *> KBHI * S -^ smallest ^ oocyteM (unyolked) new-mieM postovulator^M Figure 3 Whole L. opalescens oocytes as viewed under a dis- section microscope used for counting and classifying oocytes. Bar = 1.0 mm. result in the replacement of muscle tissue with water as it does in fishes (Woodhead, 1960). In addition to the specimens taken during the research surveys, we also estimated the fecundity of 60 L. opal- escens from the commercial catch sampled by California Department of Fish & Game (CDF&G) during 1998 and 1999. Landed specimens were not analyzed histologically because their ovarian tissues had begun to deteriorate before preservation. The 60 females were selected by dor- sal mantle length and mantle condition index to provide a wide and uniform distribution of length and mantle condi- tion. The number of oocytes in the ovaries was estimated (as described above) and the number of ova in the oviducts were predicted from oviduct weight (Fig. 5). CDF&G also provided data on the dry mantle disc weights of 1275 ma- ture females taken from the catch from December 1998 through December 1999 as random samples taken during the Southern Californian Bight market squid fishery. About 100,000 tons of market squid were landed during this sampling period . Modeling egg deposition To identify egg deposition and mortality rates most consis- tent with our current understanding of spawning biology, we developed a model to estimate the proportion of the potential fecundity deposited by a cohort in its lifetime. The mean proportion of the potential fecundity deposited is the proportion of eggs deposited weighted by the propor- 310 Fishery Bulletin 102(2) tion of the cohort that died. Both the proportion of eggs deposited and squid that died were expressed as negative exponential functions. The cumulative eggs deposited up to elapsed time t (days I for a mature female L. opalescens is the difference of two terms: ESPl = EP- EYDt where ESP/ is the total eggs deposited by one female up to time t, EP is the potential fecundity, and EYDl is the standing stock of oocytes in the ovary plus the standing stock of ova in the oviduct remaining in the body at time t. If we assume that EYDl declines at an exponential rate from EP: EYDl = EP e~ut, where v is the daily rate of eggs deposited, then ESPt = Ej, ( l-e_1 0. We constructed the cumulative egg deposition curve as Qspt~ ESPl IEP= l-e~vt. Assuming the mortality (survival) curve for the squid is e~zt , where z is adult daily total mortality rate iz=m+f, where m is natural and f is fishing mortality), we computed the mean fraction of the potential fecundity deposited (QSPt): S 80 70 60 50 40 - 30 20 10 W= 0.000051 Z_28086 ,2=0.964 n = 42 50 60 70 n 1 1 1 1 1 1 100 110 120 130 140 150 160 80 90 Dorsal mantle length (mm Figure 4 Female squid whole body weight ( W) as a function of dorsal mantle length (L) for the 158 females with fecundity analyses. The line expresses the length-weight relation of females before weight losses associated with spawning and was fitted to the combined data for immature females (solid triangles), mature preovulatory females (solid circles), and mature females judged by their mantle condition to be new recruits to the spawning ground (solid circles). Open circles indicate females that have spawned. 2000 o y = 245x " -x" -o 1500 > o pseudo r2= 0.98 ^S^ • c | 1000 Number of o o o • St** I I I I I I I I I I i i i i 0 12 3 4 5 6 7 Oviduct weight (g) Figure 5 Number oi ova in each oviduct shewn as a function of the oviduct weight; n equals 91 mature females, pseudo r- = 1 - residual ss/ total ss. | ze-zta-e-vt)dt Qsp dt (!) = 1- zil-e' ) (z + y)(\-e ) z + v for large /n where tmax is the total elapsed time (days). The mean fraction of the potential fecundity that remains in the average female (standing stock of oocytes and ova) over her lifetime is 1 - Qsp, and mean QSP is always less than one because of mor- tality. The mean duration of the spawning period in days is computed as the elapsed time correspond- ing to the mean fraction of eggs deposited (QSP: Eq. 1 and by setting QSP=l-e-'''): ^sf=ln(l- QP )/(-«). (2) We evaluated various rates of adult daily total mortality (z) and egg deposition (r) using these models to determine the combination of rates that would provide estimates of fecundity nearest to our observed field data. Modeling the effect of fishing effort on egg escapement In theory we could manage the market squid fish- ery by monitoring egg escapement, that is, the frac- tion of the fecundity realized by the average female. Under such a management scheme, egg escapement would be maintained at a specified level by chang- ing fishing effort whenever escapement of eggs fell below it. In this section we develop a model to explore the relative effects of fishing effort on egg escapement. We use this model to discuss some of the biological issues related to using egg escape- ment as a management tool. In the modeling process, we follow one cohort of spawners. The elapsed time 0 is defined as the time when squid start spawning. The total escapement of eggs for a given elapsed time (tk in days) is the sum of three sources of egg escapement: Ec, the total number of eggs deposited by mature females in the catch; EM, the total number of eggs deposited by mature females dying of natural causes; and EA, the total number of eggs deposited by females Macewicz et al.: Fecundity, egg deposition, and mortality of Lo/igo opalscens 311 alive and not taken by the fishery up to time tk, and tk < tmax. The egg escapement rate, ReJk, up to time tk is the sum of the three sources of egg escapement divided by the total number of eggs that would have been spawned if no fishery existed (E): Ec + ESI + EA (3) Egg escapement rate at the maximum elapsed time Umax) is Re,tn EC + EM (4) where tk = tn Because there are no survivors at time tmax, no eggs can be deposited and EA is zero. Each term in Equation 3 can be expressed as functions of the mean cumulative number of eggs deposited up to time tk, ESP t. =EP- EYD tk = EP ( 1— e-"'* ), and total mortal- ity (z) of the cohort; z includes both natural morality (m) and fishing mortality if). For practicality, we considered cases when tk = tmax, where EA is zero. For formulas of any tk, see appendix. The total number of eggs deposited by the females in the catch iEc) is 'max Ec= j E^tN0e- m+l V fdt -EPN0 J (l-e-l")e-""+'"fdt o E~N0f {m + f )( m+f + v) (5) (6) where EP - the mean number of oocytes in the ovary per mature female prior to spawning; and Nn = the number of mature females at time 0. EPN0m ( m + f X m + f + v ) (9) The total eggs that would be deposited for the cohort without fishing mortality is 'max 'max E= \ E~^p~tN0me~mtdt = E~pN0 J Tl-e"1'' )me-""dt (10) o o E = EPN0 ■ (11) m + v where tmax (days) = the maximum elapsed time; and time 0 = the time at the onset of egg deposition. Egg escapement based on Equation 4 is f ■ + m- R.. ( m + /')(/?? + f + v) ( w + f)(m + f + v) (12) m + f + v Thus, egg escapement reduces down to a simple ratio, involving three daily instantaneous rates: natural mortality (m), egg deposition (v), and fishing mortality (/"). R„, =1 when there is no fishing and thus i?„„ (7) where EP and EYD = sample estimates from the catch; and Nc = the total number of spawners in the catch. The total number of eggs deposited by L. opalescens prior to death due to natural mortality iEM) is EM=jESPlN0e-'"-'"mdt = (8) EpN0J(l- •")e-"' mdt Results Oocyte maturation and production Immature ovaries contain many small unyolked oocytes with a pronounced peak at about 0.15 mm in size distribu- tion ( Fig. 6A). As development continues and vitellogenesis begins, the peak diminishes and shifts to a larger size class of unyolked oocytes (Fig. 6B). Just before the onset of spawning, the size distribution of oocytes becomes relatively fiat without pronounced modes (Fig. 6C) and remains so through the rest of the spawning period ( Fig. 6, D-F). The standing stock of oocytes declines throughout the spawning period. The minimum size of oocytes in the ovary gradually increases after the onset of yolking, indicating that new oocytes are not produced. We saw no primary oogonia in our histological sections of mature ova- ries, another indication that new oocytes are not produced in mature ovaries. Knipe and Beeman (1978) reached the 312 Fishery Bulletin 102(2) A No yolk in oocytes 86 mm 15 g 5646 Total oocytes 0 ova 0.719 = C 2.0 2.5 3.0 300 100 B Begun yolking 112 mm 39 g 44 10 Total oocytes 0 ova 0.711 = C JUiiLl i i - 1 1 1 1 1 1 1 1 1 > < 2.5 3.0 C Before 1st spawn - no POF 122 mm 39 g 3724 Total oocytes 0 ova 0.694 = C 0.5 1.0 1.5 2.0 600 500 400 300 200 100 0 600 500 400 300 200 100 0 D 2 stages of POFs 137 mm 50 g 2988 Total oocytes 88 ova 0.667 = C ..Illllllllllllll.ll.lh.lJ.. I..II. IllMl.... 1.0 1.5 2.0 2.5 3.0 E 3 stages of POFs 133 mm 45 g 1 642 Total oocytes 1 446 ova 0.574 = C ■■lllllllllllH I ■■■■•■ "iHl--! ■ 100 0 2.5 3.0 0 0.5 Oocye major axis diamter (mm F Diver caught - Dying 1 36 mm 33 g 1 487 Total oocytes 0 ova 0.544 = C lillllllllilililn.iiiinl.lii.,liiiii..i 1.0 1.5 2.0 2.5 3.0 Figure 6 Oocyte-size distribution for six female Loligo opalescens. Dorsal mantle length (mm), body weight (g), the total number of oocytes in the ovary, and the number of ova are indicated for each specimen. (A) Female that is immature. (B and C) Females that are considered to be mature and preovulatory because neither has postovulatory fol- licles (POFs) in their ovaries nor ova in their oviducts. Although the oocytes have just begun yolking in the ovary of female B, female C has well-yolked oocytes and is close to its first ovulation. (D-F) Females are mature spawning females and their ovaries contained postovulatory follicles. Female F was caught by a scuba diver and appeared to be dying. same conclusion from their histological analysis of L. opal- escens ovaries. Thus, potential fecundity in L. opalescens probably becomes fixed near the onset of spawning. Not all oocytes are deposited, however, because all spawning females had some oocytes and many oocytes were counted in the ovary of a dying female (Fig. 6F). In L. opalescens the migration of the oocyte nucleus begins early in the maturation process shortly before the onset of vitellogenesis, whereas in fishes, migration is near the end of vitellogenesis. The follicle of a migratory- nucleus-stage oocyte (late stage IV) of L. opalescens has a very large granulosa cell layer (in relation to the size of the oocyte) and is highly folded and perhaps fully developed. Subsequent maturation of the oocyte seems to consist primarily of the massive addition of yolk and fluid and the consequent stretching and unfolding of the follicle, ending with the formation of a chorion. Apparently, the formation of the chorion compacts the yolk because many mature oocytes (endpoint of stage V) have a smaller major axis than advanced yolked oocytes prior to chorion formation. Thus, maximum oocyte size is not a good proxy for oocyte maturation in L. opalescens and is not an indicator of the time remaining before spawning of the next batch. More importantly, the ovary of L. opalescens seems well adapted for rapid oocyte vitellogenesis, maturation, and spawning because nuclear migration and follicle cell proliferation is completed at an early stage. Ovulation appears to occur in small batches. The distri- bution of oocyte sizes in spawning L. opalescens was flat (e.g., Fig. 6, D-F) and lacked the separate and distinct mode of hydrated oocytes that is typical in fishes. Batch sizes of mature oocytes ranged from 5 to 246 and averaged 50 (n =72 Macewicz et al.: Fecundity, egg deposition, and mortality of Loligo opalscens 313 females). The maximum number of mature oocytes (246) was never close to the maximum number of ova ( 1726 ) in the oviduct. In addition, spawning females with 900 or more ova in their oviduct had in every case three or more distinctly different stages of postovulatory follicles in their ovaries (Table 2). Thus the oviduct is probably filled by a series of ovulation bouts separated by enough time to produce dis- tinct age classes of degenerating follicles in the ovary. Potential fecundity (EP) Potential fecundity (EP) is the standing stock of oocytes of all stages in the ovary of a mature female just prior to the first ovulation. Finding females at this point in their reproductive cycle was difficult because nearly all specimens had already ovulated. The ovaries of 94% of the 247 mature females, from our research cruises, con- tained postovulatory follicles, indicating that they had recently ovulated and would not be suitable for estimating EP. As can be seen in Figure 7A, spawning females had fewer oocytes in their ovaries than did mature preovula- tory females. The relation between fecundity and squid size is best expressed in terms of dorsal mantle length (L) because L. opalescens lose weight during spawning (Figs. 4, 7C). The data from thirteen mature preovulatory females were used to establish the relationship between potential fecundity and L: EP = 85.62L - 6715, [r* = 34.3%] where L = dorsal mantle length in mm. (14) Because the constant was not significant (P=0.146) and the coefficient was (P= 0.036), we forced the regression through zero which resulted in the equation EP = 29.8L. (15) Thus, the average female (129 mm) according to Equation 15 had a potential fecundity of 3844 oocytes (SE = 317). Clearly it would be preferable if the sample size for the estimate of potential fecundity were larger because thirteen females may not accurately represent theL. opalescens stock. Although the landed catch provides an unlimited supply of specimens, histological detection of postovulatory follicles is not possible because of deterioration of the ovaries. An alternative approach is to use mantle condition of mature females from the catch as a proxy for the preovulatory state. As can be seen in Figure 7C, the mantle condition index (C) of mature females declines as oocyte maturation continues and females deposit eggs. The mature preovulatory females (n=ll, two discs were lost) had a mean C of 0.73 mg/mm2 (SE = 0.02). We believe that the twenty-two mature females from the landed catch with C&0.7 mg/mm2 had not begun to deposit eggs (Table 3). Because many of them had ovu- lated, we combined our estimates of the standing stock of oocytes (EY) with those of ova (ED) to calculate total fecun- dity (EY + ED = EYD), and then regressed total fecundity on length. Although the regression was not significant, the average total fecundity of 3890 oocytes (Table 3) was within Table 2 Percentage of s the number of of ages (stages (POFs)intheii pawning female market squid classed by eggs in their oviducts and by the number of degeneration) of postovulatory follicles ovaries. Number of Number Percentage of females eggs in the oviduct of females 1 or 2 ages of POFs s3 ages of POFs 0 1 100 0 1-300 36 22 78 301-600 20 35 65 601-900 10 20 80 901-1200 7 0 100 1201-1500 2 0 100 1501-1800 2 0 100 5% of the potential fecundity of 4083 oocytes computed by substituting the mean length of the twenty-two females (137 mm) in Equation 15. The close agreement between these two values increases our confidence that the potential fecundity equation is accurate despite the low /;. On the other hand, this rough comparison is not a substitute for increasing the sample size of specimens analyzed histologically, because females from the catch may have spawned some of their ova before they were captured. Maximum fecundity (EP — ER) Few if any L. opalescens live to realize their full potential fecundity (EP). The literature on L. opalescens indicates that females that were described as "spawned out," dying, or dead had oocytes in all stages of development except the earliest previtellogenic stage (Knipe and Beeman 1978). In addition, all the spawning females that we collected had some oocytes in their ovaries. Thus, the maximum fecundity that L. opalescens might be expected to realize is the potential fecundity less an estimate of the number of oocytes that might be left in the ovary at death (residual fecundity [ER]). To estimate residual fecundity we exam- ined the relationship of the standing stock of oocytes in the spawning period with mantle condition index (C), size of the smallest oocyte (D), and dorsal mantle length (L). The standing stock of oocytes in ovaries of mature fe- males declines rapidly with decreasing mantle condition, between a C of 0.8 and 0.6 mg/mm2, and more gradually over lower mantle conditions (Fig. 7C). A curvilinear rela- tionship also exists between oocyte standing stock and the size of the smallest oocyte (Fig. 7B). Thus the number of past spawnings (decline in oocyte standing stock) appears to be inversely correlated with C and directly correlated with the extent of ovarian maturation as measured by D. To quantify how the standing stock of oocytes changes during the spawning period we fitted a nonlinear model to the fecundity data of 75 mature spawning females (Fig. 7) from our research cruises: 314 Fishery Bulletin 102(2) 12000 r 10000 8000 - 6000 4000 - 2000 0 Hrr- 0 60 \f^H^. -H-++ 80 T" t ~T 100 120 Dorsal mantle length (mm) — I — 140 160 2000 _ B _ o 0000 _ o _ o 8000 - 6000 4000 2000 0 I I I I I + -H- I I + I I I 0.0 6000 5000 4000 3000 - 2000 1000 0.8 0.2 0.4 0.6 Major axis diameter (mm) ot smallest oocyte 1.0 0.3 0.4 0.5 — r~ 0.6 -I 1- 0.7 "i r 0.8 — I r 0.9 1.0 Mantle condition index (mg/mm2) O Immature * Mature preovulatory + Mature spawning a Diver caught Figure 7 The number of oocytes in ovaries of 98 Loligo opalescens as a function of dorsal mantle length (A) and the diameter of the major axis of the smallest oocyte (Bl. In (C), the number of oocytes is plotted as a function of mantle condition index (the dry weight per surface area of a mantle tissue disci for 87 mature females (4 discs were lost and the 7 immature were not included I. Line in A expresses potential fecundity as a function of length (£^=29.8/,) for the 13 mature preovulatory females (solid circles); open circles represent immature females; plus signs represent spawning females; and the triangle represents a dying mature female. ER = 30283e'_1 24W ■ B 19('- 0.024i + 0.059LC) where C = mantle condition index; D = size of the smallest oocyte; and L = dorsal mantle length. (16) Substituting into the model (Fig. 8) the maximum ob- served D (0.771 mm) and the minimum observed C (0.323 mg/mm2) from our research survey data set, we estimated that a female L. opalescens with L of 129 mm may have a minimum residual fecundity of 834 oocytes (CV=0.12). Macewicz et al.: Fecundity, egg deposition, and mortality of Loligo opa/scens 315 Table 3 Mean fecundity, gonad weight, and dorsal mantle length fo ports December 1998 to December 1999. ■ 60 mature female market squid (Loligo opalescens) sampled at the Mantle condition (mg/mm2) ndex ( Fecundity mean numberl Mean gonad weight Cg) Dorsal mantle length ( mm ) Number of females Oocytes in ovary (EY) ' Ova in oviduct (ED) Total (EYD) Class Mean Mean Range 0.347- -0.499 0.432 1134 231 1365 2.215 132 106- -146 22 0.500 -0.699 0.613 2072 522 2594 4.959 125 102- -154 16 0.700- -0.951 0.824 2589 1301 3890 8.988 137 106- -160 22 0.347- -0.951 0.624 1917 701 2618 5.397 132 102- -160 60 A 129-mmL. opalescens with a potential fecundity of 3844 oocytes would have a maximum fecundity of 3010 eggs (3844-834 eggs) or about 78% of the potential fecundity. Very few females would be expected to deposit 78% of their potential because this maximum is based on extreme values for both mantle condition index and ovarian matu- ration. In a much larger set of mantle samples from the catch (Table 4), only 1.5% of the females had values of C less than 0.35 mg/mm-. Clearly very few squid live to deposit 78% of their potential fecundity. Another approach is to count the number of oocytes remaining in the ovaries of females presumed, from their behavior and appearance, to be dying. Although L. opal- escens has been observed to be dying or dead on the bot- tom on video from a remotely operated vehicle (Cossio1), capturing such females was not attempted at the time. A female L. opalescens (136 mm) believed to be dying was opportunistically collected by a diver 6 July 2000 on the La Jolla Canyon spawning grounds (McGowan, 1954). There were no ova and the ovary contained 1487 oocytes — substantially more oocytes than our estimate of the mini- mum residual fecundity. In fact, the female had deposited only about 63% of her potential fecundity. Role of body reserves We used weight relationships to evaluate the extent to which body reserves might be used to support the repro- duction of spawning female L. opalescens. In these crude energetic calculations we did not include metabolism, conversion efficiencies, or caloric values of tissues. We used the average dry weight of squid eggs, length to body weight conversion, potential fecundity equation, and the conversion factor from wet to dry mantle weight. We assumed preovulatory mantle condition index (C) for an average mature female of 130 mm was 0.798 mg/mm2, the mean for values (/!=41) of C > 0.700 mg/mm2 in the Table 4 Distribution of mantle condition index for 1275 mature female L. opalescens sampled from the landed catch from December 1998 to December 1999. Mantle condition index ( mg/mm2 1 Mature females Number Percentage 0.263-0.299 4 0.3 0.300-0.349 15 1.2 0.350-0.399 29 2.3 0.400-0.449 54 4.2 0.450-0.499 91 7.1 0.500-0.549 128 10.0 0.550-0.599 207 16.2 0.600-0.649 210 16.5 0.650-0.699 216 16.9 0.700-0.749 137 10.7 0.750-0.799 94 7.4 0.800-0.849 53 4.2 0.850-0.899 18 1.4 0.900-0.949 10 0.8 0.950-0.999 6 0.5 1.000-1.043 3 0.2 1 Cossio, A. 2000. Personal commun. Southwest Fisheries Science Center, National Marine Fisheries Service. 8604 La Jolla Shores Dr., La Jolla, CA 92037 our fecundity data set. We calculated that the potential fecundity of a 130-mm L. opalescens (i.e., 3874 encapsu- lated eggs) has a dry weight of 6.86 g which is equivalent to 64.8% of the whole-body dry weight (10.58 g) of that female just before spawning. If mantle condition is reduced in proportion to the dry weight of all the eggs, our hypo- thetical female would have a C of about 0.281mg/mm2 (0.798x[(10.58-6.86)/10.58]). This end point (C=0.281, egg=0) and the beginning point for the mature preovula- tory female (C=0.798, eggs=3874) create a hypothetical 316 Fishery Bulletin 102(2) 2000 1500 1000 500 L= 129mm Er - C= 0.323 mg/mm2 30283e<'1 24D-6.19C-0.024L+0.059f.C) Maximum Observed D (0.771) b i I .Yolking Begin Ovulation t — i — i — i — i — i — rn — I — I — I — I — I I I I I I I I 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Maior axis of smallest oocyte (D) (mm) 2000 - 1500 1000 - 500 - L = 129mm D= 0.771mm Minimum Observed C (0.323) 0.2 0.3 0.4 T — I — 0.8 0.5 0.6 0.7 0.8 Mantle condition index (C) (mg/mm: "l 1 1.0 Figure 8 Changes in the standing stock of oocytes predicted by Equation 16 (equation also given at top of panel) when major axis of small- est oocyte (D) is varied and mantle condition index (Cl held con- stant (upper panel), and when C is varied and D held constant llower panel). The major axis size of oocyte when yoking begins and when ovulation begins is also indicated, as are the maximum observed D and minimum observed C. Substitution of the latter two values into the equation yields the standing stock of oocytes of females close to the end of their reproductive activity and is consid- ered to be a minimum estimate of residual fecundity. line that expresses oocyte standing stock for the average mature female of 130 mm as a function of mantle condi- tion. In addition to the hypothetical line, we plotted the total standing stock of oocyte and ova (EYD) and mantle condition index for all 147 mature females used for direct fecundity determinations (Fig. 9). Our hypothetical line, based on direct proportionality between egg dry weight and body dry weight, follows the general trend in the data, indicating that energy reserves in mantle tissue may largely support the production and spawning of eggs. Of course, actual energy costs would be higher because metabolism, other somatic tissue, and conversion efficiency of mantle tissue to eggs are not considered. The lowest observed C in the fecundity data set was 0.323 and the lowest C observed in the 1275 mature females from the landed catch was 0.263. Using the above preovulatory C (0.798 mg/mm2), we determined that these values of C are equivalent to 60% and 67% losses in body dry weight for these individuals. Fields (1965) suggested body wet weight declined by as much as 50% , which is consistent with our results. These rough calculations support the long held belief that oocyte maturation is supported primarily by body reserves. Some feeding occurs during spawning; L. opal- escens has been observed feeding under lights at night on the spawning grounds (Butler2). Maxwell and Hanlon (2000) observed L. pealeii feeding between egg-laying bouts when they were held in the laboratory. Feeding be- tween spawning bouts by the more robust spawners that may migrate on and off the grounds each day seems quite 2 Butler, J. 2000. Personal commun. Southwest Fisheries Science Center. National Marine Fisheries Service, 8604 La Jolla Shores Dr., La Jolla, CA 92037. Macewicz et al.: Fecundity, egg deposition, and mortality of Lohgo opalscens 317 7000 > o ■n 6000 t CO W 5000 >N 4000 o o 3000 <1) F 2000 -j r "Hi 1000 o - 0 -70 A A A ° *A 4 ~° o a ^ o ^ °^ (18) where C = mantle condition index; and G = gonad (ovary and oviduct) weight. 5000 r 4000 - o 3000 - 2000 1000 EY = 220.453e<1 99C + 000791.) for L= 129 mm Mature Females Collected Dec. 1998-Dec.1999 n= 1275 Mean C (0.625) ±2SE 1 — 1 — 1 — r 0.2 0.3 0.4 1 — 1 — r~ 0.5 0.6 ~~ 1 — 1 — r 0.7 0.E ~\ T~ 0.9 1 1 1.0 Mantle condition index (C) mg/mm2 The predicted fecundity related well to the observed with a pseudo r2 of 0.60 (df=143). We also used generalized additive models to estimate fecundity (GAM, pseudo r2=0.64), as well as regression on the first principal component which explained 867r of the total variation (pseudo r2=0.55). Although the GAM gave a slightly higher pseudo r2 than the parametric nonlinear regression, we chose the later for easier interpretation and implementation. A pattern existed in the residuals from our model (Fig. 13); the model overestimated some fecundities at high mantle condition and underestimated fecundity at low mantle condition. This pattern in the residuals is probably related to the differences in density and size of oocytes in the ovary. Regardless of the minor problem with the residuals, this proxy (Eq. 18) for the standing stock of oocyte and ova is preferred because it gives a much more precise estimate at the minor additional cost of preserv- ing and subsequently determining the combined weight of ovary and oviduct. Although formalin weight of ovary and oviduct are not presently monitored in the fishery, it is a variable that could be added to fishery protocols at a minor increase in cost. Another benefit of this more pre- cise approach using EYD is that oviduct is included in the estimate. If an estimate of the removal of fecundity by the fishery is needed, ova must be included. Because ova are not included in Equation 17, to add them requires using the ovi- duct classification system (Table 1) to estimate the average number of ova — a system that is imprecise but cheap. One could, of course, use Equation 17 for EY and either count the ova in the oviduct or weigh the oviduct, but that would take more work than applying Equation 18 for EYD. Discussion Potential fecundity Our estimate of Loligo opalescens potential fecundity is based on a regression of the standing stock of oocytes on Figure 12 Standing stock of oocytes in the ovary (£>•) as a function of mantle condition index (C) for a 129-mm mature female L. opalescens as predicted by Equation 17 (equation also given on top of panel; L is dorsal mantle length). Dashed lines are ±2SE. The mean EY for the females taken in the fishery was 2221 oocytes. dorsal mantle length for mature preovulatory females having yolked oocytes in their ovaries. The accuracy of this approach depends upon the assumption that these females are at the point in life when the standing stock of oocytes in their ovaries is equivalent to their potential lifetime fecundity. This key assumption would not hold if some of the mature squid classed histologically as pre- ovulatory had in fact spawned. We do not know how long postovulatory follicles are distinguishable from atretic structures in the ovary of L. opalescens and, as far as we know, the rate of degeneration has not been determined for any loliginid. We know from our work on anchovy (Hunter and Goldberg, 1980; Hunter and Macewicz, 1985a), although it is not a cephalopod, that postovulatory follicles are distinguishable from atretic structures in the ovary of anchovy for about two to three days after spawning when the water temperature is about 16°C. This means that for undetected spawning to occur in L. opalescens, the inter- val between ovulation periods would likely need to exceed three days. This may be a minimum estimate because L. opalescens spawn at lower temperatures (9-13 C, Butler2) than do anchovy. Definitely a laboratory study on the rate of degeneration is necessary because postovulatory follicles in fish degenerate slower at lower temperatures (Fitzhugh and Hettler, 1995). In addition to the absence of postovulatory follicles, the oviduct must be empty for a spawning act to be undetected. Undetected ovulation and spawning seems unlikely because females with multiple stages of postovulatory follicles were common (87% of 247 mature females), females with only old postovulatory fol- licles were not detected, and the average life span on the spawning grounds may only be a few days. Atretic losses of oocytes are another possible bias in estimating potential fecundity. Atresia (degeneration and resorption of an oocyte and its follicle) appears to be a 322 Fishery Bulletin 102(2) 3000 2000 1000 0 CD > o -1000 + (0 -2000 / *.. • • T* . I* •• • * 0.3 0.4 0.5 — i 1 1 1 — 0.6 0.7 0.8 0.9 1.C Mantle condition index (mg/mm2) 3000 1- 2000- 1000- 0 -1000 -2000 • • • V * • • . . L*J" ^ — ^* *a wm ^ M • • ■ 0 0.0006 r >. 0.0004 - 0.0002 ~1 r 2 1 1 1 r 6 8 Gonad weight (g) 10 - 1 1 1 12 14 0.0b -2000 -1000 1000 2000 3000 Residuals Figure 13 Residual plots of number of oocytes and ova from the equation EYD = 378.28e(2 33C + 0 2447°-° 24CGl (Eq. 18) where standing stock of oocytes and ova (EYD ) are predicted from mantle condition index ( C) and gonad weight (G ). Bottom panel shows probability density of residuals. normal part of ovarian maturation in L. oplaescens, as it is the case for L. v. reynaudii (Melo and Sauer, 1998). Our evidence for this is that the standing stock of oocytes in immature female L. opalescens declines sharply as their ovaries mature (D increases, Fig. 7B). Clearly a narrow window of opportunity exists for an unbiased estimate of the potential fecundity of L. oplaescens. If the count is made too early in the ovarian maturation process, the count will either be low because extensive primary oogo- nia production may be still be occurring (64-mm female, Fig. 7A) or too high because additional oocytes will be absorbed before the female reaches maturity. If the count is made too late, it will be impossible to find a female that has not ovulated. Our selection criteria "presence of yolked oocytes" (which roughly begins at a oocyte size of about 1.1 mm) filtered out the very high counts of oocytes associ- ated with immature ovaries. From the practical standpoint, dealing with atretic losses that may continue into the spawning period is much less important for L. opalescens than for L. v. reynaudii (Melo and Sauer, 1998; Sauer et al., 1999) or L. pealeii (Maxwell and Hanlon, 2000). In these squid, where the spawning period may last weeks or months, atresia may seriously bias potential fecundity estimates. In the pres- Macewicz et al.: Fecundity, egg deposition, and mortality of Loligo opolscens 323 ent study all atretic losses would be attributed, of course, to ovulation and spawning but the chances of this being a major error seem low. Because we counted atretic as well as normal oocytes, atretic losses would be erroneously attributed to spawning only if atresia had proceeded to the point that the atretic structure could not be identified as that of an oocyte in whole-mount preparations under a light microscope ( 64x power). The time at stage for atretic oocytes in L. opalescens ovaries, as well as other squid, is unknown. The duration of alpha-stage atresia of yolked oocytes in anchovy is about a week at 16°C (Hunter and Macewicz, 1985b) and we suspect for the larger L. opal- escens yolked oocyte that the alpha-stage duration may be even longer. The disappearance of unyolked atretic oo- cytes, as an oocyte-like structure that would be counted, is more difficult to dismiss because so little is known about this atretic stage and its duration. If our estimate of the average longevity of spawning female is only about 1.67 days, then atretic losses of even small unyolked oo- cytes is probably not an important bias. It would be useful if a way could be found to estimate oocyte resorption rates in squid although it may be very difficult. It seems more important to validate our preliminary estimate of the average longevity of spawning squid, because if true, any concerns regarding atresia could be dismissed. Mature females without postovulatory follicles in their ovaries made up only 6% of the 247 females examined histologically. The rarity of these females in our collections reduced the precision of our potential fecundity estimate. Only thirteen of the fifteen females classed as a mature preovulatory female were usable for estimating potential fecundity, further reducing the sample size. Such a small sample size not only results in a low precision but raises the concern that the sample may not be representative of the stock as a whole. The fact that the average total fecundity of females with high mantle condition from the catch was close to the predicted value based on the thir- teen females, indicates that the latter estimate may not be biased. Clearly a larger sample size is needed, particularly if egg escapement is used to monitor the fishery. It would be helpful, in obtaining more samples, if we knew the reason for the apparent rarity of mature preovulatory L. opalescens females. One possibility is that females might pass rapidly from the initial vitellogenesis to ovulation, perhaps in the course of a single day or some fraction of it, and ovulation might begin sometime in the evening when L. opalescens are the most vulnerable to fishing. Another possibility is that mature preovulatory females aggregate in regions not heavily fished by either our trawl or the fishery. Egg escapement A practical suggestion from this study is the idea of man- aging spawning-ground loliginid fisheries by monitoring the fecundity of the catch and computing the fraction of the potential fecundity spawned. Monitoring the escapement of eggs from the fishery is an attractive approach for Loligo opalescens because costs are moderate, unlike the high cost for monitoring egg beds that cover many locations offshore and occur at any time of the year, and because traditional fishery assessment models are difficult to apply or inappropriate at the present time (PFMC, 2002). To proceed with escapement fecundity as a management tool, it would be necessary to set a target level for egg escapement and to relate escapement to egg-per-recruit analysis so that fishing effort could be adjusted to alter egg escapement rates. Conceptual work along these lines has been completed (Maxwell3) As mentioned earlier, as a practical matter in applying the egg escapement method, one would need to use QSP, the mean fraction of the potential fecundity escaping (Eq. 1), as a proxy for the more comprehensive and more use- ful measure of egg escapement Re tmaz, the fraction of the expected lifetime fecundity deposited (Eq. 12). Obviously, QSP will always be lower than Re-tm„ because the denomi- nator of QSP (the fraction ESPIEP) is potential fecundity which will always be larger than the denominator for Retmal, which is expected lifetime fecundity (E). Although quite a different value, QSP is a useful proxy for Re tnai. If natural mortality (m ) and egg deposition rates (v) are con- stant, changes in fishing mortality will result in changes in QSP that are proportional to the change in ReJmiI. However, changes will not be proportional if either v or m varies. If there is reason to believe that m and v are varying significantly, the use of QSP as a proxy for Retm!Lll should be undertaken with caution. A point of concern in applying this method is that it may be difficult to substantially change escapement of eggs by regulating fishing effort. Our model indicated that egg es- capement may be relatively insensitive to changes in fish- ing mortality if natural mortality rates are as high as we believe them to be. Of equal importance to management is the need to protect egg beds from damage by nets and to monitor the catch to prevent any change that might result in the capture of significant numbers of female L. opal- escens before they begin to deposit eggs. Thus the fraction of the catch that is immature females must be monitored if the stock is managed by using the egg escapement method. For simplicity, our calculations of escapement were based on only mature females because immature females were only 2.6% of the females in the catch (1998-99) and their inclusion had little effect on param- eter estimates. Egg escapement would decrease with an increase in the fraction of immature in the catch. As none of the fecundity of a captured immature female escapes the fishery, a relatively small increase in the fraction of immature animals in the catch can have significant consequences. From the standpoint of fishery management, the most important unanswered question regarding the reproduc- tive biology of L. opalescens is "how long do they remain on the spawning grounds?" or the equivalent question "what 3 Maxwell, M. R.. L. D. Jacobson, and R. Conser. Unpubl. data. Managing squid stocks using catch fecundity in an eggs-per-recruit model. Southwest Fisheries Science Center, National Marine Fisheries Service, 8604 La Jolla Shores Dr., La Jolla, CA 92037. 324 Fishery Bulletin 102(2) is the daily natural mortality of the spawners?" Loligo opalescens have only one spawning period in their life time (McGowan, 1954; Fields, 1965; Butler et al., 1999) but how long that period lasts remains unknown. Melo and Sauer (1999) concluded that the spawning period of L. v. reynaudii consisted of more than one spawning bout but neither the number of bouts nor the duration of each spawning period is known. In a laboratory study of L. pealeii (Maxwell and Hanlon, 2000), the number of bouts varied from one to ten, the interval between bouts was highly variable, and the life span after the first spawn- ing bout was from 3 to 50 days. Our best guess for L. opalescens under fishing conditions was an average life on the spawning grounds of only 1.67 days and a maximum longevity of about 6 days. These estimates were based on a simple exponential model, constrained by various proxies for egg deposition rate, longevity, and the fraction of the potential fecundity in the catch (the only directly mea- sured value). We believe that two of our estimates, 36% of the potential fecundity is deposited in the first 24 hours of spawning and minimum residual fecundity is about 22% of the potential fecundity, are on relatively firm ground but our estimate of the maximum longevity on the spawn- ing grounds as 6 days is speculative. New information on mortality is needed because, over a wide range of daily mortality rates, our model yields values that are consis- tent with observed average fraction of potential fecundity in the catch. Because direct measurement of mortality on the spawning grounds may be difficult, it may be useful to develop some indirect approaches. For example, a lab- oratory study could be designed to generate an energy- based model that converts squid mantle tissue loss to deposited eggs. This mantle-to-egg conversion rate could be used to assign an age (time elapsed after first egg depo- sition) to modes of mantle condition from fishery samples. Mortality could then be computed by following modes of mantle condition through time. Acknowledgments This study was a cooperative project between the Cali- fornia Department of Fish & Game (CDF&G) and the National Marine Fisheries Service (NMFS) from start to finish. We worked closely with CDF&G personnel throughout the study with port-sampling data, cruise time, and partial financial support was provided by CDF&G. We worked particularly closely with M. Yaremko, A. Henry, and D. Hanan of CDF&G. J. Welsh assisted in the fecundity work. Others that contributed include J. Butler, T. Kudroschoff, N. Smith, A. Preti, K. Lazar, A. Cossio, and at sea K. Barsky, T. Bishop, S Charter, R. Dotson, D. Fuller, C. Graff, D. Griffith, P. Hamdorf, A. Hays, B. Horandy, M. Levy, I. Taniguchi, J. Ugoretz, and L. Zeidberg. We wish to thank the crews of the research vessels Jordan and Mako. We especially wish to thank diver J. Hyde who observed, collected, and photographed squid in La Jolla Canyon. M. Maxwell and two anonymous review- ers read the manuscript and provided constructive com- ments. R. Allen and H. Orr improved our illustrations. Literature cited Alheit, J. 1993. Use of the daily egg production method for estima- ting biomass of clupeoid fishes: a review and evaluation. Bull. Mar. Sci. 53:750-767. Arkhipkin, A. 1993. Statolith microstructure and maximum age of Loligo gahi (Myopsida: Loliginidae) on the Patagonian shelf. J. Mar. Biol. Assoc. U.K. 73:979-982. Arkhipkin, A., and N. Nekludova. 1993. Age, growth and maturation of the loliginid squids Alloteuthis africana and A. subulata on the west African shelf. J. Mar. Biol. Ass. UK. 73:949-961. Bower, J. R., and Y. 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Age and growth estimation of the European squid, Loligo vulgaris, based on statolith microstructure. J. Mar. Biol. Assoc. U.K. 72:271-280. PFMC 2002. Report of the Stock Assessment Review (STAR) panel for market squid. Appendix 3. In, Status of the Pacific Coast coastal pelagic species fishery and recom- mended acceptable biological catches: stock assessment and fishery evaluation — 2002, 17 p. Pacific Fishery Management Council. Portland, OR Quinn, T. J., and R. B. Deriso. 1999. Quantitative fish dynamics. Biological resource Mmanagement series, 542 p. Oxford Univ. Press, New York, NY. Sauer, W. H. H., Y. C. Melo, and W. de Wet. 1999. Fecundity of the chokka squid Loligo vulgaris rey- naudii on the southeastern coast of South Africa. Mar. Biol. 135:315-319. Semmens, J. M., and N. A. Moltschaniwskyj. 2000. An examination of variable growth in the loliginid squid Sepioteuthis lessoniana: a whole animal and reduc- tionist approach. Mar. Ecol. Prog. Ser. 193:135-141. Woodhead, A. D. 1960. Nutrition and reproductive capacity in fish. Nutri- tion Society Proceedings 19:23-28. Yang, W. T., R. F. Hixon, P. E. Turk, M. E. Krejci, W. H. Hulet, and R. T. Hanlon. 1986. Growth, behavior, and sexual maturation of the market squid, Loligo opalescens, cultured through the life cycle. Fish. Bull. 84:771-798. 326 Fishery Bulletin 102(2) Appendix I Terms EP potential fecundity (standing stock of oocytes in the ovary of mature females prior to spawn- ing) ESPl the total eggs deposited on the bottom up to time t. (t in days) Ec total number of eggs deposited by mature females in the catch or total number of eggs escaped EM total number of eggs deposited by mature females prior to death due to natural mortal- ity EA total number of eggs deposited by females alive and not caught by fishery E total number of eggs that would have been spawned during a squid's lifetime if no fishery existed EY standing stock of oocytes in the ovary ED standing stock of ova in the oviduct EYD total fecundity, the sum of both the number of oocytes in the ovary and ova in the oviduct EYD tlt stocking stock of oocytes in the ovary plus those ova in the oviduct after spawning has begun and up to the elapsed time tk, where tk ls £ 'max tmax maximum elapsed time with the time 0 being the time when mature females are about to ovulate or total elapsed time (in days) of spawners on the spawning ground ER standing stock of oocytes remaining in ovary at death m daily adult natural mortality rate f daily fishing mortality rate for adults v daily egg deposition rate Qsr i = EsPt IEp = 1- ervt fraction of potential fecundity deposited up to time t e~zl mortality (survival) curve JV„ number of mature females at time 0 Nr total number of spawners in the catch Re tk egg escapement rate = ratio of eggs deposited to total number of egg which would be spawned if there was no fishery, at a given elapsed time Ret egg escapement rate up to the maximum elapsed time Umax) Appendix II For any elapsed time tk, formulas for Ec, EM, EA and E: Ec = EpNJ EM = EPN0m 1-e -lm+f)tk ^_e-(m+f+v)t), m + f m+f+v 1-e -{m+f)tk -. _ -im+f+v)tk m + f m+f+v EA = (EP-EYD.)Nk = AT0e-""+/ "* EP ( 1 - e"1"' ) V -mtJ , m e 1 e E = EPN0 The derivation for EA is straight forward and the deriva- tions for £r, EM, and E, similar among one another, are as follows: where Ec=\ ESP,dC, = J Ep~a- 100 ml trawls in Aleutian and Gulf of Alaska waters. S. ciliatus is commonly found in asso- ciation with S. alutus (Pacific ocean perch), S. polyspinis (northern rockfish), and S. variabilis (dusky rockfish). Less frequently, S. uariegatus (harlequin rockfish), S. zacentrus (sharpchin rockfish), and S. proriger (redstripe rockfish) are also captured with S. ciliatus. A large (320 mm; UW 47417) S. ciliatus was found in the stomach of a Pacific cod (Gadus macrocephalus) collected in the Aleutian Islands. Females captured in summer (May- July) trawl surveys are most often ripe with eyed larvae. Near-term females and males were observed in July in shallow waters off southeast Alaska in contrast to individuals of S. variabi- Table 8 Factor loadings for principal component (PC) analysis of meristic characters for shallow water Sebastes ciliatus and S. variabilis. PCI PC2 Lateral-line pores -0.5933 0.1677 Gill rakers -0.4274 -0.6851 Dorsal-fin rays -0.5742 -0.0777 Anal-fin rays -0.3667 0.6191 Pectoral-fin rays 0.0325 -0.3364 lis, which were all immature at this time (Orr, personal observ.). Etymology The specific name ciliatus is derived from the Latin word "cilium" for "eyelid" or "eyelash" and alludes to the numer- Orr and Blackburn: Resurrection of Sebastes variabilis and redescnption of Sebastes ciliatus 339 Figure 4 Distribution of Sebastes ciliatus based on material examined (open circles) and recent National Marine Fisheries Service survey data (closed circles) for the years 1999 to 2002. Each symbol may represent more than one capture. ous accessory scales (similar to fringing eyelashes) that are found on the posterior field of the larger scales in most species of Sebastes (Tilesius, 1813). Remarks Tilesius (1813) based his description of Epinephelus cil- iatus on a single specimen collected in the North Pacific "bordering Kamchatka and America," probably during the Krusenstern expedition of 1803-06 (Bauchot et al., 1997; Svetovidov, 1978, 1981; Pietsch, 1995). Although the illustration of the specimen was published (Tilesius, 1813; Fig. 2B), the specimen itself has since been lost, probably before the transfer of the Kunstkammer collection to the Zoological Museum of the Academy of Sciences, St. Peters- burg ( Svetovidov, 1978, 1981). Because S. ciliatus may easily be confused with other dark-colored Sebastes and S. variabi- lis, we have herein designated UW 43493, collected in Lynn Canal of southeast Alaska, as the neotype of S. ciliatus. The illustration of the holotype of E. ciliatus Tilesius (1813) depicts a uniformly dark individual of Sebastes, and most of its reported meristics and other characters are consistent with both S. ciliatus and S. variabilis. However, its lateral-line pore count is low at 43, and although falling well within the range found in the material examined of S. ciliatus, the count is represented in only one individual of S. variabilis examined. Along with its low lateral-line pore count, a moderate symphyseal knob is illustrated, similar to that of S. ciliatus, excluding its identification as S. melanops, a common and similarly colored Sebastes found within the geographic range of S. ciliatus. The anal-fin posterior margin of the specimen illus- trated shows a moderate posterior slant, and tips of the posteriormost rays extend well past those of the anterior rays. Sebastes ciliatus may have an anal fin with a slight posterior slant, unlike S. variabilis, but it is never as pronounced as the illustration indicates. However, this character is not found in any other dark-colored species of Sebastes presently known from the Aleutian Islands and northern Gulf of Alaska west of Kodiak Island. Sebastes entomelas has an anal fin with a strong posterior slant to its posterior margin, but the northernmost record of this species is Kodiak Island (Allen and Smith, 1988; Love, 2002 ; Mecklenburg et al., 2002 ) where it is rare ( RACE Di- vision6). Sebastes entomelas also has a much higher count of lateral-line pores (50-60; Love et al., 2002). One syntype of Perca variabilis was sent by Martin H. K. Lichtenstein (1780-1857), the director of the Berlin Zoological Museum in 1813, to Georges Cuvier at the Mu- seum National d'Histoire Naturelle in Paris, and has been preserved as MNHN 8670 (Svetovidov, 1981; Fig. 2 A). Although originally from the collections of Carl Heinrich Merck (1761-1799; Svetovidov, 1981; Blanc and Hureau, 1968; Bauchot and Desoutter, 1986) and thus contempora- 340 Fishery Bulletin 102(2) neous with Tilesius's material, this specimen probably did not serve as the example for Tilesius's (1813) illustration. The illustration is of the left side of a whole fish, whereas MNHN 8670 is the dried skin and head of the right side. Counts and measurements taken from the original descrip- tion and compared with the specimen indicate that it is improbable that the left side of this individual was the subject of the illustration. The counts provided by Tilesius (1813) include D XIII, (soft rays not given); A III, 8; PI 18; lateral-line pores 43. MNHN 8670 differs in counts of anal- fin rays (9) and in lateral-line pores (49). Although the sym- physeal knob is reduced and the anal-fin margin is strongly slanted posteriorly in the Tilesius illustration, MNHN 8670 has a strong symphyseal knob and a perpendicular anal-fin margin with a distinctly pointed tip (Fig. 2, A and B). Sebastes variabilis (Pallas, 1814) Dusky rockfish Figs. 1-3, 5; Tables 1-8 Perca variabilis Pallas, 1814:241 (original description, three? specimens; lectotype hereby designated, MNHN 8670, dried skin, sex unknown, 343.7 mm. "mari Ameri- cam borealum"; other syntypes apparently lost). Sebastes variabilis: Cuvier, in Cuvier and Valenciennes, 1829:547 (new combination). Sebastichthys ciliatus: Jordan and Jouy, 1881:8 (in part, new combination). Sebastodes ciliatus: Jordan and Gilbert, 1883:658 (in part, new combination). Sebastosto/nus ciliatus: Eigenmann and Beeson, 1894:388 (in part, new combination). Sebastes ciliatus: Westrheim, 1973:1230 (in part, new combination). Material examined A total of 253 specimens, 48.0-430.8 mm, including the lectotype listed above, were examined. See Appendix for additional catalog numbers and locality data. Diagnosis A species of Sebastes with the following combination of character states: body light yellow to greenish brown to gray, typically greenish brown, with orange flecks vari- ously present on sides, particularly light ventrally above anal-fin base and on ventral pectoral-fin rays; peritoneum light gray to jet black; symphyseal knob strong; extrin- sic swimbladder muscle with a single section of striated muscle, lacking anterior fascia, otherwise of type I (a-z) of Hallacher (1974); lateral-line pores 43-54, lateral-line scales 47-63; pectoral-fin rays 16-19; anal-fin rays 7-9; dorsal-fin rays 13-16; vertebrae 28-29 (11-12 + 16-18). Description D XIII-XIV, 13-16; A III, 7-9; PI 16-19, 7-11 simple; lateral-line pores 43-54, scales 47-63; gill rakers 32-37 110-11 + 22-26); vertebrae 28-29 ( 11-12 + 16-18) (one of ten specimens with 27 vertebrae, with one caudal vertebra bearing two neural and two haemal spines). Meristic fre- quency and statistical data are presented in Tables 2-4. Morphometric data and statistics are presented in Tables 1 and 4. Body relatively deep, especially at nape, depth at pelvic-fin base 29. 2-40. 91 SL: profile of dorsal margin of head steep to nape above anterodorsal margin of gill slit, flattening to dorsal-fin origin. Mouth large, with posterior end of maxilla extending beyond pupil to or beyond posterior rim of orbit, maxilla length 42.7-54.01 HL; symphyseal knob strong with blunt tip, lower-jaw length 52.8-62.71 HL; mandibular pores of moderate size. Cranial spines weak, in large adults covered by flesh, head smooth. Nasal spines invariably present; parietal ridge invariably present and small spine typically pres- ent; postocular and tympanic spines typically absent or obsolete in adults (weak postocular spines present on at least one side in 29.71 and weak tympanic spines present on at least one side in 50.61 of specimens examined) are typically present in juveniles. Interorbital region wide, 22.5-30.41 HL, strongly convex; parietal ridges weak, and area between ridges slightly convex; preopercular spines 5, directed posteroventrally; two opercular spines, upper spine directed posteriorly, lower spine directed posteroventrally; posttemporal and supracleithral spines present; lachrymal spines rounded, small; dorsal margin of opercle nearly horizontal; lower margin of gill cover with small spines: posteroventral tip of subopercle and anteroventral tip of interopercle rugose or with 1 or 2 small spines. Dorsal-fin origin above anterodorsal portion of gill slit; dorsal fin continuous, gradually increasing in height to spine IV or V and decreasing in height to spine XII; spine XIII much larger, forming anterior support of soft dorsal fin; membranes of spinous dorsal fin moderately incised, less so posteriorly; soft dorsal fin with anterior rays lon- gest, posterior rays gradually shortening. Anal-fin spine II shorter than III (5.8-13.6 vs. 9.5-15.61 SL), anterior rays longest on soft rayed portion of anal fin, posterior rays gradually shortening, posterior margin perpendicular to body axis or with slight posterior slant, anterior ray tips directly ventral to posterior tips, anterior tip of anal fin typically pointed. Pectoral fins with ray 10 longest, extending to or slightly anterior to vent, fin-ray length 23.5-31.01 SL, fin base to ray tip length 24.2-35.11 SL; fin-base width 9.4-11.21 SL. Pelvic fins extend about 601 of distance from pelvic-fin base to anal-fin origin, falling well short of vent, ray length 19.2-29.21 SL, spine length 44.9-70.71 ray length. Caudal fin slightly emarginate, length 15.4-26.91 SL. Vent positioned below dorsal-fin spine 10, 2.2-7.01 SL from anal-fin origin. Lateral body scales with many (ca. 5-7) accessory scales in posterior field. Maxilla and underside of mandible com- pletely scaled; suborbital region scaled; branchiostegal rays scaled. Gill rakers long, 11.6-19.91 HL, and slender on first arch, longest raker in joint between cerato- and hypobran- chials, length of preceding and succeeding rakers progres- sively shorter; rudiments absent. Pseudobranchs 36-38. Orr and Blackburn: Resurrection of Sebastes variabilis and redescription of Sebastes aliatus 341 Figure 5 Distribution of Sebastes variabilis based on materia] examined (open circles! and recent National Marine Fisheries Service survey data (closed circles) for the years 1999 to 2002. Each symbol may represent more than one capture. Body color in life variable (Fig. ID); adults typically light, greenish-tan (Fig. IB), often darker gray dorsally (Fig. ID), rarely lighter yellow overall (Fig. ID); invari- ably lightening ventrally to pinkish-white on head, belly, anal-fin base, and caudal peduncle; a clear demarcation between darker dorsum and light ventrum above anal-fin base; vague darker mottling tapering from origin of soft dorsal-fin ventrally and forward narrowing across lateral line, faint darker mottling also present farther posterior at soft dorsal-fin base, mottling most evident in tan individu- als; brown to orange "flecks" present on sides of body on posterior fields of scales, appearing as darker speckling in juveniles. Head similar in background color to body, two prominent bars extending from orbit to preopercle, a prom- inent bar along anterior margin of maxilla in darker indi- viduals (these bars obsolete in light individuals). Median fins and pelvic fins uniformly gray, lighter in light-bodied individuals. Pectoral fins brown to grayish pink; lower rays pink. Peritoneum light gray to jet black, typically dark gray; stomach, pyloric caeca, and intestines pale. See Figure 1 ( A-D) and previously published color figures of Kessler (1985; "Sebastes sp., light dusky rockfish"), Kramer and O'Connell (1986; "S. ciliatus, light"), Kramer and O'Connell (1988, 1995; "S. ciliatus, light specimen"), Orr et al. (1998, 2000; "S. ciliatus, light dusky rockfish"), Orr and Reuter (2002; "light dusky"), Mecklenburg et al. (2002; "light phase"). Juveniles in life (Fig. 1C) lighter than adults, with dor- sum light-brown to tan, background covered with orange- brown speckles, often with distinct dark band at base of soft dorsal fin; head brassy; ventrum pink on lower jaw. breast, and base of anal fin, lightening to white on belly. Largest specimen examined 430.8 mm (527.7 mm fork length [FL], 541.3 mm TL; UW 44253). Maximum size reported 590 mm FL (RACE Division6). Distribution and natural history Sebastes variabilis is recorded from a single specimen off Hokkaido, Japan (Shinohara et al., 1994), and from other specimens collected from the east coast of Kamchatka to Cape Ol'utorskii (at 60°N) in the western Bering Sea. along the Aleutian Islands to 60°N in the eastern Bering Sea, through the Gulf of Alaska south to Johnstone Strait, British Columbia (Peden and Wilson, 1976; Richards and Westrheim, 1988; Fig. 5), and to central Oregon (based on a recently collected single specimen [UW 46575] ). The ear- lier record of Schultz (1936) and Alverson and Welander ( 1952 ) from Washington at Neah Bay was reidentified by Westrheim (1968) as S. entomelas. Although the depth of collection for material examined ranges from 6 to 370 m, and the species is recorded at depths to 675 m, large adults are commonly found along the edge of the continental shelf at depths of 100-300 m, 342 Fishery Bulletin 102(2) where the species is the target of commercial fisheries in the Gulf of Alaska. During trawl surveys, it is most com- monly associated with S. alutus and S. polyspinis, and at greater depths with S. aleutianus (rougheye rockfish) throughout its range in Alaskan waters (Reuter, 1999; Ackley and Heifetz, 2001). Females and males captured during summer ( May- July ) trawl surveys ranged widely in maturity state. Occasional ripe females were observed, although most females were maturing (Orr, personal observ. ). A high percentage of females caught in trawl surveys during early April off southeast Alaska were releasing larvae, indicating that parturition occurs in the spring (Lunsford7). During July in shallower waters (ca. 40 m) of southeast Alaska, all S. variabilis collected were immature. Etymology The specific name variabilis is presumed to be a reference by Pallas (1814) to the wide range of body color in the species. Remarks Pallas ( 1814 ) described Perca variabilis from at least three specimens probably collected by Merck during the 1786-94 Billings expedition to the Russian Far East, including the Aleutian Islands, eastern Bering Sea, and northern Gulf of Alaska (Schmidt, 1950; Svetovidov, 1978, 1981; Pierce, 1990). One specimen was more completely described and used by him to obtain a set of counts and measurements. The other specimens were used to describe variation in the species, as in the following excerpt translated by the authors from the Latin text of Pallas (1814): "Body colored according to life and sex, varied, sometimes dark blue, belly white, fins blackish; female red below; those older wholly red or even purplish...." Pallas (1814) ultimately based the name P. variabilis on the supposed variability in color in this species. Jordan and Evermann (1898) examined an individual from the Pallas collection, recognized by him as the "sum- mer variety" of P. variabilis (ZMB 8145). They identified this "summer variety" as Sebastes aleutianus Jordan and Evermann (Jordan, 1884, 1885; Jordan and Evermann, 1898), a species easily distinguished from both S. varia- bilis and S. ciliatus by its full complement of eight pairs of strong cranial spines. These specimens have since been lost, probably during the destruction of the Berlin Zoo- logical Museum during World War II (Paepke and Fricke, 1992). Although Jordan and Gilbert (1883) wrote that S. proriger was also confounded with S. ciliatus, Jordan (1885) and Jordan and Evermann (1898) corrected this statement, noting that only S. ciliatus and S. aleutianus were included within the material described as E. ciliatus and P. variabilis by Tilesius and Pallas. 7 Lunsford, C. 2002. Personal commun. National Marine Fisheries Service, Aukc Bay Laboratory, Alaska Fisheries Science Center, 11305 Glacier Highway, Juneau, AK 99801- 8626. Although MNHN 8670 (Fig. 2A) is from the Pallas col- lection (Svetovidov, 1978, 1981), it is apparently not the specimen used for the complete description. In his original account, Pallas (1814) listed the following meristic data (modified to standard notation): D XIII, 15; A III, 7; PI 17 (8 simple). Although the dorsal-fin ray count is identical with that of the MNHN 8670 specimen, both anal- and pectoral-fin ray counts differ. All elements are well pre- served and easily counted. Comparisons of proportions are more difficult to inter- pret because measurements had not been standardized at the time of the original description. However, of those measurements that can be readily compared, the following significant differences were found, providing additional evidence that this individual was not the specimen used for the primary description: total length (391.6 mm in Pallas [1814; "longitudo majoris speciminis"] vs. 414.0 mm taken from MNHN 8670), head length (101.6 mm ["capi- tis a summa maxilla ad operculum angulum"] vs. either 96.6 mm [standard head length] or 110.2 mm [head length to tip of lower jaw]). The specimen used by Pallas for the detailed meristics and morphometries is presumed lost (Svetovidov, 1978). Ayres ( 1854) misidentified S. melanops from the vicinity of San Francisco Bay as S. variabilis. Giinther (I860) and Ayres (1862, 1863) placed S. variabilis of Ayres into the synonymy of S. melanops. Comparisons Sebastes variabilis is most similar to S. ciliatus; the latter is distinguished by its uniformly dark-blue to black color. Sebastes ciliatus is invariably dark at the base of the anal fin and on the lower pectoral rays, areas of lighter color in S. variabilis even in those individuals that have an overall dark body. The peritoneum of S. ciliatus is always jet black, unlike the usual gray peritoneum of S. variabilis, which however may often be dark or occasionally jet black. In combination with these color differences, a low count of 39-42 lateral-line pores will distinguish S. ciliatus from S. variabilis, although the total range of counts overlaps considerably. The extrinsic morphological features of the swimblad- der of both S. ciliatus and S. variabilis are of type I (a-z) of Hallacher (1974) in which the anterior muscle mass originates from the occipital region of the neurocranium. attaches to the pectoral girdle near the insertion of Baude- lot's ligament, passes between the epineural and pleural ribs of vertebrae 3 and 4, passes ventral to the pleural rib of vertebrae 5, and continues posteriorly as three tendons that insert on the pleural ribs of vertebrae 8, 9, and 10. In S. ciliatus and not S. variabilis, the anterior striated muscle mass is separated into two sections by a thin fascia, similar to the condition reported in S. paucispinis alone among species of Sebastes (Hallacher, 1974). The morpho- logical features of the complex differ significantly in S. paucispinis, however, in that the striated muscle does not attach to the pectoral girdle but bypasses it to insert by a single tendon into the posterior portion of the swimblad- der. Only five specimens each of S. ciliatus and S. varia- Orr and Blackburn: Resurrection of Sebastes variabilis and redescription of Sebastes ciliatus 343 bills were dissected in the present study to examine these muscles. Additional material should be examined to assess the intraspecific variability and systematic significance of this character complex. Typical habitats of these two species also differ. Adult S. ciliatus are found in nearshore shallow habitats at maximum depths of 160 m and are abundant in protected coves on the outer coast of Alaska. Sebastes variabilis, in contrast, is found along the continental shelf margin at depths to 675 m. However, adult S. variabilis have also been collected in nearshore waters as shallow as 40 m ( UW 43494). In areas of sympatry, such as the inside waters in Lynn Canal of southeast Alaska and Monashka Bay of Kodiak Island, S. variabilis is found at greater depths in stronger current, whereas S. ciliatus is found, often with S. melanops, among kelp (Macrocystis) on rocky ledges (Blackburn and Orr, personal observ. ). Other uniformly dark colored species of Sebastes, such as S. melanops and S. mystinus, may also be confused with S. ciliatus and darker individuals of S. variabilis, al- though both may be distinguished on the basis of color and morphological features. The body of S. melanops is dark bluish-black, has black speckling on the dorsum and lat- eral surfaces, and a distinctly white ventrum (in contrast with the slightly lighter ventrum of S. ciliatus (Fig. IE]). Unlike S. ciliatus, in which the peritoneum is jet black, S. melanops has a white peritoneum. In S. melanops, five or six faint light blotches slightly larger than the orbit are present on the dorsum about midway between the lateral line and the dorsal-fin base. These blotches are especially prominent underwater, and in Alaska easily distinguish S. melanops from both S. variabilis and S. ciliatus, which lack blotches (Lauth8; see color figures of Love, 2002, and Stewart and Love, 2002). The symphyseal knob in S. melanops is obsolete, consisting only of a fleshy pad at the tip of the mandible, unlike the distinct bony knob of S. ciliatus and S. variabilis. Mandibular pores of S. melanops are obsolete, as compared with the larger, readily appar- ent pores of S. ciliatus and S. variabilis. Vertebral counts also differ, from 28-29 in S. ciliatus and S. variabilis to 26 in S. melanops. Sebastes melanops ranges from southern California to Atka Island in the Aleutian Islands (Meck- lenburg et al., 2002) and the southern Bering Sea, where its presence is documented by a single recent collection (UW 47037). Most previous reports from the Bering Sea may be of S. ciliatus. Sebastes mystinus is also similar to S. ciliatus but may be distinguished by the four distinct dark bars across its head and nape contrasting with its general body color of light mottled bluish-gray. The mouth of S. mystinus is smaller than that of S. ciliatus, and the maxilla extends only to the middle of the pupil rather than to the posterior portion of the orbit as in S. ciliatus. Like S. melanops and most Pacific Sebastes, S. mystinus has 26-27 vertebrae, compared to the 28-29 vertebrae of S. ciliatus and S. s Lauth, R. R. 1998. Personal commun. Resource Assess- ment and Conservation Engineering Division, Alaska Fisheries Science Center, Natl. Mar. Fish. Serv., NOAA, 7600 Sand Point Way NE, Seattle, WA 98115. variabilis. Sebastes mystinus has been recorded from Sitka Harbor, Alaska (Kramer and O'Connell, 1995), to Punta Santo Tomas, northern Baja California (Hobson, 2002). Earlier reports from Kodiak Island, the Aleutian Islands, and Bering Sea are undocumented ( Quast and Hall. 1972 ; Kramer and O'Connell, 1995; Mecklenburg et al., 2002) and probably refer to S. ciliatus. Sebastes polyspmis is commonly caught in trawls and may be confused with S. variabilis, especially when pre- served. It can be distinguished from S. variabilis by its modal count of 14 dorsal-fin spines and light (pink or white when live) oblique band across the lower rays of the pecto- ral fin, which remains prominent when recently preserved. In life, the overall color of S. polyspinis is reddish-orange to pink, overlaid with gray-green mottling and fine green spots. Evermann and Goldsborough (1907) considered the then undescribed S. polyspinis within the range of varia- tion of "S. ciliatus" because at least one lot (USNM 6243) was misidentified by them as "S. ciliatus." They probably also confused S. melanops with S. variabilis, or possibly S. polyspinis, describing the color in life of S. melanops from Alaska as "olive-brown, blotched with dirty red." Sebastes melanops never has a trace of red, whereas the most com- mon color pattern of S. variabilis could be adequately described by this phrase. In the western Pacific, two species, the dark-colored S. taczanowskii and the light-colored S. schlegelll, may be confused with S. ciliatus and S. variabilis, respectively. Both may be distinguished from S. ciliatus and S. varia- bilis by modal counts of pectoral-fin rays (15 in S. tacza- nowskii and 17 in S. schlegelii vs. 18 in both S. ciliatus and S. variabilis) and vertebrae (26 in both S. taczanowskii and S. schlegelii vs. 28-29 in S. ciliatus and S. variabilis). Sebastes schlegelii may also be distinguished by its typi- cal dorsal-fin spine count of 12 (vs. 13 in S. ciliatus and S. variabilis). Implications for fisheries management The dusky rockfish (S. variabilis) and the dark rockfish (S. ciliatus) have been subjected to two distinct fisheries separately managed by U.S. federal and Alaska state agencies: S. variabilis is captured in the offshore trawl fishery; S. ciliatus, in the nearshore jig fishery. Although the offshore fisheries for dusky rockfish only inciden- tally catch the dark rockfish and are managed for dusky rockfish, the nearshore fishery is not managed for dark rockfish, and instead the species has been routinely mis- identified as black rockfish (S. melanops). Sebastes ciliatus has been found to comprise up to 25<7, the number of schools, Nr the school size distribution, Pt(k), and the average school size, k r Note thati?, =Ntkr Simula- tions were run until there were no fish left. Encounter rates The encounter rates, m and w, were based upon random movements in statistical mechanics (Tolman, 1979) in which the encounter rate (£/) of objects of type i with objects of type 7 is described by Methods U=(G, + GO Dp, {v? + v/)"3, (1) Simulation of individuals in ecology and population dynamics (individual-based models) have become increas- ingly popular (McCauley et al., 1993). However, it is often difficult to understand the dynamics of large individu- ally based models (Pascual and Levin, 1999). Thus, it is important to obtain models that describe dynamics of groups that incorporate individual behavior (Flierl et al., 1999). The models that are developed here include an individually based model (simulation model) and an ana- lytical model that describes "mean-field" dynamics of the individuals behavior. Simulation model The recruiting fish of a year class may be modeled as a network offish in which a fish "links" to other fish to form schools. (Note that in this context it is assumed that a "school" includes aggregations consisting of a single fish). Thus, the process of aggregation is a process of adding links to nodes (aggregation of schools). Similarly, mortal- where G, = the size of the detection space at which object detects object type./'; Z), = the density of objects of type i; and u, = the velocity (in three-dimensional space) at which object i moves in the environment. For these simulations the G parameters were scaled to one and the velocity parameters (v's) were collapsed into two encounter rates: ,11 for mortality encounters (scaled to unity) and a for aggregation encounters. Mortality rate In the simulations, mortality of fish is perpetrated by mortality agents. If the mortality agents randomly encounter schools of fish, then the probability of a successful mortality (the removal of a fish from the system) is proportional to the school size k. Under these conditions Equation 1 reduces to Equation 2 with G=G, ■UV? +V3. )<={!■■ (2) m = 2^ENk, Powers: Recruitment as an evolving random process of aggregation and mortality 351 where E = the density of mortality agents; and k = the encounter rate of fish with mortality agents. Note that on average Equation 2 reduces to m = 2iiElNlkl= 2iiEt Rt = -dRIdt. Hence, if the density of mortality agents is constant throughout the recruitment period, then mor- tality is density independent and mortality is proportional to abundance. An alternative interpretation of Equation 2 is that the mortality agents randomly encounter fish and that all encounters result in a successful mortality. The mortality model (Eq. 2) will be referred to as mdl (for den- sity-independent). It is not expected that mdi is the most realistic, but rather it provides a basis for comparison. A second mortality alternative is where mortality agents randomly encounter schools, whereupon they always per- petrate a successful mortality: mN = 2iiEtNt. This model, like mdi, assumes that the density of mortality agents are constant throughout the recruitment period. For purposes of simulation, the density of mortality agents at the onset of the recruitment process was speci- fied to be unity (£0=1). For the two mortality models, mdi and mN, this meant that £=1 throughout a simulation. More realistic density-dependent mortality models are immediately suggested. The first is a density-dependent model in which the ratio of mortality-agent density to the number of schools remains constant throughout the re- cruitment period, i.e., EtINt remains constant throughout the recruitment period. This leads to mdN = 2f.iNzk, where EtINt was set equal to one. In this model the ratio of mor- tality agents to schools is constant, agents and schools randomly encounter one another, and the probability of a successful mortality (given there is an encounter) is pro- portional to the number offish that are in the school that is encountered (mortality success is related preferentially toward larger schools). A second density-dependent model is where the mortal- ity agent density is proportional to the number offish (Etl Rt is a constant set equal to one, mdR=2iiR2=2iiN2k2). In this model the ratio of mortality agents to the number of fish in the population is constant; agents and schools randomly encounter one another; and the probability of a successful mortality (given there is an encounter) is pro- portional to the number offish that are in the school that is encountered (mortality success related preferentially toward larger schools). Another interpretation of this model is that agents randomly encounter fish, at which time the fish suffers mortality at a probability independent of school-size characteristics. A third density-dependent model depicts mortality- agent density proportional to school size (Etlkt is a con- stant set equal to one, mdk=2\.i Nk'1). In this model the ratio of mortality agents to mean school size is constant, agents and schools randomly encounter one another, and the probability of a successful mortality (given that there is an encounter) is proportional to the number offish that are in the school that is encountered. Another interpreta- tion of this model is that agent density is proportional to the number of schools, agents encounter schools prefer- entially according to school size, and the probability of a successful mortality (given that there is an encounter) is proportional to the number of fish that are in the school that is encountered. Subsequently it will be shown that the first density- dependent model is related to a Ricker-like stock-recruit- ment model and the second model is exactly equivalent to a Beverton-Holt model. Definitions of the mortality models are summarized in Table 1. Note that in the ini- tial applications of these mortality models, it is assumed that a mortality encounter results in mortality of one fish. More detailed mortality models in which a number of fish greater than one are removed by mortality may be implemented in the future. Clearly, these would be more biologically realistic in many instances. However, the emphasis of this study is on the possible scaling behavior of school-size distributions. Barabasi and Albert (1999) showed that the scaling behavior of a growing random network is independent of the number of randomly se- lected links at each time step. With this analogy, simple increases in mortality per encounter are not expected to alter the scaling behavior of the school-size distributions. Therefore, the one-fish-per-mortality-encounter approach was used in these initial simulations. Aggregation rate Similar to mortality-rate encounters, aggregations were investigated as 1) random attachment of two unique schools (wN=2aNiN-D) and 2) preferential attachment of two unique schools i and./' (w =2aN(N-\)klkJ; [Table 1]). Note, the trivial alternative where there was no attach- ment, (a=0), results in equivalence between the mortal- ity models mdN, and mdR\ whereas mdl becomes a simple proportional mortality rate. Thus, results of models with o=0 are uninteresting in the context of this study and are not presented. Initial conditions Each simulation was conducted with one of two alternative initial conditions. The first alterna- tive was one of complete disaggregation in which simula- tions were initiated with S fish, S schools, and one fish in each school (NQ=S, kQ=l). The second alternative initial condition was constructed from the population dynamics of a typical fish population. The main assertion of this alternative is that the eggs or larval fish produced by one female during spawning constitutes one school at the onset of the recruitment process. Thus, the fecundity per female at age is a measure of initial school size and the abundance of females at age is a measure of the frequency of schools of that size. More precisely, the initial condition was constructed for a population of females greater than five years of age (age of maturity), where their fecundity at age, F ' is proportional to weight at age determined from a von Bertalanffy growth equation with parameters K=0.2 and L^ = 10, and an allometric parameter of 3: (F =1000 [(l-exp(-age( 0.2)))] 3). Abundance at age, Aage, was computed with an instantaneous mortality rate of 0.2: [A =Zexp(-0.2(age-5))]. The scalarXwas obtained from the approximate solution to S =2Fage Aage, where F and A were integer values and S was the initial number of fish 352 Fishery Bulletin 102(2) Table 1 Summary of definitons of the mortality models used in this study. Model Definition Mortality rates': mdi = 2ftNk m ix = 2fiN2k mdR = 2fiN2kk mdk=2ijNkk mN = 2iiN density-independent density-dependent, mortality agents proportional to A'' density-dependent, mortality agents proportional to R density-dependent, mortality agents proportional to k random encounters with schools Aggregation rates: wN = 2ctN(N-l) random encounters with schools wpa = 2aN(N-l)klkJ preferential attachment of schools i andj Initial conditions: Disaggregated Aggregated N0 = S,k0=l (see text and Table 2) Mean field equivalents usee in analytical model (see text): mdi = 2fiNk m(/v = 2^iNk2 mJR = 2fiN2k2 mM = 2iiNk2 wpa = 2NiN-l)k2 Key to figures of simulation results Figure 1: disaggregated md, Wpa a = 10-6 S = 106 Figure 2: disaggregated mJ, WN « = io-6 S=106 Figure 3: aggregated "hi, ™pa a = IO"6 S = 108 Figure 4: aggregated "IJN Wpa a =1.5xl0-6 S = 2 x 106 Figure 5: aggregated mdN wN «= 1.5xl0-6 S = 2 x 106 1 In all simulations, ft was set equal to 1. Table 2 The aggreg ated in itial school-size distribution , when S = 1,000,000 . Per capita female fecundity at age is a measure of school size, num ber of female; at age is a measure of freq jency of sc hools. See text for details of computation. School size Freq . of schools Freq. x size School size Freq of schools Freq. x size 252 348 87,696 857 47 40.279 341 284 96,844 882 38 33.516 427 233 99,491 903 31 27,993 508 190 96,520 920 25 23.000 581 156 90,636 934 21 19,614 596 1 596 946 17 16,082 646 128 82,688 955 14 1.3,370 703 104 73,112 963 11 10,593 751 85 63,835 970 9 8730 793 70 55,510 975 7 6825 828 57 47,196 979 6 5874 Sum of freq. x size = S = = 1,000.000. of a simulation. Then one school of an appropriate magni- tude, M, was added such that the M +^Fll/,vA„.,,, was exactly equal to S. Note that under this construction the school sizes in the distribution do not vary with S (except for the one school of size M), whereas the frequency of schools by size do. An example of the initial distribution with the use of this construction is given in Table 2. Analytical models Analytical models of aggregation and recruitment are presented, where the models are developed from first prin- ciples and the parameters have an interpretation in the physics and biology of the recruitment process. Hopefully, the nature of the parameters can guide model selection, Powers: Recruitment as an evolving random process of aggregation and mortality 353 and the estimates may provide a theoretical framework for empirical research on recruitment processes. Noting that R,=Ntkt, the recruitment dynamics depicted in the simulations may be modeled by using Equations 3-6 in which recruitment is dependent on the particular mortality and aggregation models that are chosen (m and w; Table 1): dR,l = -m = d(N,k, "/dt=(dkl Idf'N, +(dN, I dt"k, (3) dNJdt -2aNt{Nt-l)\l dN, I dt = -w - mxPu dk,/dt = mft, [( k, - 1)1 ( N, - 1) -< m - m^Pv )l N,+ wkt / 1 i-l + wYJP,Pk-,INl (6) = m2PuINt - miPua-Pu >/< AT, -1) k = l -w1PuINl , where Ph , = the proportion of schools with k fish in them at time t . Also, mk and wk denote encounter rates appropriate to schools of size k, whereas unsubscripted m and w denote mean field dynamics and, thus, the kt t's are replaced by kt's (see Table 1). The first term in Equation 4 denotes the reduction in number of schools due to aggregation events; the second term denotes a reduction due to mortality events on schools with one fish in them. Similarly, the first term in the mean school-size equation ( Eq. 5 ) describes the change in mean school size due to mortality events on schools of size equal to one; the second term is due to mortality events on schools of size greater than one; and the third term is due to aggregation events. Finally, the first term in Equation 6 describes the change in probability of school size k due to mortality; the second term describes loss due to aggregation; and the third describes gain due to aggre- gation. Of particular importance is Pl t: when Px , is zero, the loss of schools occurs only due to aggregation. When Pj , is positive, then loss of schools is accelerated due to mortality (Eq. 4). The goal is to obtain solutions to Equations 3-6 as functions of a, ju, and the initial conditions. If one can be assured that there will not be a school composed of one fish during a particular recruitment period (Pj ,=0), then Equation 6 is eliminated, the Pj ( terms drop out of Equa- tions 4 and 5, and a numerical or analytical solution to the differential equations can be obtained, which is com- putationally feasible for use in fitting to stock-recruitment data. For example, when there is preferential aggregation (w ) and mortality agents are proportional to schools (mdN), the equations reduce to dktldt-- ldN I N, + w k,( Nj_-1)= -2/jNtk, + 2aNtkt3 Analytical solutions were obtained for some of the mor- tality and aggregation models when P1(=0 throughout the recruitment process (Appendix 1). In particular for m„ !Randwpa- Rt = SHl+2iitS) (7) Nt=NQ+(a/n"[S-S/e%* %itS"] (8) ^=^/[l+2^S + 2ctfS^]. (9) which is the Beverton-Holt stock-recruitment model expanded to include equations for the number of schools and the mean school size. Interestingly, Equation 9 indi- cates that monitoring the school-size distribution two or more times during a recruitment procession would yield estimates of the stock-recruitment parameters without having direct measures of the number of surviving fish. Equation 7 predicts recruitment by using one parameter. j.1. , the rate of mortality encounters during the recruitment period. However, spawning stock biomass is often used as a surrogate for the number of initial stock, S. Thus, another parameter is needed to convert spawning stock biomass to S in Equation 7. In that case the recruitment model becomes Rt = aS/(l+2utaS), where a is another parameter related to fecundity. The additional parameter will be needed for all the models discussed here, if spawning stock biomass is the measure of initial stock. The assumption that Plt=0 for all t of a recruitment period may not be justified in all situations. An approxi- mation was developed (Appendix 2) to be applied when the initial conditions are disaggregated and when there is preferential attachment. In this circumstance, the dif- ferential equation (Eq. 6) when k = l is replaced by dPu/dt = -wPu/N, + m ( 1 - Pu)INt. (10) Results Simulations Several hundred simulations were conducted under vari- ous initial stock sizes (S), aggregation parameters (o), ini- tial aggregation conditions, and mortality and aggregation models (m and w). An example set of results are presented in Figures 1-5 (a key to figures is in Table 1). A typical example of the evolution of the school-size distribution is given in Figure 1 for the disaggregated initial condition, a=10"6, S=106, mortality model mdi and aggregation model w . In this example both the mortality and aggregation models exhibit preferential attachment, and the school-size distribution approaches scale-free be- havior P(k)~k->, although y evolves over time. Eventually, a so-called "giant cluster" is formed by the aggregation process, in which all the fish attach to one school. This has 354 Fishery Bulletin 102(2) 1 ,000.000 o f = 0.1 1 + f=0.40 » f=0.95 A O V \ sw luminal iih i School size -r 1,000,000 -- 5,000,00 ( Figure 1 Simulated dynamics of school-size distributions with mdl as the mortality model and w as the aggregation model. This simulation started with disaggregated initial conditions (JVn = Sl, where S=106. The aggregation parameter was oc=10-6. The top panel shows school-size distributions (in log-log scale) at selected times (/). The lower panel gives the mean school size (kbar) and school abundance (AM versus time. been shown to be an analog of Bose-Einstein condensation (Bianconi and Barabasi, 2001; Dorogovtsev and Mendes, 2002) and gelation (Krapivsky et al., 2000). Greater mix- ing rates Cot's) and larger densities (N's) accelerate the aggregation process and the formation of the giant cluster. The average size, k, increases over time from the disag- gregated initial condition until a giant cluster is formed. The number of schools declines over time because of both aggregation and the mortality of fish in schools that only have one fish in them. When there is random aggregation beginning from a disaggregated initial condition (a=10-6, S=10K, mdi, wN ; Fig. 2), the school-size distribution exhibits exponential behavior P(k)~exp(-/Jt), with A evolving over time. This is equivalent to the Erdos and Renyi (1960) results for random graphs. A comparison of Figure 2 with Figure 1 shows the difference between preferential attachment and random attachment, i.e., the difference between scale-free and exponential models. Aggregated initial conditions (Figs. 3-5) result in a transition from the initial distribution to either scale- free or exponential distribution. During the transition, the size of the smallest school gradually becomes smaller until there is a finite probability of schools with one fish in Powers: Recruitment as an evolving random process of aggregation and mortality 355 1.000.000 - o + A + +• o ( = 0.03 + (=0.33 a (=0.62 >. o c B- 1,000 - 1 - A O + A O + A O + A -t- A + A + A * — I 1 School size J3 -r 1.000,000 Figure 2 Simulated dynamics of school-size distributions with mdl as the mortality model and wN as the aggregation model. This simulation started with disag- gregated initial conditions (N0 = S), where S=106. The aggregation parameter was a=10~6. The top panel shows school-size distributions (frequency in log) at selected times ((). The lower panel gives the mean school size (kbar) and school abundance (JV) versus time. them. At this point the reduction in the number of schools is accelerated because of the mortality of fish that are in "schools" in which they are the only member, and because of the loss of schools attributed to aggregation. Model comparisons Numerical integration of Equations 3-5 matched the sim- ulation results (Fig. 6, when P1(=0), indicating that the mathematical model describes the simulation dynamics. The numerical techniques are sufficiently efficient to be used in a curve-fitting context. Evaluations of the approxi- mation (Appendix 2) indicate that the approximation may be useful for predictions of recruitment, when compared with the simulations. However, the components of recruit- ment, kt and Nr were biased (Fig. 7). Further research is needed to develop estimates of P1 1 and, more generally, P(k) under other models and initial conditions. Recruitment was compared between mortality models and aggregation models (Fig. 8). If the mortality model was either mdl or mdR, then the mortality rate was not af- fected by the school-size distribution: random attachment and preferential attachment perform equally as well in terms of survival at a given time. But if mortality encoun- ters proportional to school density (.mdN) were imposed, then there were better survival rates with preferential at- tachment than with random attachment (Fig. 8, A and B). Conversely, mortality encounters proportional to school 356 Fishery Bulletin 102(2) o f=0 + f = 0.35 A f= 1.50 1.000 School size T 2.000 Figure 3 Simulated dynamics of school-size distributions using mdl as the mortal- ity model and w as the aggregation model. This simulation started with aggregated initial conditions (S = 106). The aggregation parameter was a=10~6. The top panel shows school-size distributions lin log-log scale) at selected times (t). The lower panel gives the mean school size Uibar) and school abundance IN) versus time. size (mdk) led to poorer survival with preferential attach- ment (Fig. 8, C and D). Discussion Koslow (1992), Rickman et al. (2000), and others have commented on the inherent variability in stock-recruit- ment data and the limited predictive power of determin- istic stock-recruitment models. Therefore, there is no expectation that one could select the models described here over other stock-recruitment models on the basis of fits to data. Although the aggregation-mortality models may be fitted to stock-recruitment data, the real useful- ness is as a guide to selection of stock-recruitment models used in management, as a mechanism for integrating research on recruitment processes, and as a tool for explor- ing the structure of recruitment variability. The aggregation-mortality models were introduced by using an analogy with evolving random networks ( Barabasi and Albert, 1999 ) and were shown to be analyti- cally equivalent (Appendix 2). Modeled fish are subjected to competing forces of organization (aggregation) and decay (mortality), as in a network in which links to nodes in the network are created, destroyed, and rewired (Albert and Barabasi. 2002). An important finding of Barabasi and Powers: Recruitment as an evolving random process of aggregation and mortality 357 o f= 0 + t=5t a f= 2f School size 0002 [ Figure 4 Simulated dynamics of school-size distributions using mJX as the mortal- ity model and w as the aggregation model. This simulation started with aggregated initial conditions (S=2xl06). The aggregation parameter was o=1.5x 10"6. The top panel shows school-size distributions (in log-log scale) at selected times (t). The lower panel gives the mean school size (kbar) and school abundance (N) versus time. Albert (1999) was that scaling of the aggregate-size dis- tribution was dependent on the type of aggregation, spe- cifically preferential attachment. Bonabeau and Dagorn noted the generic occurrence of scaling of aggregation distributions in nature (Bonabeau and Dagorn, 1995) and this scaling of aggregation distributions motivated the development of the models presented here. The emphasis of the aggregation models was on prefer- ential attachment and on comparison of model results with results for models with random attachment strategies. The preferential attachment rule used in the simulations was that aggregation rates were proportional to the size of the school encountered. But, what is meant by preferential attachment and does preferential attachment occur in nature? Clearly, a fish, school or mortality agent has no global knowledge of the proportional size of a school that is encountered. However, preferential attachment in these models is a metaphor for aggregation strategies that are weighted toward larger school sizes. Indeed, studies of networks have shown that attachment may be proportional to a power of school size and still produce scale-free prop- erties (Albert and Barabasi, 2002). Also, network studies have shown that scale-free distributions occur when a wide number of attachment criteria are included, such as the "fitness" of the object being encountered and the attractiveness of local conditions (Bianconi and Barabasi, 358 Fishery Bulletin 102(2) 1,000 i 0 0 O O 0 o o (=0 + f=5f -6 a f= 1f -6 o o o o >, 0 'OT(mo •wpa>- Mortality proportional to schools: mdN Random aggregation: wN Rt =Se -2(1/ J feo_ |s-(S- v? -2a* 364 Fishery Bulletin 102(2) N, S-(S-k0)e' k, =RtINt = k^e** [S-(S-kf))e-2,"\' Mortality proportional to school size: mdk Random aggregation: wN and Albert (1999). Dorogovtsev and Mendes (2000) and Albert and Barabasi (2002). When the aggregation model is preferential attachment (w ) (ignoring for the moment the nonstationarity of N and P), then the partial differential of a school of size klt with respect to Rt has been shown by Dorogovtsev and Mendes (2000) to asymptotically be dkuim, = P,ik,,IR,), (AD where ft, is the net rate of decay per each mortality event, i.e., R, l + 2pSi-^(S-Ml-e~2°*] a fit l-wpalm. With specific-mortality models, fi, is (A2) Nt = k,=R,/N,=- S-iS-k0)e' S-iS-kpte -2at l + 2^t-^S-k0)[l-e~ a Random mortality encounters: mN Random aggregation: wN V i„ \ S can be determined by using continuum theory (Dorogovtsev and Mendes, 2000; Albert and Barabasi, 2002) leading to scale-free degree distributions. Therefore, when preferential attachment and nonrandom mortality are used, then the model may be couched as a scale-free network in the manner of Barabasi Equation A3 is equivalent to the results of Dorogovtsev and Mendes (2000), Krapivsky et al. (2000), and Albert and Barabasi (2002) and suggest that ft, may be a useful approximation for determining the power-law tail of the school-size distribution (Appendix Fig. 1). The simulation results showed the dynamics of Pkt. When the aggregated initial condition was imposed, at the start of the simulations there were no schools with only one fish in them (P,, = 0). Eventually, as the number of schools and fish declined, P„ became positive. Finally, as the distribution became scale-free, -i)Px,l Hk became negative and remained so throughout the remainder of the simulation or until a single giant cluster was formed (Ap- pendix Fig. 1). Conversely, if the initial conditions began with schools being disaggregated, then dPuldk began as a negative number and remained so until either a giant cluster formed or there were no more fish remaining. An approximation is suggested by the above results for circumstances when the initial conditions are disag- gregated and when there is preferential attachment: the differential equation dP,. , Idt when k = 1 ( Eq. 6 ) is replaced by dPuldt -irPy,IN, + ;?Ml-Pu)/JV,. (A4) Powers: Recruitment as an evolving random process of aggregation and mortality 365 1 1 ,000 School size (no. of fish) Appendix Figure 1 School-size distribution at selected times. (A) School-size distribution of a simulation starting with a disaggregated initial condition, S=106, n=10_6, where mortality is density- independent (»!(/l) and there is preferential attachment (w^). (B) Distribution starting with an aggregated initial condition, S=2xl06, «=1.5xl0~6, where mortality is density-dependent proportional to schools imdN) and with preferential attachment (w a). The dotted lines are the predictions of y = 1 + 1//3 from Equation A2, horizontally offset for viewing. 366 Abstract— We examined the diets and habitat shift of juvenile red snapper (Lutjanus campechanus) in the north- east Gulf of Mexico. Fish were col- lected from open sand-mud habitat (little to no relief), and artificial reef habitat (1-m3 concrete or PVC blocks), from June 1993 through December 1994. In 1994, fish settled over open habitat from June to September, as shown by trawl collections, then began shifting to reef habitat — a shift that was almost completed by December as observed by SCUBA visual surveys. Stomachs were examined from 1639 red snapper that ranged in size from 18.0 to 280.0 mm SL. Of these, 850 fish had empty stomachs, and 346 fish from open habitat and 443 fish from reef habitat contained prey. Prey were identified to the lowest possible taxon and quantified by volumetric measure- ment. Specific volume of particular prey taxa were calculated by dividing prey volume by individual fish weight. Red snapper shifted diets with increas- ing size. Small red snapper (<60 mm SL) fed mostly on chaetognaths. cope- pods, shrimp, and squid. Large red snapper (60-280 mm SL) shifted feed- ing to fish prey, greater amounts of squid and crabs, and continued feeding on shrimp. We compared red snapper diets for overlapping size classes (70- 160 mm SL) offish that were collected from both habitats (Bray-Curtis dis- similarity index and multidimensional scaling analysis). Red snapper diets separated by habitat type rather than fish size for the size ranges that over- lapped habitats. These diet shifts were attributed to feeding more on reef prey than on open-water prey. Thus, the shift in habitat shown by juvenile red snapper was reflected in their diet and suggested differential habitat values based not just on predation refuge but food resources as well. Diet shifts of juvenile red snapper (Lutjanus campechanus) with changes in habitat and fish size Stephen T. Szedimayer Marine Fish Laboratory Department of Fisheries Auburn University 8300 State Highway 104 Fairhope, Alabama 36532 E-mail address: sszedlmas'acesag auburn.edu Jason D. Lee Barry Vittor & Associates 8060 Cottage Hill Rd. Mobile, Alabama 36695 Manuscript approved lor publication 4 November 2003 by Scientific Editor. Manuscript received 20 January 2004 ..1 NMFS Scientific Publications Office. Fish. Bull. 102:366-375 (200 1 1 Larval red snapper (Lutjanus cam- pechanus) spend approximately 26 days in the plankton, prior to metamorpho- sis and first appearance on benthic substrate. For the most part the fish settle on open substrate, where peaks in recruitment are observed in August and September, after which they may move to more structured habitat some- time within the first year ( Szedimayer and Conti, 1999). The apparent advan- tage of this habitat shift would be increased food resources and protec- tion from predators. To help clarify the value of increased food resources on reef habitats, comparisons of diets from the two habitats are necessary. Also, because many fish species shift diets with increasing size (Sedberry and Cuellar, 1993; Burke, 1995; Rooker, 1995; Lowe et al., 1996), we need to distinguish possible ontogenetic diet differences from shifts that are due to habitat. Previous red snapper diet studies have focused on larger individuals and on small sample sizes for fish <250 mm SL (Camber, 1955; Moseley, 1966; Bradley and Bryan, 1975). Camber (1955) described the diets of 15 "small red snapper" from Campeche Banks, and reported that 14 of the 15 stom- achs contained small penaeid shrimps. Moseley ( 1966) described the diets of 45 "juvenile red snapper" collected off the coasts of Texas, and 28 off Louisiana. Louisiana fish fed on fishes, shrimps. detritus, and stomatopods, and Texas fish fed on shrimps, crabs, and mysid shrimps. Perhaps the most comprehensive red snapper diet study to date has been that of Bradley and Bryan ( 1975) which described the diets, by season, of trawl- collected (open sand-mud habitat) and hook-and-line reef "rough bottom ar- eas" fish off the Texas coast. They de- scribed the diets of 258 open-habitat and 190 reef red snapper and found that juvenile red snapper (25-325 mm FL) were dependent on shrimp, crabs, and other crustaceans and that adults (325-845 mm FL) were dependent on fish, crabs, and other crustaceans. They described a change in juvenile red snapper diet as fish size increased, "young red snapper depend almost exclusively upon invertebrates," and showed a gradual increase in verte- brate prey with growth. However, they did not separate out the proportions of their "juvenile" red snapper that were collected from reef versus open habi- tat. Thus, the shift from open to reef habitat is still poorly understood. If and when this shift occurs and whether this shift is accompanied with a diet shift that is independent of fish-size effects needs to be defined. The purpose of the present study- is to describe the diet of red snapper off the coast of Alabama — from the juvenile stage (just after settlement I to one-year old fish. We examined overall Szedlmayer and Lee: Diet shifts of Lutjanus campechanus 367 ontogenetic shifts in red snapper diet with increasing size and possible changes in diet with habitat shifts from open substrate to structured habitat (artificial reefs). Materials and methods Red snapper were collected from open-flat substrate (sand and mud) and reef habitats (artificial reefs; Fig. 1). The open habitat was located approximately 6 km south of Mobile Bay, Alabama (30'06'N, 88°03'W), and ranged in depth from 12 to 20 m. Previous studies showed very high concentrations of age- 0 red snapper from these areas (Szedlmayer and Shipp, 1994; Szedlmayer and Conti, 1999 ). The artificial reef habitats were located in the Hugh Swingle artificial reef area, approximately 20 km south of Mobile Bay, AL, and ranged in depth from 18 to 23 m (Szedlmayer and Shipp, 1994; Szedlmayer, 1997). We collected fish from open substrate by trawl (7.62-m head rope, 2.54-cm mesh, 2-mm codend mesh). Samples were taken every two weeks from June to December 1994; however, time between samples was longer in the winter because of poor weather. Each trawl was fished for 10 min, and all age-0 and age-1 red snapper collected were placed on ice, returned to the laboratory, and frozen for later analysis. Bottom dissolved oxygen, salinity, and temperature were sampled with a Hydrolab Surveyor II at each location (Szedlmayer and Conti, 1999). Prior to diet analysis, red snapper were thawed, weighed to the nearest 0.1 g, and measured to the nearest 0.1 mm SL. The whole fish was preserved in 10% formalin if SL was <50 mm, whereas for larger fish, stomachs were re- moved and preserved. After 48 hours in formalin, stomach samples were transferred to 75% isopropyl alcohol. Concrete block and PVC artificial reefs (1 m3) were placed in the Hugh Swingle reef area in August 1992 and July 1993 (Szedlmayer, 1997). "Reef is used here for defining these artificial habitats. Reefs were not sampled for a minimum of 3 months after placement. Red snapper were collected from June 1993 through December 1994. Fish were collected from these reefs by SCUBA divers first placing a drop net (3.0 m radius, 1.3 cm square mesh) over the reef and then releasing rotenone into the enclosed area. Reef fish were placed on ice in the field and trans- ported back to the laboratory. Approximately 12-18 h after collection all reef fish were weighed to the nearest 0.1 g and measured to the nearest 1.0 mm. Stomachs were fixed in 10% formalin, and after 24 h transferred to 75%' isopropyl alcohol. Red snapper size classes were also esti- mated by SCUBA visual surveys in July and August 1994. On each visual survey, divers counted red snapper by 50- mm size intervals. Bottom dissolved oxygen, salinity, and temperature were sampled with a Hydrolab Surveyor II during each survey. All stomachs were dissected and contents placed in petri dishes. All prey were counted and identified to the lowest possible taxon. Volume was calculated by using an adapta- tion of the method described by Hellawell and Able (1971). Each prey taxon from each stomach was placed into a glass well of a known depth. A cover slide was placed on the well, Mobile Bay X If FL Gulf of Mexico O Gulf of Mexico O o o 30.00 N □ □ DnaDnr 0 5 10 Kilometers 80.00 W Figure 1 Collection sites for red snapper ( Lutjanus campechanus) in the northern Gulf of Mexico. Open circles are open habitat trawl sites, and gray squares are 1-m3 concrete or PVC artificial reefs. depressing the prey taxon to a known depth (e.g., 1 mm). The prey were video taped with a high-8 Sony camera and images were digitized with Image Pro 2.0 software (Media Cybernetics. Silver Spring, MD). Image size was calibrated to 0.01 mm by a stage micrometer. The surface area of each preparation was measured by using Image Pro software. Volume was calculated by multiplying the surface area by the known depth. Specific volumes for par- ticular prey taxa were calculated by dividing prey volume by individual fish weight (mnv'Vfish wt g). Comparisons of diet shift by increasing fish size were made by grouping prey taxa into ten prey groups and by calculating specific volume for 10-mm-size intervals of red snapper. A dissimilarity index (Bray-Curtis) was calculated from specific volumes of individual prey taxa, for overlap- ping size classes of red snapper both within and between habitats: Bray-Curtis = IW^-Yj/IiY^+Y^), where Y = specific volume of jth species, and j and k are the samples being compared (Field et al., 1982). The dissimilarities were then used in a multidimensional scaling analysis (MDS; Schiffman et al., 1981). The MDS provided a two- dimensional "map" of the distances between samples (fish 368 Fishery Bulletin 102(2) 6 4 2 0 8 £ 6 ! 4 O 0 2 1 0 O E 4 E g 2 ! o o 6 6 C yj 4 0_ o 2 0 6 4 2 0 Trawls - 1 1 28 Jun-1 Jul -d Reefs = 5 25 May-2 Jun ELi Trawls = 31 10-19 Jul Visual Reefs = 13 1-25 Jul L Trawls = 32 31 Jul-12 Aug Visual Reefs = ! 9 Aug J_L Trawls = 26 Reefs = 2 •23 Aug 23 Aug 1 id1 ^t Trawlj Trawls = 32 9 SeP Reefs = 3 6 Sep JTlhffl Trawls = 25 25-28 Sep Reefs = 4 28 Sep-6 Oct II Trawls = 25 12-21 Oct Reefs = 4 19-24 Oct Jktft^f^n Trawls = 26 30 Oct-9 Nov Reefs = 3, 9-18 Nov ^bjMkffl Trawls = 15 12-16 Dec Reefs = 6 5-8 Dec IT>^ Trawls = 26 9-12 Jan £\ Reefs = 3 25Mar-15 Apr 100 200 100 Standard length (mm) 200 Figure 2 Movement patterns for age-0 red snapper iLutjanus campechanus) from the northern Gulf of Mexico in 1994. Black bars represent trawl samples, grey bars represent reef drop-net samples, and white bars represent SCUBA visual surveys of concrete reefs. size and habitat type) in Euclidian space based on the Bray-Curtis index. Thus, comparisons of red snapper diets were based on all prey taxa, yet independent of capture habitat and fish size. Results In the sampling areas during the summer and fall of 1994, salinity ranged from 30 to 35 ppt. Dissolved oxygen was 7 ppm in the early summer, decreased to 3 ppm in July and August, and increased to 7 ppm in the fall. Temperature was 22°C in June, increased to 28°C in late August, then dropped to just below 20°C by December. No significant differences were detected between trawl and reef sites for these environmental measures U-test, Ps0.05). Red snapper showed a clear shift in habitat during their first few months of life (Fig. 2). Fish first recruited to open habitat at the end of June, at sizes <40 mm SL. Fish con- tinued to recruit to open habitat until early September, at which time they were larger ( 30 to 100 mm SL ) and began shifting to more structured habitat. By mid-October most age-0 fish had moved to reef habitat. During the initial settlement no new recruits were collected or visually ob- served on the artificial habitats (Fig. 2). Overall, only red snapper <160 mm SL were collected from open habitat, whereas only red snapper >70 mm SL were collected from reef habitat. Size overlapped from 70.0 to 160 mm SL be- tween habitats (Fig. 3). A total of 1639 red snapper stomachs were analyzed: 570 from open substrate and 1069 from reef habitat. Prey were found in 789 (48'< ) of the total stomachs examined, 346 (61%) from the open habitat and 443 (41%) from the reef habitat (Fig. 3). Trawl-collected red snapper were mostly collected from site one, but sample sizes were also large (>30 with prey) at two other sites (Table 1). Total red Szedlmayer and Lee: Diet shifts of Lut/anus campechanus 369 Open Habitat n=346 Reef Habitat n=443 Empty n=850 QD=- ■i~~ r i T i i 60 80 100 120 140 160 180 200 220 240 260 280 300 Size class (mm SL) Figure 3 A comparison of red snapper {Lutjanus campechanus) length frequencies between open and reef habitats in the northern Gulf of Mexico. Gray bars = empty stomachs from both habitats. Table 1 Number of red snapper (Lutjanus campechanus) stom- achs sampled in), and number of stomachs containing prey from open and reef habitat in the northeast Gulf of Mexico. Open trawl sites Reef habitats n n with prey n n with prey 356 223 108 53 45 21 17 5 75 58 198 115 57 33 55 31 37 11 249 71 50 23 14 1 89 45 11 5 209 74 35 10 22 4 12 6 snapper collected from the reefs varied by site (from 11 to 249 fish), but large samples were collected from at least 6 different reefs (Table 1). Large sample sizes were collected during most months over open habitat, with the exception of November 1994 (n=12), and for most months (6 out Table 2 Number of red snapper (Lutjanus campechanus) stomachs sampled (re), and number containing prey, by month and year, from open and reef habitat in the northeast Gulf of Mexico. Open habitat Reef habitat Month r with Month n with and year n prey and year n prey Jul 1994 56 43 Jun 1993 94 50 Aug 1994 169 109 Oct 1993 370 169 Sep 1994 187 98 May 1994 141 37 Oct 1994 97 52 Jun 1994 46 37 Nov 1994 16 12 Aug 1994 41 8 Dec 1994 45 32 Sep 1994 155 86 Oct 1994 76 28 Nov 1994 65 12 Dec 1994 81 16 of 9) from reef sites (Table 2). Only red snapper stomachs containing prey were used in our analyses. Red snapper diets showed 55 different prey identi- fied to the lowest possible taxon. In general, red snap- per diets were dominated by fish (43%), squid (29.5%), shrimp (16.4%), and crabs (4.4%; Table 3). Specifically, the "shrimp" group included Mysidacea (mysid shrimps), Stomatopoda (mantis shrimps), Penaeidea (penaeid 370 Fishery Bulletin 102(2) Table 3 Specific volume (mnvVfi sh weight g) for prey taxa from red snapper (Lutjanus compel ha n us 1. % = percent specific-voli lme of total volume, Habitat = prey habitat. General prey groups are noted in quotation marks, unid. = unidentified. Prey taxa Total volume Percent Lowest taxon Specific volume Percent Habitat Osteichthyes "fish" 5408.2 43.5 unid. fish 3465.9 27.9 Halichoeres spp. 650.4 5.2 reef Blenniidae 279.2 2.2 reef Serranidae 278.1 2.2 reef Serranus subligarius 240.8 1.9 reef Centropristis ocyurus 207.3 1.7 reef Engraulidae 117.9 0.9 open Ophichthidae 100.6 0.8 open Cynoglossidae 35.2 0.3 open Triglidae 20.8 0.2 open Ophichthus sp. 10.8 0.1 open Cephalopoda "squid" 3665.6 29.5 Loliginidae 3665.6 29.5 open Natantia "shrimp" 2033.7 16.4 unid. shrimp 544.6 4.4 Sicyoninae 359.6 2.9 reef Hippolytidae 345.7 2.8 reef Penaeidae 264.5 2.1 open Alpheidae 131.1 1.1 reef Sergestidae 24.2 0.2 open Luciferinae 22.6 0.2 open Ogyrididae 8.8 0.1 open Stomatopoda "shrimp" Squillidae 221.8 1.8 open Mysidacea "shrimp" Mysidacea 109.8 0.9 open Reptantia "crabs" 550.8 4.4 Portunidae unid. crab 302.0 143.0 2.4 1.2 mixed Diogeninae 51.6 0.4 open Leucosiidae 20.7 0.2 reef Xanthidae 16.7 0.1 reef Porcellanidae 7.3 0.1 reef Chaetognatha 199.6 1.6 Sagitta spp. 199.6 1.6 open Polychaeta 130.1 1.0 Polycheata 75.4 0.6 mixed Polychaeta Onuphidae 34.0 0.3 open Maldanidae 19.9 0.2 open Ascidiacea "tunicate" 121.0 1.0 Ascidiacea 121.0 1.0 reef Calanoida "copepod" 118.2 1.0 Calanoida 113.3 0.9 open Octopodidae 93.6 0.8 Octopus sp. 93.6 0.8 reef unid. 79.5 0.6 unid. 79.5 0.6 Amphipoda 13.8 0.1 Amphipoda 9.4 0.1 mixed Ostracoda 6.1 0.0 Ostracoda 6.1 0.0 open shrimps), and Caridea (caridean shrimps). In addition, all Squillidae were probably Squilla empusa, according to Hopkins et al., (1987). Among fish, many were uniden- tified due to digestion, but if proportions of unidentified fish are similar to identified fish, then dominant fish prey included Halichoeres spp., (5.2c/r ), Blenniidae (2.2%), and Serranidae (2.2%). Two prey fish were identified to species: Serranus subligarius (1.9%), and Centropristis ocyurus (1.7%). Among the squid taxon, one genus dominated: Lolli- guncula spp., (29.59! I, but all squid were either L. brevis or Loligo pealeii (Hopkins et al., 1987). Among shrimp, dominant taxa included Sicyoninae (2.9%), Hippolytidae (2.8%), Penaeidae (2.1%), Squillidae (1.8%), and Alpheidae (1.1%). Among crabs, dominant taxa were mostly Por- tunidae (2.49; ). Other groups showing greater than 1.0% included Chaetognatha [Sagitta sp. 1.69J ), and Ascidiacea or tunicates (1.0%; Table 3). Szedlmayer and Lee: Diet shifts of Lutjanus campechanus 371 16 48 45 41 37 41 33 35 37 59 67 37 40 37 41 28 28 35 29 13 15 15 5 3 0 20 40 60 80 100 120 140 160 180 200 220 240 Size class (mm SL) Figure 4 Stomach contents by specific-volume for ten higher taxonomic groups over 10-mm size classes of red snapper {Lutjanus campechanus) from both open and reef habitats in the northern Gulf of Mexico. Numbers on the upper axis are the number of red snapper that contained prey for each respective size class. Red snapper shifted diets with increasing size. For red snapper <60.0 mm SL, diets were dominated by shrimp, chaetognaths, squid, and copepods. Large red snapper (60-280 mm SL) shifted to feeding on fish prey, greater amounts of squid and crabs, and continued feeding on shrimp (Fig. 4). The diets of juvenile red snapper changed as they moved from open to reef habitats. Fish collected had overlapping sizes of 70.0 to 160.0 mm SL from both open and reef habitats, and the MDS analysis for this size range showed a clear separation of diets between the two habitats (Fig. 5). Two points that were outliers (R75, T155 1 were biased because they represented only one fish each, and the third outlier (R85) was difficult to explain. The clear separation of red snapper diets shown by the MDS analysis can be attributed to several prey shifts that accompanied habitat shifts. For prey crabs, open-habitat red snapper diets were dominated by Xanthidae, and smaller amounts of Paguridae, Portunidae, Diogeninae, and Pinnotheridae (Fig. 6), whereas diets of red snapper from reef habitats shifted to a dominance by Portunidae and Diogeninae ( Fig. 7). For prey shrimp, open habitat red snapper diets were dominated by Penaeidae and Mysida- cea (Fig. 8), whereas diets from reef habitats shifted to a dominance of Sicyoninae, Hippolytidae, Alpheidae, and Squillidae (Fig. 9). For prey fish, open-habitat red snap- per diets were dominated by Engraulidae (although most were unidentified; Fig. 10), whereas diets from reef habitat clearly reflected prey fish from reef habitats and included Blenniidae, Serranidae, and three prey fish identified to genera, Centropristis spp, Halichoeres spp., and Sen-anus spp. (Fig. 11). -3-2-1 0 1 2 3 X(unitless) Figure 5 Multidimensional scaling of diets for red snapper (Lutja- nus campechanus) based on the Bray-Curtis dissimilarity index computed for specific volume of prey taxa both within and between habitats for overlapping size classes (70.0 to 159.9 mm SL). The letter and number accompanying each point indicates the habitat and size class that each point represents (e.g., T = trawl, R = reef, 75 = 75 mm SL size class). Circles were drawn by hand. Axes are unitless. Discussion The present study provides a substantial sample size (ft = 1639) for red snapper diet analysis and a relatively 372 Fishery Bulletin 102(2) 16 48 45 41 37 41 32 30 20 15 6 3 3 "I — I 1 r Crabs Open habitat n T 1 1 1 1 — UNIDcrab l l Paguridae Xanthidae Portunidae Diogeninae I I Pinnotheridae a j=u t — T — T — T — T — T — T — T — ~l 1 1 f — T 0 20 40 60 80 100 120 140 Size class (mm) Figure 6 Crab prey from open habitat. Stomach contents by spe- cific volume over 10-mm size classes of red snapper iLut- janus campechanus) from the northern Gulf of Mexico. Numbers on the upper axis are the number of red snapper that contained prey for each respective size class. 16 48 45 41 37 41 32 30 20 15 8 6 3 3 0 4 0 1 1 1 ' 1 — UNID shrimp Lucifennae Mysidacea I I Ogyndidae ^^1 Penaeidae Sergestidae Sicyoninae I I Squillidae "Shrimp" Open habitat - II 1 I I I nW 20 40 60 80 100 120 140 Size class (mm) Figure 8 "Shrimp" prey from open habitat. Stomach contents by specific volume over 10-mm size classes of red snap- per (Lutjanus campechanus) from the northern Gulf of Mexico. Numbers on the upper axis are the number of red snapper that contained prey for each respective size class. 5 17 44 59 31 37 34 40 28 28 35 29 13 15 15 5 3 5 3 - m E I I I I 1 I 1 I I 1 I I I 1 1 UNIDcrab Crabs 1 1 Xanthidae Reef habitat ^m Portunidae I I Diogeninae 1 1 Pinnotheridae fSSg Porcellanidae |::::::| Leucosiidae i i i ^E, "5 2- > 12 - < 11 - , Cl b - 70.0 mm SL fed on fishes and to a lesser ex- tent crabs, shrimps, and stomatopods. Similar diet shifts were also shown for several fish species of Puget Sound. For example in pile perch {Rhacochilus vacca), striped seaperch iEmbiotoca lateralis), and quillback rockfish (Sebastes maliger), the smallest juveniles preyed on open- habitat plankton and benthic fauna, and medium-size and larger fish (>121 mm) of all three species shifted their diets to include reef-associated prey ( Hueckel and Stayton, 1982). However, at larger sizes these three species were not totally dependent on reef-associated prey. We have examined red snapper diets based on specific volume of food. Although many other studies have used an index of relative importance (IRI; Pinkas et al., 1971: Cortes, 1997), we were specifically interested in the nutri- tional value of particular prey, and prey separation into open-habitat or reef-habitat. With IRIs these separations would be more difficult to define, e.g., pelagic prey with high numbers might be considered more important, but actually provide little nutritional value to red snapper diets (Macdonald and Green, 1983). Future studies on the effects of red snapper predation on prey distributions may be better suited for using IRIs. In summary, red snapper diets from open habitat showed prey taxa associated with open sand-mud sub- strate and the planktonic environment. Open-habitat prey, such as chaetognaths, are known to be pelagic as well as benthic, as are sergestid shrimp, calanoid copepods, my- sids, and stomatopods (Williams, 1968; Manning, 1969; Gosner, 1978; Stuck et al., 1979; Alldredge and King, 1985; Lindquist et al., 1994 ). Red snapper shifted diets to reef-associated prey with their habitat shift, and this diet shift was independent offish size. These diet shifts were clearly apparent for both fish and shrimp prey but less so for crab prey. As shown with marine fish species from Puget sound, red snapper diets from reef habitat were not restricted to reef-associated prey. For example, squids were an important prey over both open and reef habitats in the present study and our findings agree with those of Bradley and Bryan (1975). The squids Loligo sp., and Lolliguncula sp. are both abundant in nearshore coastal waters and are not typically associated with reef structure (Gosner 1978; Laughlin and Livingston, 1982; Hopkins et al., 1987). Availability and ease in capture could be a key as to why squid are important for red snapper over size ranges of 40 to 240 mm SL. This flexibility in feeding habits allows red snapper to take advantage of prey from wide-ranging habitats, but clear shifts to additional reef prey supports the hypothesis that reef structure provides new prey resources. Acknowledgments We thank Joseph Conti, Kori M. Heaps, and Frank S. Rikard for help in field collections and invertebrate identification. This study was funded by NOAA, NMFS. MARFIN grant number USDC-NA47FF0018-0. This is a contribution of the Alabama Agricultural Station. 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A critical review of methods of studying fish feeding based on analysis of stomach contents; application to elas- mobranch fishes. Can. J. Fish. Aquat. Sci. 54:726-738. DeMartini. E. E.. F. A. Parrish, and D. M. Ellis. 1996. Barotrauma-associated regurgitation of food: impli- cations for diet studies of Hawaiian pink snapper. Pristi- pomoides filamentosus (family Lutjanidaei. Fish. Bull. 94:250-256. Field, J. G.. K. R. Clarke, and R. M. Warwick. 1982. A practical strategy for analyzing multispecies dis- tribution patterns. Mar. Ecol. Prog. Ser. 8:37-52. Gosner, K. L. 1978. A field guide to the Atlantic seashore, 329 p. Hough- ton Mifflin Company, Boston, MA. Hellawell. J. M.. and R. Able. 1971. A rapid volumetric method for the analysis of the food of fishes. J. Fish Biol. 3:29-37. Hopkins. T. S., J. F Valentine, and L. B. Lutz. 1987. An illustrated guide with key to selected benthic invertebrate fauna of the Northern Gulf of Mexico. Mis- sissippi Alabama Sea Grant Publ. 087(10). Biloxi. MS. Hueckel. G. S., and R. L. Stayton. 1982. Fish foraging on an artificial reef in Puget Sound, Washington. Mar. Fish. Rev. 44: 38-44. Laughlin, R. A., and R. J. Livingston. 1982. Environmental and trophic determinants of the spatial/temporal distribution of the brief squid {Lolligun- cula brevis) in the Apalachicola estuary (North Florida, USA). Bull. Mar. Sci. 32:489-497. Lindquist. D. G.. L. B. Cahoon, I. E. Clavijo, M. H. Posey. S. K. Bolden, L. A. Pike, S. W. Burk, and P. A. Cardullo. 1994. Reef fish stomach contents and prey abundance on a reef and sand substrata associated with adjacent artificial and natural reefs in Onslow Bay, North Carolina. Bull. Mar. Sci. 55:308-318. Lowe, C. G„ B. M. Wetherbee, G. L. Crow, and A. L. Tester. 1996. Ontogenetic dietary shifts and feeding behavior of the tiger shark, Galeocerdo cuvier, in Hawaiian waters. Environ. Biol. Fish. 47:203-211. Macdonald, J. S., and R. H. Green. 1983. Redundancy of variables used to describe importance of prey species in fish diets. Can. J. Fish. Aquat. Sci. 40:635-637. Manning. R. B. 1969. Stomatopod Crustacea of the Western Atlantic, 380 p. Univ. Miami Press. Miami, FL. Moseley, F. N. 1966. Biology of the red snapper. Lutjanus aya Bloch, of the northwestern Gulf of Mexico. Publ. Inst. Mar. Sci. Univ. Texas 11:90-101. Nelson. B. D., and S. A. Bortone. 1996. Feeding guilds among artificial-reef fishes in the northern Gulf of Mexico. Gulf Mex. Sci. 2:66-80. Pequegnat, L. H.. and R. W. Heard. 1979. Synalpheus agelas, new species of snapping shrimp from the Gulf of Mexico and Bahama Islands (Decapoda: Alpheidae). Bull. Mar. Sci. 29:110-116. Pinkas, L., M. S. Oliphant, I. L. R. Iverson. 1971. Food habits of albacore. bluefin tuna, and bonito in California waters. Calif. Fish Game 152:1-105. Rooker, J. R. 1995. Feeding ecology of the schoolmaster snapper, Lut- janus apodus iWalbum). from southwestern Puerto Rico. Bull. Mar. Sci. 56:881-894. Schiffman, S. S., M. L. Reynolds, and F W. Young. 1981. Introduction to multidimensional scaling, 413 p. Ac- ademic Press Inc.. New York, NY. Sedberry. G. R., and N. Cuellar. 1993. Planktonic and benthic feeding by the reef associated vermilion snapper. Rhomboplites aurorubens (Teleostei, Lutjanidae). Fish. Bull. 91:699-709. Stearns, S. 1884. On the position and character of the fishing grounds of the Gulf of Mexico. Bull. U.S. Fish. Comm. 4:289-290. Stuck, K. C, H. M. Perry, and R. W. Heard. 1979. An annotated key to the Mysidacea of the north cen- tral Gulf of Mexico. Gulf Res. Rep. 6: 225-238. Sweatman, H. P. A. 1993. Tropical snapper (Lutjanidae) that is piscivorous at settlement. Copeia 1993:1137-1139. Szedlmayer. S. T. 1997. LHtrasonic telemetry of red snapper, Lutjanus campechanus, at artificial reef sites in the northeast Gulf of Mexico. Copeia 1997:846-850. Szedlmayer, S. T, and K. W. Able. 1993. Ultrasonic telemetry of age-0 summer flounder. Para- lichthys dentatus. movements in a southern New Jersey estuary. Copeia 1993:728-736. Szedlmayer, S. T., and J. Conti. 1999. Nursery habitats, growth rates, and seasonality of age-0 red snapper. Lutjanus campechanus, in the northeast Gulf of Mexico. Fish. Bull. 97:626-635. Szedlmayer, S. T, and R. L. Shipp. 1994. Movement and growth of red snapper. Lutjanus campechanus, from an artificial reef area in the northeast- ern Gulf of Mexico. Bull. Mar. Sci. 55: 887-896. Williams, A. B. 1968. Substrate as a factor in shrimp distribution. Lim- nol. Oceanog. 3:283-290. 376 Abstract— We measured growth and movements of individually marked free-ranging juvenile white shrimp [Litopenaeus setiferus) in tidal creek subsystems of the Duplin River, Sapelo Island. Georgia. Over a period of two years, 15.974 juvenile shrimp (40-80 mm TL) were marked inter- nally with uniquely coded microwire tags and released in the shallow upper reaches of four salt marsh tidal creeks. Subsequent samples were taken every 3-6 days from channel segments arranged at 200-m intervals along transects extending from the upper to lower reach of each tidal creek. These collections included 201,384 juvenile shrimp, of which 184 were marked recaptures. Recaptured shrimp were at large an average of 3-4 weeks (range: 2-99 days) and were recovered a mean distance of <0.4 km from where they were initially marked. Mean residence times in the creek subsystems ranged from 15.2 to 25.5 days and were esti- mated from exponential decay func- tions describing the proportions of marked individuals recaptured with increasing days at large. Residence time was not significantly correlated with creek length (Pearson = -0.316, P= 0.684 I, but there was suggestive evidence of positive associations with either intertidal (Pearson r = 0.867, P=0.133) or subtidal (Pearson /-=0.946, P=0.054) drainage area. Daily mean specific growth rates averaged 0.009 to 0.013 among creeks; mean absolute growth rates ranged from 0.56-0.84 mm/d, and were lower than those previously reported for juvenile penaeids in estuaries of the southeast- ern United States. Mean individual growth rates were not significantly different between years (/-test, P>0.30) but varied significantly during the season, tending to be greater in July than November. Growth rates were size-dependent, and temporal changes in size distributions rather than tem- poral variation in physical environ- mental factors may have accounted for seasonal differences in growth. Growth rates differed between creeks in 1999 U-test, P<0.015), but not in 1998 (r-test, P>0.5). We suggest that spatial varia- tion in landscape structure associated with access to intertidal resources may have accounted for this apparent inter- annual difference in growth response. Manuscript approved for publication 30 September 2003 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Scientific Publications Office. Fish. Bull. 102:376-388(20011. Individual growth rates and movement of juvenile white shrimp (Litopenaeus setiferus) in a tidal marsh nursery* Stacey Webb Florida Department of Environmental Protection Water Quality Standards and Special Proiects Program 2600 Blair Stone Road, M.S. 3560 Tallahassee, Floida 32399 E-mail address: stacey fekervcudep state. fl. us Ronald T. Kneib UGA Marine Institute Sapelo Island, Georgia 31327 In 2001, shrimp became the most popu- lar seafood in the United States when per capita consumption (1.54 kg) sur- passed that of canned tuna (1.32 kg) for the first time in recorded history (NOAA1). Although 77% of the catch is from the Gulf of Mexico, commercial fisheries in Atlantic coastal states of the southeastern United States also depend heavily on penaeid shrimp pop- ulations. Of the three most common estuarine-dependent penaeid species (Litopenaeus setiferus, Farfantepenaeus aztecus, and F. duorarum)2 harvested in the South Atlantic Bight, white shrimp Litopenaeus setiferus domi- nate, comprising >70% of the catch in the region (North Carolina to the east coast of Florida) and 75-87% in South Carolina and Georgia (NMFS:I). Concerns over the possibility of de- pleting the resource as early as the 1930s led to intensive studies of the life cycle (Lindner and Anderson, 1956; Williams, 1984). The white shrimp has an annual life cycle that can be divid- ed into offshore (oceanic) and inshore (estuarine) phases. Adults spawn in Atlantic waters in spring and the post- larvae migrate into estuaries, aided by flood tides and wind-generated currents (Lindner and Anderson, 1956; Wenner et al., 1998). Postlarvae penetrate into the shallow upper reaches of the nurs- ery habitat where juveniles achieve a substantial portion of their adult body mass before moving into deeper creeks, rivers, and sounds where they approach maturity and emigrate seaward to spawning areas (Muncy, 1984; Wil- liams, 1984). Given the commercial importance and early interest in this species, sur- prisingly little research has focused on the juvenile stages within tidal marsh nursery habitats ( Minello and Zimmer- man, 1985; Zein-Eldin and Renaud. 1986; Knudsen et al., 1996; McTigue and Zimmerman, 1998). Seasonal mi- grations and ontogenetic movements of white shrimp between coastal ocean spawning grounds and estuarine nurs- eries are well known ( Dall et al.. 1990 1. as are the sometimes extensive migra- tions of adult shrimp along the Atlantic coast, primarily to the south during fall and early winter, and northward in late winter and early spring (Lindner and Anderson, 1956; Shipman, 1983). Within the estuary, juvenile white ■Contribution 921 of the Univerisity of Georgia Marine Institute, Sapelo Island, GA. 1 NOAA (National Oceanic and Atmo- spheric Administration. 2002. Shrim p overtakes canned tuna as top U.S. sea- food. Website: http://www.noaanews. noaa.gov/stories/s970.htm. I Accessed: 28 August 2002.] - These species were all previously included in the genus Penaeus, but the subgenera were elevated to genera by Perez-Farfante and Kensley (1997). :! NMFS (National Marine Fisheries Service). 2002. Unpubl. data. Web- site: http://www.st.nmfs.gov/stl/commer- cial/index.html. lAccessed 29 August 2002.1 Webb and Kneib: Individual growth rates and movement of Litopenaeus setiferus in a tidal marsh nursery 377 shrimp, in contrast to other penaeid species, are found across a wider range of environmental conditions and habitats (Kutkuhn, 1966) and often make tidal excursions between subtidal and vegetated intertidal habitats to for- age (Mayer, 1985; Kneib. 1995; 2000). However, relatively little is known about movements within subtidal creeks of the primary nursery areas, and the degree to which indi- viduals exhibit fidelity to a particular tidal creek drainage system is unknown. Direct measurement of juvenile shrimp growth rates within the nursery have also been rare. Most growth estimates for free-ranging juvenile shrimp are based on analyses of size-frequency data, which can be misleading (Loesch, 1965). Shrimp grow rapidly while in the estua- rine nursery throughout the summer and early fall, and juveniles approach adult, or commercially harvestable size within 2-4 months after immigration to the estuary (Kutkuhn, 1966; Williams, 1984). Mean absolute growth rates of 0.7-1.1 mm/d are commonly reported for many penaeids (Dall et al., 1990). However, growth studies are difficult to compare because the rate of growth may vary between years and among seasons, as well as with size, age, and sex of individuals (Perez-Farfante, 1969). Growth estimates for Litopenaeus setiferus range widely, from 10 to 65 mm/month (Williams, 1984). Previous estimates were based on a variety of approaches including experimental studies in aquaria and ponds (Pearson, 1939; Johnson and Fielding, 1956), size distributions from tagging studies of adults (Lindner and Anderson, 1956), length-frequency distributions of juveniles in field samples (Gunter, 1950; Williams, 1955; Loesch, 1965; Harris, 1974; Mayer, 1985), mark-recapture of uniform size ranges of subadults and adults (Klima, 1974), and mark-recapture of shrimp in marsh ponds (Knudsen et al., 1996). Many estimates of growth for small (<80 mm TL) juvenile L. setiferus have been extrapolated from mark-recapture studies of larger (>100 mm TL) individuals (Lindner and Anderson, 1956; Harris, 1974; Klima, 1974). However, there is a paucity of empirical data on growth rates of small, free-ranging juvenile white shrimp within natural estuarine nursery habitats. The purpose of the present study was to provide reliable data on growth and movements of individual ju- venile white shrimp within a natural estuarine nursery environment and to initiate an assessment of spatial variation in habitat quality in relation to tidal marsh landscape structure. Recent innovations in tagging techniques have pro- duced an effective way to obtain information on individual organisms through the use of sequentially numbered bi- nary-coded microwire tags (Northwest Marine Technol- ogy. Inc. Shaw Island, WA). Microwire tags were first used in tagging experiments by Jefferts et al. (1963) and have since been used successfully to tag a variety of crustaceans including prawns (Prentice and Rensel, 1977), crayfish (Isely and Eversole, 1998), blue crabs (van Montfrans et al., 1986; Fitz and Weigert, 1991), and lobsters (Krouse and Nutting, 1990; Uglem and Grimsen, 1995). Results of these studies and others generally show that tag reten- tion rates are high and tagging has little effect on the growth or survival of the fishes and crustaceans in which microwire tags have been used. In a laboratory study involving 240 juvenile white shrimp, Kneib and Huggler (2001) confirmed that tag retention was high (-98%), growth rates between tagged and control individuals were not significantly different, and the best location (based on tag retention and survival) for tag injection was in the muscle tissue of the abdomen. This type of tag allows for identification of individuals because each tag is etched with a unique number encoded in binary form. In addi- tion, the tag is completely internal and inconspicuous, thus eliminating problems associated with external tags (e.g., streamer-type tags) that might interfere with molting or increase predation risk (Garcia and LeReste, 1981; van Montfrans et al., 1986; Isely and Eversole, 1998). Materials and methods Study area All samples were collected from four tidal creek subsys- tems associated with the Duplin River on the west side of Sapelo Island, Georgia. The Duplin River tidal drainage ( -11 km2 ) includes almost 10 km- of tidal salt marsh that is inundated twice daily by unequal tides with a mean range of 2.1 m (Wadsworth, 1980). Smooth cordgrass iSpartina alterniflora) is the dominant vegetation in the intertidal marshes of this area. Seasonal water temperatures aver- age between 10°C and 30°C, and salinity is characteristi- cally polyhaline, ranging from 15 to 30 ppt (Kneib, 1995). Freshwater flow into the system is intermittent and originates largely from local upland runoff and indirect flows by several interconnected tidal channels from the Altamaha River about 8 km to the southwest (Ragotzkie and Bryson. 1955). Tidal creeks included in this study were Post Office Creek (PO) and Stacey Creek (SO in 1998, and the East and West forks (EF. WF, respectively) of the upper Duplin River in 1999 (Fig. 1). Logistical constraints precluded sampling shrimp populations from more than two creek systems within the same year, and different pairs of creeks were chosen in each of the two years to broaden the spatial coverage of the study. High-resolution black and white photographs (1:16000 scale) from an aerial survey of the region in December 1989 were used to measure broad- scale structural characteristics of the creek systems, in- cluding areal extent of the intertidal and subtidal portions of each drainage. The metrics and methods of extracting the information from the photographs are fully described elsewhere (see Webb and Kneib, 2002). Field sampling Shrimp were collected by cast net along the shallow (<1 m depth) edges of the subtidal portion of each creek system during low tide. Preliminary studies showed that 1.52-m diameter nets with ca. 1-cm mesh size collected the range of juvenile shrimp sizes (40-80 mm) targeted for mark- ing in this study. All samples were collected within 2-3 hours of low tide to ensure that the shrimp popula- 378 Fishery Bulletin 102(2) Figure 1 Map of the salt marsh estuary in the vicinity of Sapelo Island, Geor- gia, showing locations of the tidal creek subsystems within the tidal drainage of the Duplin River. tion was restricted to the tidal creek channels and had no refuge in the intertidal vegetation. A series of stations, at intervals of approximately 200 m, was established along the length of the subtidal portion of each creek from the upper reaches to the mouth, so that the number of stations within a creek depended on the navigable length of the subtidal channel. There were 13 stations in PO, 11 in SC, 9 in EF, and 7 in WF. Salinity, water temperature, and dissolved oxygen were measured near the surface (<1 m depth) at the mouth of the tidal creek on each day of sampling by using a YSI model 85 meter ( YSI, Inc. Yellow Springs, OH). Juvenile shrimp were marked with uniquely coded microwire tags (1.1 mm longx0.25-mm diameter, Northwest Marine Technology [NMT], Inc. Shaw Island, WA), which were injected into the muscle tissue of the first abdominal segment. We used a hand-held multishot injector (NMT) that was designed to cut, magnetize, and inject sequentially coded tags from a continuous stain- less-steel wire spool. Each tag was etched with six lines of binary code that could be read under a microscope (25x) and translated into a set of numbered coordinates. Only three of the coded lines were required to identify a unique individual. A master line contained a distinguishing sequence code that was necessary to properly interpret codes on data lines designated D3 and D4. The numeri- cal values associated with these coded lines were entered into a sequential tag conversion computer program (GR [Growth Rate], version 1.3, Northwest Marine Technology. Inc. Shaw Island, WA) that output the unique tag number corresponding to those coordinates. A reference tag was archived for every shrimp marked in order that the code on either side of the tag injected into a shrimp was known with certainty. This was necessary to ensure positive identification of recaptured individuals be- cause the injector was designed only to cut tags to a known length (1.1 mm) and did not distinguish between the be- ginning and end of sequential codes and often cut tags that included a portion of two adjacent codes. Prior to their re- lease, marked shrimp were passed across a magnetometer (NMT) which signaled the presence of the ferromagnetic tag with an audible tone and flashing light. All shrimp collected after these marking sessions were scanned in the same manner and when a tag was detected, it was removed from the recaptured shrimp, cleaned and read under the microscope. The two reference tags bracketing the recovered tag were then located in the archive set to determine the date, location, and initial length at release of the marked shrimp. Thus the growth rate, time at large, and distance between points of release and recapture could be determined with certainty for individual shrimp. Webb and Kneib: Individual growth rates and movement of Litopenaeus setiferus in a tidal marsh nursery 379 Shrimp were marked and released only in the upper reaches of each tidal creek. During the marking process, small batches of shrimp (<50 individuals) were collected and held in insulated plastic coolers. Water in the coolers was exchanged each time a new batch of shrimp was col- lected. Only active individuals, 40-80 mm total length (TL, tip of the rostrum to end of the telson) and in appar- ently good condition, were candidates for marking. The marking process required a minimum field crew of two researchers. One measured shrimp and recorded data, while the other injected tags and released marked shrimp at the edge of the tidal creek. We attempted to mark 1000 shrimp during a 3-day pe- riod in each tidal creek near the beginning of every month from July to November in both years. It was not possible to tag shrimp in both creeks simultaneously; therefore shrimp were marked and released in week 1 during the first week of the month, then in creek 2 during the second week of the month. The remainder of the month was spent collecting marked shrimp (Fig. 2). Inclement weather and mechanical problems with the tagging equipment sometimes prevented us from achieving the goal of tag- ging 1000 shrimp per creek within the first week of each month. In August 1998, the tag injector malfunctioned on the first day of tagging in SC and was unavailable for sev- eral weeks while it was being refurbished. Consequently, sampling was suspended in SC during that time. Although the same problem occurred while we attempted to mark shrimp from the EF in September 1999, we continued sampling in an attempt to recapture shrimp tagged in previous months. A total of 6 sampling events after marking were planned in each creek per month (Fig. 2). A sample consisted of the combined contents of 10 haphazard casts of the net along the edge of the tidal creek within each station per sampling date. Shrimp populations were usually sampled at 3-day intervals for 21 days beginning from the midpoint of the marking period. A consistent exception was the second sample in the series, which occurred at a 6-day interval to accommodate the marking effort in the second creek and to keep the sampling effort consistent in all creeks. Inclem- ent weather interrupted the sampling schedule on occasion and when unfavorable conditions persisted for more than 3 days, some of the planned sampling events after marking were cancelled; some months were represented by fewer than 6 sampling events. Sampling was terminated when, as a result of normal seasonal emigration from the nursery areas, shrimp densities declined to the point that they could no longer be consistently collected from the tidal creeks by cast net (19 November 1998 and 21 November 1999). Catches of marked shrimp from each station were re- tained in separate plastic bags, placed on ice, and trans- ported to the laboratory. A subsample of shrimp from each station (every tenth individual) was measured (TL, mini to construct size distributions. If a sample included fewer than 100 shrimp, all were measured. Sex was not deter- mined. All individuals were scanned for the presence of tags and when a marked shrimp was detected, it was mea- sured (TL, mm) before the tag was removed and stored in a plastic vial for reading at a later date. For each recapture. Marking — • •• • coo o o • Creek 1 O Creek 2 Postmarking OOO sampling o 10 15 20 25 Days of the month 30 Figure 2 Schedule for monthly marking and postmarking field schedule for the juvenile white shrimp study. we recorded date, creek, station of recapture (i.e., distance from original release site) and size offish. Daily instantaneous (specific) growth rates (mm/[mm/ d] ) were calculated as [(In L2- In Lj)/t], where L2 = total length (mm) of an individual on the date of recapture; L1 = initial total length (mm) on the date of tag- ging; and t = number of days at large. Daily absolute growth rates (mm/d) also were calculated (L2-L1/t) to facilitate comparison with estimates from previous studies. Displacement (distance moved) was determined by comparing the location of recapture with the original location at marking. Residence time within a tidal creek was determined from a plot of the proportion of recaptured individuals against time-at -large for each creek system. First, using the Regression Wizard in the com- puter program SigmaPlot® (version 8.0, SPSS, Inc. Chicago, ID, we fitted the data to an exponential decay function: (y=a x e~bt) , where y = the proportion of total recaptures; t = time at large; and a and b = the estimated parameters. Constraints imposed on the fit were a = l (because the proportion of total recaptures could not exceed 1) and fe>0 (because this was an exponential decay function). Mean residence time for shrimp in each creek was then estimated from the area below the fitted curves describing the proportion of recaptures with time at large. This was calculated with the macro function "area below curves" included in the "Toolbox" menu selection of SigmaPlot® (vers. 8.0, SPSS, Inc. Chicago, ID which uses the trapezoi- dal rule to estimate the area under curves. Statistical analyses Most of the data analyses used statistical procedures in version 8.0 of the computer software package Systat® 380 Fishery Bulletin 102(2) (SPSS, Inc. Chicago, ID. When parametric tests were performed, residuals were analyzed to determine whether the data met the required assumptions (Sokal and Rohlf, 1995). Levene's test was used to evaluate conformity to the assumption of variance homogeneity among groups. When this assumption was violated, the data were trans- formed and retested. If the assumptions were still not met, then an appropriate nonparametric test was applied (e.g., Kruskal-Wallis one-way analysis of variance ). Two sample /-tests were used to compare spatial and temporal varia- tion in water temperature, salinity, and dissolved oxygen between creeks within a year and between years. August was omitted in comparisons of data between creeks in 1998, and between years because sampling in SC was suspended during August 1998. Regression analyses were performed to determine whether there were significant linear relationships between initial shrimp length and growth rates within each tidal creek. One-way ANOVA (controlling for the covariate initial length) was used to test for differences in growth rates between creeks within each year. A similar approach (controlling for initial size) was used to test for monthly (seasonal) differences in growth rate within years. If growth rates did not differ significantly between creeks, the data were pooled within year, otherwise creeks were treated separately. Only indi- viduals at large for a month or less (to ensure that growth was representative of individual months) were included in the analyses. Shrimp at large for fewer than 3 days were excluded from the statistical analyses to reduce certain antici- pated biases associated with estimating growth rates. These included 1) measurement error (assumed to be at least 1 mm), which would likely represent a substantial proportion of the growth rate estimate when absolute change in size was small; 2) increments of growth associ- ated with molting (Dall et al., 1990), which could either underestimate growth for shrimp that had been at large for a short time or had not molted since they were tagged or overestimate growth if shrimp were recaptured shortly after the first molt following marking; and 3) size-specific growth, where shrimp marked at a relatively small size and smaller shrimp exhibit a higher relative growth, so that early recaptures could represent larger than average growth rates. Results Physical parameters Average water temperature, salinity, and dissolved oxygen (measured at the mouth of each tidal creek) were similar between creeks within years (see Table 1 in Webb and Kneib, 2002). In 1998, temperature ranged from 18.9 to 33.4°C, salinity from 18.2 to 28.0 ppt, and dissolved oxygen from 1.4 to 11.3 mg/L. In 1999, temperature ranged from 15.0 to 33.4°C, salinity from 23.9 to 32.5 ppt, and dis- solved oxygen from 0.8 to 7.1 mg/L. Temperature followed expected seasonal patterns each year; mean values were highest in summer and declined toward autumn. Results of /-tests with separate variance estimates showed no significant differences between years in either mean tem- perature ( = 0.14, df=134.0, P=0.80) or dissolved oxygen (£=1.82, df=115.9, P=0.07) but mean salinity was sig- nificantly (£=11.63, df=122.7, P<0.01) higher in 1999 (28.1 ppt) than in 1998 (24.8 ppt). Cumulative rainfall was 83.9 cm/yr in 1998 and 82.9 cm/yr in 1999 (Garbisch4). These values were indicative of drought conditions because they were well below the long-term mean annual precipitation value of ca. 132 cm/yr reported for Sapelo Island between May 1957 and March 2003 (Southeast Regional Climate Center5). Growth Shrimp collections during recapture efforts ranged from 20,077 to 78,724 individuals, but the proportion of marked individuals recaptured was low in both years, averaging just over 1% (Table 1). However, the recaptures included 184 individuals for which growth rates and net movements within the nursery were known precisely. Daily absolute growth rates of individuals, which ranged from 0.25 to 2.5 mm, averaged 0.86, 0.78, 0.84, and 0.61 mm at PO, SC, WF and EF, respectively. The mean values are on the low end of the range reported in previ- ous studies of juvenile Litopenaeus setiferus with other methods and conducted in different locations (Table 2). Daily specific growth rates were size-dependent in both years. Negative linear relationships between growth rate and initial size (i.e., smaller shrimp grew relatively faster) was the prevalent trend in all creeks (Fig. 3). No sig- nificant difference (£=1.19, df=74, P=0.237) in growth was detected between PO and SC, where mean (±SD) specific (instantaneous) daily growth rates were 0.014 ±0.006 and 0.012 ±0.007, respectively. In 1999, shrimp exhibited sig- nificantly U=2.12, df=56, P=0.038) higher mean specific growth rates in the WF ( 0.014 ±0.008) compared to the EF (0.010 ±0.006) of the Duplin River. The physical environ- ment was similar at these sites (Webb and Kneib, 2002), and there was no significant difference (£=1.43, df=81, P=0.156) in the mean final sizes of shrimp recaptured from these sites. However, the mean (±SD) initial size of marked shrimp at EF (61.3 ±8.3) was significantly U=2.20, df=81, P=0.031) larger than at WF (56.0 ±10.9); therefore a lower specific growth rate was to be expected at EF. On a finer temporal scale, seasonal variation in growth rates occurred in both years, more rapid growth early in the season, and a general increase in the mean size of in- dividuals as the season progressed were evident i Fig. 4). The earlier observation that specific growth rate declined with size (Fig. 3) opens the possibility that seasonal variation in growth rates could be explained simply by 4 Garbisch, -J. Unpubl. data. Univ. Georgia Marine Institute Flume Dock Monitoring Station, NOAA, Sapelo Island Nat ional Est ua vine Research Reserve. Univ. Georgia Marine Institute. Sapelo Island, GA 31327. 5 Southeast Regional Climate Center. Unpubl. data. Website: http://water.dnr.state.sc.us/water/climate/sercc/elimateinfo/ Instmical/historicaLga.html. [Accessed 21 November 20031. Webb and Kneib: Individual growth rates and movement of Litopenaeus setiferus in a tidal marsh nursery 381 004 0.03 0.02 0.01 % 0.1 EF r2 = 0.24 P=0.06 40 50 70 80 0.04 PO r2 = 0.47 0.03 • • P=<0.001 0.02 • £••• • • • «>v • • • --*V 0.01 ••. • 0.00 , ■% . . . 30 40 50 60 70 80 90 30 40 50 60 70 80 90 0.04 0.03 0.02 0.01 0.00 sc r2 = 0.16 P=0.04 30 40 50 60 70 80 90 Initial size (TL, mm) Figure 3 Scatter plots and linear regression results for the relationships between individual specific growth rates and initial sizes of recaptured juvenile shrimp in each tidal creek subsystem: EF = East Fork of Duplin River, WF = West Fork of Duplin River, PO = Post Office Creek, SC = Stacey Creek. Table 1 Monthly summary of the number of shrimp tagged. number collected in subsequent sampling, and the number of tagged shrimp recaptured in each tidal creek. Collection site and sampling process July August September October November Total Post Office Creek Number tagged and released 1004 1077 1025 1000 779 4885 Number collected after tagging 7477 13.851 4730 8384 1298 35,740 Number of tags recaptured 13 16 16 22 1 68 Stacey Creek Number tagged and released 719 91 804 862 623 3099 Number collected after tagging 5877 0 5466 6376 2358 20,077 Number of tags recaptured 2 0 12 16 3 33 East Fork of Duplin River Number tagged and released 812 1000 0 1024 1003 3839 Number collected after tagging 23,080 14,490 11,020 14,804 3449 66,843 Number of tags recaptured 7 11 0 4 4 26 West Fork of Duplin River Number tagged and released 1008 1000 447 696 1000 4151 Number collected after tagging 32,130 17,926 10,954 10,600 7114 78,724 Number of tags recaptured 27 16 3 4 7 57 382 Fishery Bulletin 102(2) Table 2 Summary of estimated mean if reported in other units. daily absolute growth rates for juvenile Litopenaeus setiferus. Growth rates were converted to mm/d Reference Location Growth rate (mm/d) Method and notes Gunter, 1950 Gulf of Mexico. Texas 0.8-1.3 Size frequency in field samples, juveniles 28-100 mm Williams, 1955 coastal North Carolina 1.2 Size frequency in field samples, progression of maximum sizes of juveniles, 32-117 mm Johnson and Fielding, 1956 Florida 1.3 Pond culture, juveniles Lindner and Anderson 1956 South Atlantic Bight and Gulf of Mexico 1.0-1.3 Extrapolated for juveniles 40-80 mm from Walford plot results using field mark-recapture (disc tags) data for individuals 70-205 mm Loesch, 1965 Mobile Bay, Alabama 0.6-1.0 2.2 Size frequency from spring and summer field samples; progression of maximum sizes of juveniles 50-135 mm juveniles 15-70 mm Klima, 1974 Galveston Bay, Texas 1.4-1.8 Extrapolated for juveniles 40-80 mm from Walford plot results determined from field mark-recaptured (stain-injected) subadults (117 mm) coastal Louisiana 1.0-1.3 Extrapolated for juveniles 40-80 mm from Walford plot results determined from field mark-recaptured (stain-injected) subadults (120 mm) Mayer, 1985' Sapelo Island, Georgia 0.9-1.5 Estimated from modal size-frequency data for juveniles 20-120 mm Knudsonetal.. 1996 coastal Louisiana 0.3-0.7 Mark-recapture (injected pigments) of juveniles 45-58 mm (initial size) from coastal marsh ponds This study Sapelo Island, Georgia 0.6-0.9 Monthly mark-recapture (coded ferromagnetic tags) of juveniles 40-80 mm (initial size) from subtidal creeks ' Mean growth rates reported ir derived directly from the data Table 3 of Mayer 1 1985 ) were inconsistent with cohort data in Figure 8 of that thesis; rates reported here were Doints shown in Figure 8 of Mayer's thesis. changes in the average size of shrimp within the nursery over time. We tested this hypothesis by comparing mean growth rates among months after controlling for initial length as a covariate. For these analyses, the 1998 data from PO and SC were pooled because there was no evidence of a differ- ence in growth rates between these two creeks; the 1999 data from EF and WF were analyzed separately because mean growth rates differed between these two systems. After removing the effect of initial size, there was no sig- nificant difference among months in 1998, nor in 1999 at EF, but significant differences in mean growth remained detectable among months at WF (Table 3). The findings from WF also were unusual in that the covariate (initial length) was not a significant factor in the analysis. Post hoc multiple comparisons (Bonferroni, experiment-wise <*=0.05) of mean growth rates among months (without accounting for the covariate) indicated that the specific growth rate in July (0.021) was significantly greater than thai in the other months (0.007 to 0.011). This was the only statistically significant evidence of seasonal varia- tion in growth apparently not associated with shrimp size distributions. With respect to spatial variation in growth rates of ju- venile shrimp, the most notable observation in this study was the relatively low mean growth rate observed at EF compared to the other sites. This difference could have resulted from the larger mean initial size of individuals tagged at EF (61.3 mm) compared with those at WF (56.0) in 1999. However, a similar difference in mean initial sizes of marked shrimp between tidal creek subsystems (SC, 64.2 mm; PO, 59.6 mm) in the previous year did not result in a significant difference in growth rates. When we con- sidered the structural characteristics of each tidal creek at a landscape level, the EF subsystem had the largest tidal drainage area (119.5 ha. ) compared to the other sites ( 58.6 to 104.9 ha.), but proportionally less of that area was inter- tidal drainage. There was a stronger correlation between mean growth rate (pooled across all individuals within a creek) and the proportion of the drainage area that was Webb and Kneib: Individual growth rates and movement of Litopenaeus setiferus in a tidal marsh nursery 383 Table 3 Summary of ANOVA results for the effects of month on specific growth rate of Litopenaeus setiferus after controlling for the covariate initial length. Only individuals at large between 3 and 32 days were included in the analyses. PO = Post Office Creek; SC = Stacey Creek; EF = East Fork of Duplin River; and WF = West Fork of Duplin River. Prob. = probability. 1998 (PO and SC) EF WF Source df F-value Prob. df F-value Prob. df F-value Prob. Covariate (initial size) 1 Month 4 Error 70 30.77 <0.01 1.25 0.30 1 3 11 1.42 0.26 0.74 0.55 1 4 36 1.42 4.32 0.24 <0.01 Oct (39) Nov (15) Month Figure 4 Mean daily specific growth rates and mean initial size of marked and recaptured juvenile white shrimp by month. Only individuals at large for 3 to 32 days were included. Error bars are 2 SEs and the number of observations are given in parentheses below each month. intertidal (Fig. 5A) than there was between growth and mean initial size (Fig. 5B) at the landscape level. There was almost no correlation between proportion of drainage area that was intertidal and initial mean size of marked shrimp (Pearson r=-0.18, P=1.0). Residence time and movement of marked shrimp Recaptured shrimp were at large for up to 99 days, but mean residence time for individuals marked in all four tidal creeks was between 15 and 26 days (Fig. 6). Mean residence time was greatest at EF and least at SC. During their time-at-large, net displacement (distance between mark and recapture sites) of the marked individuals ranged from 0 to 3000 m , but averaged 258-373 m in all creeks. There was no evidence of a significant relationship between time-at-large and mean net displacement (linear regression F=1.48; df=l,45; P=0.23), but movement was slightly related to shrimp size, and larger individuals showed greater displacement (Fig. 7). Variation in resi- dence time among creek subsystems was not significantly 0.016 A 0.014 • Pearson r= 0.090 wp # P=0.10 po«/- 0.012 ■ ^^^ • SC CO ^ 0.010 ■ ^-^ g EF * S 0.008 I £ 0.84 0.86 0.88 0.90 0.92 %_ Intertidal area/total drainage area ™ 0.016 B J 0.014 0.012 A WF """"■ •— ^* PO ~~-C^_^^ SC A 0.010 Pearson r= -0.55 P = 0.45 A£F 56 58 60 62 64 Mean initial size (TL, mm) Figure 5 Correlations between mean daily specific growth rate in each tidal creek (PO=Post Office, SC = Stacey Creek, EF=East Fork of Duplin River, WF=West Fork of Duplin River). (A) The proportion of the tidal drainage area that is intertidal; IB) mean initial size of marked shrimp. correlated with length of the creek mainstem (Pearson r=-0.32, P=0.684), but there was evidence of positive associations with the amount of intertidal (Pearson r=0.87, P=0.133) and subtidal (Pearson r=0.95, P=0.054) drainage areas within each subsystem. Most shrimp (939r) were recaptured in the same tidal creek subsystem in which they were originally marked, but there was some evidence of movement among creeks and between the subtidal and intertidal components of the shrimp nursery within creeks. The marked individual at large for the longest time (99 days) was recaptured at the same station where it was originally marked (net 384 Fishery Bulletin 102(2) Mean residence time (area under each curve) Post Office Creek (20.4 days) Stacey Creek (15.2 days) 100 Mean residence time (area under each curve): i East Fork (25.5 days) i West Fork (20.4 days) 40 60 Days at large 100 Figure 6 Estimates of mean residence times of marked shrimp in (A) tidal creeks sampled in 1998 and ( B ) creeks sam- pled in 1999. Estimates are based on the area under the curves describing the proportion of recaptured marked shrimp in each creek system that still remained to be captured after the indicated number of days-at-large. displacements 0 m). In contrast, one shrimp marked at PO demonstrated a net displacement of 3 km when it was recaptured at SC after 61 days at large. Nine shrimp (at large from 18 to 49 days) marked at WF were recaptured at EF. and two (at large 19 and 45 days) tagged at EF were recaptured at WF. It was not possible to determine pre- cisely when these shrimp moved out of the creek in which they were tagged or how long they were present in the creek subsystem where they were ultimately recaptured. For the growth rate analyses, it was assumed that most growth occurred while the shrimp were in the creek and where individuals were marked. The mean (±SD) final size (mm, TL) of individuals that moved between creek subsystems was significantly (separate variance estimate <=2.62, df=16.9, P=0.018) larger (78.9 ±7.4) than that of the group tagged and recaptured in the same subsystem (71.3 ±13.1); the initial mean size of the two groups was nearly identical (57.5 ±10.8 and 57.7 ±10.4, respectively). Two shrimp (at large 7 and 17 days) tagged at EF were recaptured at high tide in flume weirs located 25 m into 1000 800 600 400 200 F=7.10, df = 1.37 P = 0.01, r2 = 0.16 40 50 60 70 80 90 100 110 Final size (TL. mm) Figure 7 The effect of shrimp size at recapture on mean dis- tance between mark and recapture locations (dis- placement!. Summary results from the linear regres- sion ANOVA performed on the data are shown. Values of mean displacement were based on data from 2-11 individuals within each size. the interior of the intertidal marsh drained by that tidal creek subsystem. The flume weir samples were part of an ongoing study (Kneib, unpubl. data) to determine nekton use of the intertidal marsh surface (see Kneib, 1991, 1997; Kneib and Wagner, 1994). Discussion Growth Mean growth rates of juvenile white shrimp measured in this study (0.6-0.9 mm/d) were near the lower end of the range of estimates previously reported for juvenile white shrimp along the U.S. Atlantic and Gulf coasts (Table 2). The principal difference between the present and previous studies is that the values presented in this study were based on direct measurements of free-rang- ing individual juvenile shrimp rather than on extrapola- tions from batch mark-recaptures of larger individuals or changes in modal size frequencies. The open nature of estuarine ecosystems, prolonged seasonal recruitment to the nursery, and ontogenetic differences in mortality and movement all may confound the interpretation of size-fre- quency data (Haywood and Staples, 1993). Given that our growth values were based on actual changes in the size of individuals rather than estimated from the apparent growth trajectories of cohorts, we are confident that the mean growth rates reported here accurately reflect those of free-ranging juvenile white shrimp (40-80 mm TL) in the polyhaline portion of the tidal marsh nursery habitat of coastal Georgia. Temporal differences in observed growth rates in this study may have resulted from either variation in environ- mental conditions or spatial variation in habitat quality. Webb and Kneib: Individual growth rates and movement of Litopenaeus setiferus in a tidal marsh nursery 385 Penaeids are most abundant in tidal marsh nurseries when physical conditions (eg., temperature and salinity) appear optimal for their growth and survival (Zein-Eldin and Renaud, 1986), but environmental variability is characteristic of most estuaries and therefore is an obvi- ous starting point for explaining observed differences in shrimp growth among sites or times. Salinity was the only environmental factor we measured that showed a significant difference between years but could not be as- sociated with any interannual difference in mean growth rates. Temperature may affect the growth and estuarine dis- tribution of juvenile penaeids more than salinity (Vetter, 1983), and interactions between salinity and temperature may have even greater effects than variation in either fac- tor alone (Zein-Eldin and Renaud, 1986). Mean tempera- tures throughout our study period (with the exception of November) in both years were largely within the optimum range for growth of white shrimp which, in the laboratory, was reported to be between 25°and32.5°C (Zein-Eldin and Griffith, 1969). Higher temperatures generally contrib- ute to faster growth in young penaeids (Perez-Farfante. 1969; Muncy, 1984), and therefore it seems reasonable to expect seasonal variation in temperature to be reflected in growth rates. However, this interpretation is con- founded by the fact that growth rates also are size depen- dent (Fig. 3, Table 3) and that increases in mean size of juvenile white shrimp (Fig. 4) occurred while tempera- ture in the nursery habitat was decreasing from the July maxima. It seems likely that growth rates of juvenile white shrimp were robust over the relatively narrow range of seasonal variation in temperature and salinity observed in the present study. Alternatively, differences in growth between certain sites could be the result of spatial variation in habitat quality. This variation need not be a function of water quality, but rather a function of some structural aspect of the nursery habitat. There was a strong correlation be- tween mean growth rates and the proportion of tidal creek drainage area that was intertidal. Only four creek sub- systems were examined in ours study, and we recognize that this is an insufficient sample size to justify anything more than a suggestive hypothesis. However, evidence of relationships between the amount of intertidal habitat and penaeid shrimp production (Turner, 1977, 1992), as well as the amount of intertidal creek edge and juvenile shrimp abundance in adjacent subtidal creeks (Webb and Kneib, 2002), supports the contention that intertidal ac- cessibility is an important component of nursery habitat quality for juvenile white shrimp. We propose that the ratio between intertidal and shallow subtidal habitat may be a key feature of white shrimp nursery habitat quality. When tidally inundated, the intertidal portion of marsh creek drainage systems is used extensively by juvenile white shrimp (Kneib, 1995, 2000), most likely as a rich foraging area (Kneib, 1997), and the shallow subtidal portion functions as a low tide refuge and corridor for the seasonal migration of postlarvae and subadults between the open estuary and coastal ocean spawning grounds and the juvenile nursery (Kneib, 1997, 2000). Movement and residence time Understanding the causes of broad-scale migration of penaeids has obvious implications for predicting com- mercial catches and therefore these causes have been the focus of research on shrimp movements for decades ( Perez- Farfante, 1969; Muncy, 1984). However, finer-scale move- ments, which may affect growth and survival of juvenile shrimp within the estuary, are not as well known. Emigra- tion of white shrimp from estuaries is determined by size, maturity, and environmental conditions (Muncy, 1984), and size plays a principal role (Dall et al., 1990). In the South Atlantic Bight, larger white shrimp (>100 mm TL) begin emigrating from the nursery to commercial fishing areas in the nearshore coastal ocean in August (Lindner and Anderson 1956, Shipman, 1983). We collected few shrimp >100 mm in the tidal marsh creeks, which is con- sistent with previous observations of ontogenetic migra- tion to deeper waters. According to growth rates measured in this study, a shrimp of 40 mm TL would become large enough to emigrate from the estuary to the coastal ocean in 2-3 months (i.e., a shrimp tagged at 40 mm TL could reach 85-108 mm TL in 2.5 months). The presence of high densities of small juvenile white shrimp in the upper reaches of Georgia's tidal marsh creeks (Harris, 1974; Hackney and Burbanck, 1976; Webb and Kneib, 2002) has supported the contention advanced by Weinstein ( 1979) that these areas are primary nurser- ies for juvenile fish and shellfish. However, it has been unclear whether these aggregations represent stable res- ident populations or are composed of tidal transients that constantly migrate among creek subsystems within the broader estuarine nursery. Young shrimp are known to move short distances to avoid unfavorable physiochemical conditions (Hackney and Burbanck, 1976; Dall etal., 1990) and routinely make tidally mediated excursions between subtidal and intertidal portions of the nursery to forage or escape predators (Kneib, 1995, 1997). Our findings showed that juvenile white shrimp also tended to remain resident in the upper reaches of tidal creeks where they were originally tagged until attaining a size ( 80-100 mm ) at which they normally begin to emigrate from the nursery (Perez-Farfante, 1969). Although there was some movement between tidal creek subsystems, the high level of site fidelity demonstrated by juvenile white shrimp was remarkable given the open- ness and degree of tidal flux in the Duplin River system (mean tide range=2.1 m). Data from the chemical analysis of shrimp tissue composition also suggest limited move- ments of juvenile penaeids within estuarine nurseries. Using the stable isotopes of carbon and nitrogen from mus- cle tissues of pink shrimp (Farfantepenaeus duorarum), Fry et al. (1999) traced shrimp movements within and between seagrass and mangrove habitats of southwestern Florida. They found distinct differences among individu- als sampled from similar inshore habitat types separated by small (3-5 km) open water distances, indicating that individuals remained "resident" in specific portions of the estuary at least for several weeks. Noting a similar study in coastal Louisiana, Fry et al. (2003) suggested that 386 Fishery Bulletin 102(2) small juvenile brown shrimp (Farfantepenaeus aztecus) are more transient in suboptimal habitat (open bays and deeper channels) and exhibit less movement upon reach- ing optimal habitat (ponds and shallow channels). The only study with which we can directly compare our findings on residence time and movements was conducted by Knudsen et al. (1996) near Calcasieu Lake, Louisiana, where tidal flux was considerably lower (mean tide range <0.6 m) and the system (marsh impoundments) was less open than that in the present study. Knudsen et al. (1996) marked batches of juvenile white shrimp (45-69 mm TL) by injection of colored pigments and released them into a pair of 35-ha. impoundments, each connected to the open estuary through a narrow channel that was fitted with screen deflectors and traps designed to collect all emigrat- ing nekton. The mean time from release to emigration of juvenile white shrimp ranged from 30.2 to 59.9 days. Our estimates of tidal creek residence time for juvenile shrimp in Georgia tidal creeks was about half that reported for impoundments in Louisiana and may be explained by the differences in tidal flux and openness between the two systems. However, the values we observed were likely underestimates of the actual residence period of survivors within the creeks because they included losses due to mor- tality as well as emigration. It seems clear from the studies conducted thus far that juvenile penaeids, once having entered the estuarine nursery, tend to remain within a limited spatial range where they are exposed to local conditions for several weeks. Our findings also provide evidence of spatial variation for both residence time and growth rate of ju- venile white shrimp that is possibly attributable to struc- tural differences in tidal creek subsystems. We suggest there may be an optimal value for the ratio of subtidal to intertidal drainage area within marsh creek systems that can achieve a favorable balance between suitable habitat (space) at low tide, which tends to enhance residence time and density of juvenile shrimp, while providing sufficient intertidal foraging habitat and predator refugia at high tide to promote high rates of juvenile shrimp growth and survival. Spatially explicit information on growth rates and the extent to which individual shrimp move within their estuarine nurseries are necessary initial steps to- ward meeting the challenge of maintaining quality nurs- ery habitat for a sustainable shrimp fishery and satisfying other demands associated with human development in and around estuarine watersheds. Acknowledgments Several individuals provided field and laboratory assis- tance for this project, but we especially thank K. Feeley, J. Kneib, and J. Spicer for helping on a regular basis. The primary source of funding was a National Estuarine Research Reserve System Graduate Research Fellowship to S. Webb (NA870R0284) (Estuarine Reserves Division, Office of Ocean and Coastal Resource Management, NOS, NOAA), and matching funds provided by the University of Georgia Marine Institute. The Georgia Sea Grant Col- lege Program contributed funds for the purchase of an additional tag injector unit, which substantially improved the effectiveness of the mark-recapture program. The con- ceptual basis for this project was derived from research conducted under a grant from the National Science Foun- dation ( DEB-9629621 ), which also contributed supplemen- tal student support. Literature cited Dall, W., B. J. Hill. P. C. Rothlisberg, and D. J. Staples. 1990. The biology of the Penaeidae. In Advances in marine biology, vol. 27 (J. H. S. Blaxter and A. J. Southward, eds.), 489 p. Academic Press, London. Fitz, H. C, and R. G. Weigert. 1991. Tagging juvenile blue crabs, Callinectes sapidus, with microwire tags: retention, survival, and growth through multiple molts. J. Crust. Biol. 11(21:229-235. Fry, B., D. M. Baltz, M. C. Benfield, J. W. Fleeger, A. Gace, H. L. Haas, and Z. J.Quinones-Rivera. 2003. 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Juvenile white shrimp growth, mortality, and emi- gration in weired and unweired Lousiana marsh ponds. North Am. J. Fish. Manag. 16:640-652. Krouse, J. S., and G. E. Nutting. 1990. Evaluation of coded microwire tags inserted in legs of small juvenile American lobsters. //; Fish-marking techniques (N. C. Parker, et al.. eds.), p. 304-310. Am. Fish. Soc. Symp. 7, Bethesda, MD. Kutkuhn, J. H. 1966. The role of estuaries in the development and per- petuation of commercial shrimp resources. Am. Fish. Soc. Spec. Publ. 3:16-36. Lindner, M. J., and W. W. Anderson. 1956. Growth, migrations, spawning and size distribution of shrimp, Penaeus setiferus. Fish. Bull. 56:555-645. Loesch, H. 1965. Distribution and growth of penaeid shrimp in Mobile Bay, Alabama. Publ. Inst. Mar. Sci., Univ. Texas 10:41-58." Mayer, M. A. 1985. Ecology of juvenile white shrimp, Penaeus setiferus Linnaeus, in the Salt Marsh Habitat. M.S. thesis, 62 p. Georgia Inst. Technology, Atlanta, GA. McTigue, T A., and R. J. 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Penaeoid and sergestoid shrimps and prawns of the world: keys and diagnoses for the families and genera. Memoires du Museum National d'Historie Naturelle 175: 1-233. Prentice, E. F., and J. E. Rensel. 1977. Tag retention of the spot prawn, Pandalus platyceros, injected with coded wire tags. J. Fish. Res. Board Can. 34:2199-2203. Ragotzkie, R. A., and R. A. Bryson. 1955. Hydrography of the Duplin River, Sapelo Island. Georgia. Bull. Mar. Sci. Gulf Caribb. 5:297-314. Shipman, S. 1983. Mark-recapture studies of penaeid shrimp in Geor- gia, 1978-1981. In Studies and assessment of Georgia's marine fisheries resources, 1977-1981 (S. Shipman. V. Baisden, and H. Ashley, eds. ), p. 1-176. Georgia Dep. Nat. Res. Contr. Ser. 35. Sokal, R. R., and F. J. Rohlf. 1995. Biometry: the principles and practice of statistics in biological research, 3rd ed., 887 p. W.H. Freeman and Co., New York, NY. Turner, R. E. 1977. Intertidal vegetation and commercial yields of pen- aeid shrimp. Trans. Am. Fish. Soc. 106:411-416. 1992. Coastal wetlands and penaeid shrimp habitat. In Stemming the tide of coastal fish habitat loss ( R. H. Stroud, ed.), p. 97-104. National Coalition for Marine Conserva- tion, Savannah, GA. Uglem, I., and S. Grimsen. 1995. Tag retention and survival of juvenile lobsters, Hom- marus gammarus, marked with coded wire tags. Aquacult. Res. 26:837-841. van Montfrans, J., J. Capelli. R. J. Orth, and C. H. Ryer. 1986. Use of microwire tags for tagging juvenile blue crabs {Callinectes sapidus). J. Crust. Biol. 6:370-376. Vetter, E. A. 1983. The ecology of Penaeus setiferus: habitat selec- tion, carbon and nitrogen metabolism, and simulation modeling. Ph.D. diss.. 151 p. Univ. Georgia. Athens. GA. Wadsworth, J. R. 1980. Geomorphic characteristics of tidal drainage net- works in the Duplin River system, Sapelo Island, Georgia. Ph.D. diss., 247 p. Univ. Georgia, Athens, GA. Webb, S. R., and R. T Kneib. 2002. Abundance and distribution of juvenile white shrimp Litopenaeus setiferus within a tidal marsh land- scape. Mar. Ecol. Prog. Ser. 232:213-223. Weinstein, M. P. 1979. Shallow marsh habitats as primary nurseries for fishes and shellfish. Cape Fear River, North Carolina. Fish. Bull. 77:339-357. Wenner. E. L„ D. Knott, J. O. Blanton, C. Barans, and J. Amft. 1998. Roles of tidal and wind-generated currents in trans- porting white shrimp {Penaeus setiferus) postlarvae through a South Carolina (USA) inlet. J. Plank. Res. 20(121:2333-2356. Williams, A. B. 1955. Contribution to the life histories of commercial shrimp (Penaeidae) in North Carolina. Bull. Mar. Sci. Gulf Caribb. 5:116-146. 1984. Shrimps, lobsters, and crabs of the Atlantic coast of the eastern United States, Maine to Florida, 550 p. Smithsonian Institution Press, Washington, D.C. 388 Fishery Bulletin 102(2) Zein-Eldin, Z. P., and G. W. Griffith. Zein-Eldin, Z. P., and M. L. Renaud. 1969. An appraisal of the effects of salinity and tempera- 1986. Inshore environmental effects on brown shrimp, ture on growth and survival of postlarval penaeids. FAO Penaeus aztecus, and white shrimp. P. setiferus, popula- Fish. Rep. 57(3):1015-1026. tions in coastal waters, particularly of Texas. Mar. Fish. Rev. 48(3):9-19. 389 Does the California market squid (Loligo opalescens) spawn naturally during the day or at night? A note on the successful use of ROVs to obtain basic fisheries biology data John Forsythe National Resource Center for Cephalopods University of Texas Medical Branch at Galveston 301 University Blvd Galveston, Texas 77555-1163 E-mail address: |ohn forsythetg' utmb.edu Nuutti Kangas Roger T. Hanlon Marine Resources Center Marine Biological Laboratory Woods Hole, Massachusetts 02543 The California market squid iLoligo opalescens Berry), also known as the opalescent inshore squid ( FAO ), plays a central role in the nearshore ecological communities of the west coast of the United States (Morejohn et al., 1978; Hixon, 1983) and it is also a prime focus of California fisheries, ranking first in dollar value and tons landed in recent years (Vojkovich, 1998). The life span of this species is only 7-10 months after hatching, as ascertained by aging statoliths ( Butler et al., 1999; Jackson, 1994; Jackson and Domier, 2003) and mariculture trials (Yang, et al., 1986). Thus, annual recruitment is required to sustain the population. The spawning season ranges from April to November and spawning peaks from May to June. In some years there can be a smaller second peak in November. In Monterey Bay, the squids are fished directly on the egg beds, and the con- sequences of this practice for conser- vation and fisheries management are unknown but of some concern (Hanlon, 1998). Beginning in April 2000, we began a study of the in situ spawning behavior of L. opalescens in the south- ern Monterey Bay fishing area. The prevailing thought is that the majority of spawning activity takes place at night because fishermen have observed these squids mating under their bright lights (which are used to attract and capture squids) and be- cause television documentaries have revealed mating and spawning activ- ity in large aggregations at night. The scientific literature on reproductive behavior is sparse. There are some cur- sory observations of actively spawning L. opalescens during diver surveys of egg beds (McGowan, 1954; Fields, 1965; Hobson, 1965; Hurley, 1977). Some daytime spawning has been seen both in southern and northern California but Fields ( 1965 ) and Hixon (1983) suggested indirectly that most spawning occurs at night. Shimek et al. (1984) also suggested night spawn- ing by L. opalescens in Canada. Other loliginid squids whose natural behav- ior has been studied in the field were found to be daytime spawners (e.g., L. pealeii, L. vulgaris reynaudii, Sepioteu- this sepioidea; summarized in Hanlon and Messenger, 1996). To help resolve this issue, we con- ducted three field expeditions (28 April-8 May 2000, 10-17 September 2000, and 16-21 August 2001) using remotely operated vehicles (ROVs) de- ployed either from the RV John Martin (Moss Landing Marine Laboratory) or the commercial squid FV Lady J. The ROVs were tethered vehicles with on- board video cameras and lights. Live video signals were transmitted by the tether to shipboard VCRs where observational data were viewed and recorded. For the first field trip, a large S4 Phantom ROV was used; it was outfitted with a video camera and zoom lens with tilt capability, and the video was recorded on Hi8 format video decks. For the second and third trips, a smaller S2 inspection-class Phantom ROV on loan from the NOAA Sustain- able Seas Expeditions was used; this ROV had a customized fiber-optic teth- er and the video data were recorded on mini-digital video cassettes. Our goal was to make ROV dives each day from approximately dawn to dusk and to make a few comparable all-night sur- veys. A combination of adverse weather conditions and technical problems with the ROVs rarely allowed continuous video observations. During dives, if squids were encountered, we used video to conduct focal animal samples on females (which were paired) for as long as squids were present, or as long as we could keep track of the same individuals. Unless absolutely neces- sary to see the squids (for instance at night or at depths greater than 30 m in turbid daytime conditions), lights were not used for video taping in an effort to minimize their impact on the mating squids. Squids acclimated within minutes to the ROVs. After the expeditions, the videotapes were stud- ied and the behavioral and biological data were quantified on a multimotion playback VCR. By "mating" we refer to the peculiar mating behavior of this species that is unique among loliginid squids. The male firmly grasps the female from her ventral side and holds her for minutes or hours in a "copulatory embrace" in a nearly vertical position. Both copula- tion (i.e., transfer of spermatophores) and deposition of egg capsules occur in this posture. For example, as the female exudes a new egg capsule, the male and female lower themselves in unison to the egg bed where the female deposits the egg capsule in the sand. We have reported elsewhere on egg- Manuscript approved for publication 20 January 2004 by Scientific Editor. Manuscript received 25 January 2004 at NMFS Scientific Publications Office. Fish. Bull. 102:389-392 (2004). 390 Fishery Bulletin 102(2) No Maling Squids Mating Squids Present 29Apr00-, 30Apr00 n i May Uli 04May00 OSMayOO OtSMayOO 07May00 •Wk ^mmm-M Sunset b mw?. wmmmmr* lOSepOO 12Sep00 USepOO 16SepOO 0000 0200 0400 0600 0800 1000 1200 1400 Time of day (h) 1600 1800 2000 2200 2400 Figure 1 A summary of 154 spawning groups of Lot igo opalescens showing the daily presence or absence of mating squids on egg beds in Monterey Bay, California. The horizontal bars repre- sent the time periods when a ROV was on the bottom searching for mating squids during three different expeditions. Note three occasions in which operations were conducted continuously through the day and night, and also that spawning ceased at dusk. laying frequency (Hanlon et al., in press). Only very rarely did we observe females laying eggs while unattended by a male; in these cases the female was moribund and laying her last few egg strands. Results and discussion We examined 28 hours of videotape recorded during 50 ROV dives over 18 days (divided into three expeditions). Figure 1 illustrates the relative presence or absence of mating Lo//go opalescens throughout 24-hour periods. The large gaps in the daytime observation record were due to ROV problems. Although observation times varied daily, it is clear that normal mating and egg-laying behaviors were exclusively observed during daylight hours (ca. 0800-1800 hours but with some seasonal variation) and concluded near dusk. In all instances in Figure 1 where egg-laying extended into the early evening, these mating assemblages had formed during daylight hours and per- sisted slightly past sunset and the number of participating squids constantly decreased as sunset approached and passed. Observations were made throughout the night on three nights. Not only were no mating squids ever encountered around the egg beds at night, but generally no squids were encountered at all near the seabed, despite large aggregations that were present higher in the water column. Thus the 200-400 watt lights on the ROVs never induced any artificial spawning behavior because there were no squids present. Figure 2 provides some quantification of Figure 1. This graph is based on 154 spawning groups that were vid- eotaped and includes all three trips as well as the three "all night observations" illustrated in Figure 1. We were studying discrete groups of squids to examine mating dy- namics and thus were sometimes biased to smaller groups of squids that could be kept in view. Overall, we observed that squids were present in greatest numbers in mid to late afternoon and absent during the night. Our findings strongly indicate that the extensive egg beds produced at depths of 20-60 m in southern Monterey Bay (just beyond the kelp beds) are the result of daytime aggregations of mating Loligo opalescens. These benthic aggregations begin forming in the early morning hours and tend to be larger in the afternoon. Reproductive ac- tivity begins to wane toward sunset and comes to a near halt at sunset. We could find no evidence that egg laying occurs naturally during the night. All observations that we are aware of (mainly television documentaries) have occurred in the presence of artificial light sources near the surface provided either by fishermen or cinematographers. In the absence of artificial lighting, L. opalescens in South Monterey Bay does not aggregate into mating and spawn- ing groups at night. Thus, we conclude that all significant egg deposition in the Monterey Bay fishery is the result of daytime aggregations of squids. NOTE Forsythe et al.: Spawning patterns of Loligo opa/escens 391 Two other ascribed characteristics of L. opalescens spawning are mass aggregations at the sea floor and subsequent die-offs after squids have spawned. Mass aggregations can be detected by standard fathometers used by commercial fishermen, who report that mass aggregations on the sea floor are rare in Mon- terey Bay. During our ROV operations we en- countered only one large aggregation, which occurred on 21 August 2001. We estimated from our video recordings that there were ap- proximately 3000-4000 squid in a 50-m2 area on the sea floor and that intermittent egg lay- ing was occurring over an area of ca. 2000 m2 during a period of about 3 hours. Collectively, then, we recorded 154 very small spawning groups and one large spawning group. There was no mass die-off during or after this large spawning aggregation. Instead, we consis- tently observed in all spawning groups that females actively broke the embrace of the paired male and jetted strongly upwards away from the spawning groups and rejoined large schools in the upper water column. Thus, squids that dispersed from the egg beds were consistently in excellent condition — certainly not senescent or moribund. These observa- tions corroborate the results of other studies on loliginid squids that spawn intermittently (Moltschaniwskyj, 1995; Maxwell and Hanlon, 2000 ). Twice we encountered large numbers of dead squids on the sea floor in the early morning, but in both instances the squid fishing fleet had been working in the same area the night before and it appeared as though these mortalities were associated with the purse-seine fishery; there were few eggs in those localities. McGowan ( 1954 ), Hobson ( 1965 ), and Cousteau and Diole (1973) reported that squids died after spawn- ing in S. California. Various Loligo spp. are noted for flexible reproductive strategies (cf. Hanlon and Messen- ger, 1996) so it should not be surprising if L. opalescens occasionally engaged in large reproductive events. Our data suggest that small groups of squids (20-200 indi- viduals) generally descend during the day and lay eggs for several hours before rejoining squids in the water column. We encourage other researchers to use ROVs or SCUBA without lights and with stealthy approaches to determine the natural diurnal spawning of L. opalescens throughout its range. Given our findings that active sex- ual selection processes are occurring during the day and that there is vertical migration between the large schools of squid in the water column and the small spawning groups at the substrate, it would be prudent, at the very least, to restrict daytime fishing directly over egg beds or to create protected spawning areas in southern Mon- terey Bay. This strategy would allow the complex mating system of L. opalescens to be played out without direct disruption by fishing activity. In such a short-lived spe- cies, annual recruitment to the population is necessary; thus sufficient eggs must be laid for each new generation to ensure a viable living resource. 30 - n = 1 54 groups 25 - CO Q. =J o oi 20 - en c "c I 15 ■ ■ Q. (fi O _■ 5 10 ll E 3 ■ z - 5 1 III III! o "' III 1 Illl 0000 02O0 0400 0600 0800 1000 1200 1400 1600 1800 2000 2200 2400 Time of day (h) Figure 2 The number of squid spawning groups at the egg beds at different times of day (data pooled from three expeditions over 2 years; n= 154 groups). A group is denned as a group of two or more mating pairs. These data correspond hour by hour with data in Figure 1. Acknowledgments We are most grateful for funding on NOAA grant UAF 98 0037 from the National Undersea Research Center (West Coast). Additional funding was provided by the David and Lucile Packard Foundation and the Sholley Foundation. J. Forsythe gratefully acknowledges financial support for travel from the National Institutes of Health, National Center for Research Resources (grant P40 RR0102423- 23), and the Marine Medicine General Budget account of the Marine Biomedical Institute. N. Kangas gratefully acknowledges financial support from the Academy of Finland. We thank Sylvia Earle for loan of the Sustain- able Seas ROV and we appreciate the professional efforts of Deep Ocean Exploration and Research (DOER) who supported the ROV operations. We especially thank John Rummel who helped begin this project, and Brett Hobson who kept it going at a critical juncture. We are thankful for expert shipboard assistance from the captains and crew of the KVJohn Martin and the FV Lady J (especially Captain Tom Noto). We benefitted from discussions with Bob Leos, Bill Gilly, Annette Henry, John Butler, Teirney Thies, and Sue Houghton. Literature cited Butler, J., D. Fuller, and M. Yaremko. 1999. Age and growth of market squid iLoligo opalescens) off California duringl998. CalCOFI Rep. 40:191-195. Cousteau J.-Y.. and P. Diole. 1973. Octopus and squid: the soft intelligence, 304 p. Cas- sell, London. 392 Fishery Bulletin 102(2) Fields, W. G. 1965. The structure, development, food relations, repro- duction, and life history of the squid Loiigo opalescens Berry. Fish. Bull. 131:1-108. Hanlon, R. T. 1998. Mating systems and sexual selection in the squid Loiigo: How might commercial fishing on spawning squids affect them? CalCOFI Rep. 39:92-100. Hanlon, R.T., N. Kangas, and J. W. Forsythe. In press. Rate of egg capsule deposition, changing OSR and associated reproductive behavior of the squid Loiigo opalescens on spawning grounds. Mar. Biol. Hanlon, R. T., and J. B. Messenger. 1996. Cephalopod behaviour, 232 p. Cambridge Univ. Press, Cambridge. Hixon. R. F. 1983. Loiigo opalescens. In Cephalopod life cycles, vol. I, species accounts, 475 p. Academic Press, London. Hobson, E. S. 1965. Spawning in the Pacific Coast squid. Loiigo opal- escens. Underwater Naturalist 3:20-21. Hurley, A. C. 1977. Mating behavior of the squid Loiigo opalescens. Mar. Behav. Physiol. 4:195-203. Jackson, G. D. 1994. Statolith age estimates of the loliginid squid Loiigo opalescens: corroboration with culture data. Bull. Mar. Sci. 54:554-557. Jackson, G. D.. and M. L. Domeier. 2003. The effects of an extraordinary El Nino/La Nina event on the size and growth of the squid Loiigo opalescens off Southern California. Mar. Biol. 142:925-935. Maxwell, M. R., and R. T. Hanlon. 2000. Female reproductive output in the squid Loiigo pea- leii: multiple egg clutches and implications for a spawning strategy. Mar. Ecol. Prog. Ser. 199:159-170. McGowan, J. A. 1954. Observations on the sexual behavior and spawning of the squid, Loiigo opalescens, at LaJolla, CA. Cal. Fish Game 40:47-54. Moltschaniwskyj, N. A. 1995. Multiple spawning in the tropical squid Photolo- ligo sp.: what is the cost in somatic growth? Mar. Biol. 124:127-135. Morejohn, G. V., J. T. Harvey, and L. T.Krasnow. 1978. The importance of Loiigo opalescens in the food web of marine vertebrates in Monterey Bay, California. In Biological, oceanographic, and acoustic aspects of the market squid, Loiigo opalescens Berry (C. W. Recksiek and H. W . Frey, eds.), p. 67-98. Calif. Dep. Fish Game Fish Bull. 169. Shimek, R. L., D. Fyfe. L. Ramsey, A. Bergey, J. Elliott, and S. Guy. 1984. A note on spawning of the Pacific market squid. Loiigo opalescens (Berry, 1911), in the Barkley sound region, Van- couver Island, Canada. Fish. Bull. 82:445-446. Vqjkovich, M. 1998. The California fishery for market squid (Loiigo opalescens). CalCOFI Rep. 39:55-60. Yang, W. T, R. F. Hixon, P. E. Turk. M. E. Krejci, W. H. Hulet, and R. T. Hanlon. 1986. Growth, behavior, and sexual maturation of the market squid, Loiigo opalescens, cultured through the life cycle. Fish. Bull. 84:771-798. 393 Incidental capture of loggerhead (Caretta caretta) and leatherback (Dermochelys coriacea) sea turtles by the pelagic longline fishery off southern Brazil Jorge E. Kotas IBAMA/Acordo Projeto TAMAR- Instituto de Pesca/CPPM Programa REVIZEE-SCORE SUL Rodovia Osvaldo Reis 345 apt. 22 C Itajaf-SC 88306-001. Brazil Silvio dos Santos DTI-CNPq Programa REVIZEE-SCORE SUL Rua Ezio Testlni 320 Santos-SP 11089-210, Brazil Berenice M. G. Gallo Fundacao Pro-TAMAR Rua Antonio Athanasio 273 Ubatuba-SP 11680-000, Brazil Paulo C. R. Barata Fundacao Oswaldo Cruz Rua Leopoldo Bulhoes 1480 - 8A Rio de Janeiro - RJ 21041-210, Brazil E-mail address (for P. C. R. Barata, contact author): pbarataigialternex.com.br back sea turtles by the surface longline fishery operating off the southern coast of Brazil, within Brazil's 200 mile exclusive economic zone (EEZ) and in international waters, and present catch-per-unit-of-effort (CPUE) data and estimates of average probability of death at capture for these species. Preliminary results of incidental cap- tures of sea turtles by longliners dur- ing one longline trip in this area were presented by Barata et al.2 In the present study we provide more detailed data from additional trips, including information concerning leatherback sea turtles, as well as analyses of these data. To our knowledge, this is the first detailed report about the incidental capture of sea turtles by the Brazilian commercial longline fleet. Venancio G. de Azevedo DTI-CNPq Programa REVIZEE-SCORE SUL Av. Pavao 1 64 Caraguatatuba-SP 11676-520, Brazil Incidental capture in fishing gear is one of the main sources of injury and mortality of juvenile and adult sea turtles (NRC, 1990; Lutcavage et al., 1997; Oravetz, 1999). Six out of the seven extant species of sea turtles — the leatherback (Dermochelys coriacea), the green turtle (Chelonia mydas), the loggerhead (Caretta caretta), the hawksbill (Eretmochelys imbricata), the olive ridley (Lepidochelys olivacea). and the Kemp's ridley (Lepidochelys kempii) — are currently classified as endangered or critically endangered by the World Conservation Union (IUCN, formerly the International Union for Conservation of Nature and Natural Resources), which makes the assess- ment and reduction of incidental cap- ture and mortality of these species in fisheries priority conservation issues (IUCN/Species Survival Commission, 1995). Several studies have examined sea turtle bycatch by pelagic longline fish- eries, especially in the North Atlantic and Pacific oceans (NRC, 1990; Nish- emura and Nakahigashi, 1990; Tobias, 1991; Bolten et al., 1996; Williams et al., 1996; Lutcavage et al., 1997), but little is known about sea turtle bycatch in the South Atlantic. One of the most detailed reports on longline incidental captures in that area is that by Acha- val et al. (2000), which documents the incidental capture of loggerhead and leatherback sea turtles in the south- western Atlantic by longliners target- ing swordfish (Xiphias gladius), tuna (Thunnus obesus), and other related species. Additional references, some- times with scant detail, can be found in Weidner and Arocha ( 1999 ), Fallabrino et al. (2000), and Domingo et al.1 In this study, we report the inciden- tal capture of loggerhead and leather- 1 Domingo, A.. A. Fallabrino, R. Forselledo, and V. Quirici. 2002. Incidental cap- ture of loggerhead (Caretta caretta) and leatherback (Dermochelys coriacea) sea turtles in the Uruguayan long-line fish- ery in Southwest Atlantic. Presented at the 22nd Annual Symposium on Sea Turtle Biologv and Conservation, Miami, USA, 4-7 April 2002. [Available from A. Domingo: Direccidn Nacional de Recur- sos Acuaticos, Constituyente 1497, C.R 11.200, Montevideo, Uruguay.] Materials and methods Observations were carried out by three of the authors (JEK. SS, and VGA) during three trips aboard Brazil- flagged commercial longline vessels based in Itajai, State of Santa Cata- rina, southern Brazil (Fig. 1). The trips occurred in 1998, the first (10 sets) between 13 March and 12 April (summer-fall), the second (13 sets) between 15 June and 5 July (fall- winter I, and the third (11 sets (between 28 September and 13 October (spring), and took place between latitudes 27°30'S and 34°30'S and longitudes 36°00'W and 52°00'W (Fig. 1). The Barata, P. C. R., B. M. G. Gallo, S. dos Santos, V. G. Azevedo, and J. E. Kotas. 1998. Captura acidental da tartaruga marinha Caretta caretta (Linnaeus, 1758) na pesca de espinhel de superficie na ZEE brasileira e em aguas internacionais. In Resumos Expandidos da XI Semana Nacional de Oceanografia, Rio Grande, RS, outubro de 1998, p. 579-581. Edi- tora Universitaria-UFPel, Pelotas, RS, Brazil. [Available from FURG, Oceano- logia. Av. Italia, km 8, Campus Carreiros, C.P 474, Rio Grande, RS 96201-900, Brazil.! Manuscript approved for publication 22 December 2003 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Scientific Publications Office. Fish. Bull. 102:393-399(2004). 394 Fishery Bulletin 102(2) t — i — i — i — i — i — vr—r Atlantic Ocean J. 10 N 40 - 50 S - Ascension St. Helena Tristan da Cunha South Georgia N t j i i i i i i i i 80 W 20 10 10 20 E Figure 1 Fishing locations. Numbers 1, 2, and 3 indicate locations of the first, second, and third longline trips respectively; for each location, one or more sets were performed. Circled numbers indicate international waters outside the 200-mile Brazilian exclusive economic zone. The rectangular ocean area is limited by latitudes 25°S and 35°S and longitude 35°W. The fishing location farthest to the east is about 1320 km (713 nautical miles) from Itajai, State of Santa Catarina, Brazil, the home port of the fishing vessels. seabed in this area ranged from the continental shelf border to abyssal plains, including submarine elevations (e.g., Rio Grande). Operation depths, ranging from 170 to 4000 m, were obtained from nautical charts. The first and second trips were aboard the Yamaya III, a 20.7-m, 325-hp engine, 30-t hold capacity, 10-crew long- liner, and the third trip was aboard the Basco, a 24.4-m, 330-hp engine, 70-t hold capacity, 11-crew longliner. The vessels targeted swordfishes, sharks (mainly blue sharks, Prionace glauca) and tunas (Thunnus albacares, T. alalunga and T. obesus). Their fishing gear was the U.S. -style monofilament nylon longline, with 200- 300 m sections between buoys, and each section contained four to five gangions set 40-60 m apart. Buoy dropper length ranged between 10 and 20 m, and gangion length ranged between 13 and 20 m. Each non-offset "J" hook (Swordfish 9/0) was baited with Argentine shortfin squid (Illex argentinus) and had a yellow chemical light stick hung over it. The average number of hooks per set was 1030, 992, and 950 on the first, second, and third trips, respectively. On the first and second trips, the mainline was set off the stern by means of a line shooter so that a marked catenary was formed between buoys, allowing the hooks to operate at a greater depth. In this case, the maximum hook depth may have reached more than 40 m. On the third trip, the vessel Basco did not use a line shooter, and thus the hook depth for that trip may have been shallower. The longline gear was set around 5:30 PM, and was retrieved early in the morning. The average soak time was 7 h 30 min. For each set, the date, time, geographical position, number of hooks, and sea surface temperature were recorded. The species and condition (i.e., if the animal was alive or dead) of captured turtles were recorded; specimens with no ap- parent movement were considered dead. Incidentally captured loggerhead turtles were taken aboard and hooks and lines were then removed. Whenever possible curved carapace length (CCL) and width were measured, and the turtles were double tagged (inconel tags style 681, National Band and Tag Co., Newport, KY), according to Projeto TAMAR's (Projeto Tartaruga Marinha, the Brazilian sea turtle conservation program) standard methods (Marcovaldi and Laurent, 1996). In some cases, it was not possible to bring loggerhead sea turtles on board the fishing vessel and, because of their great size, no leatherback sea turtles were brought on board. On these occasions, the turtles were pulled close to the boat and the gangions were then cut to free the turtles with the hooks still attached to them; however the length of the line remaining on the turtle was not recorded. None of these turtles was measured or tagged, although some of the leatherback sea turtles were filmed on video. No addi- tional data and measurements, other than those presented in this study, were obtained. NOTE Kotas et al.: Incidental capture of Caretta caretta and Dermochelys coriacea by the pelagic longline fishery 395 Table 1 Data referring to fishing practices, sea surface temperature CO, and capture of loggerhead and leatherback sea turtles. CPUE = catch-per-unit-of-effort (number of captured turtles/1000 hooks). by trip. Trip Date No. of sets Average hooks/set Average sea surface temperature Loggerheads Leatherbacks Alive (tagged) Dead Condition not recorded CPUE Condition Alive Dead not recorded CPUE 1 13 Mar 98- 12 Apr 98 10 1030 13.6 84(17) 15 9 10.49 1 — 0.10 2 15 Jun 98- 5 Jul 98 13 992 21.4 28(12) 4 2.48 13 1 1.09 3 28 Sep 98- 13 Oct 98 11 950 18.9 5(5) _ _ 0.48 5 0.48 Total 34 990 117(34) 19 9 4.31 19 1 — 0.59 CPUE (number of captured turtles/1000 hooks) was calculated separately for each species. Straight carapace lengths in published data were converted to CCL by us- ing the formula in Teas (1993) to compare the CCL of captured loggerhead sea turtles to carapace length data found in the literature. To assess the significance of the difference in the proportion of dead loggerhead or leath- erback sea turtles among trips, exact tests were applied, because ordinary chi-square tests are not reliable when expected cell frequencies are too small. The test statistics were x2 = ^[(Observed - Expected )'2IExpected] , and exact probabilities were computed for all tables with marginal frequencies fixed at the observed values (Lindgren, 1993, p. 376). These probability calculations were performed by a Turbo Pascal vers. 7 program (Borland International. Scotts Valley, CA). The confidence interval for overall prob- ability of death at capture was calculated by the method in Zar ( 1996, p. 524 ). Ordinary chi-square tests and analysis of variance (ANOVA) tests followed Zar (1996) and were carried out with the software Systat vers. 9 (SPSS Inc., Chicago, IL). In the statistical tests, type-I error a was equal to 0.05. In the construction of Figure 2, to avoid overlapping of data points, the temperatures (but not the CPUEs) were jittered, that is, a small amount of uniform random noise was added to the temperature measure- ments (Cleveland, 1993). Results From a total of 34 sets and 33,650 hooks, 145 logger- head (CPUE = 4. 31/1000 hooks) and 20 leatherback (CPUE = 0. 59/1000 hooks) sea turtles were captured. There was a significant difference in loggerhead CPUE among the trips (chi-square test, x2=137.3, P<0.001), but the proportion of dead loggerhead sea turtles was not sig- nificantly different among the trips (exact test, P= 0.656). The average probability of death at capture for loggerhead sea turtles for the three trips was 0.140 (95% confidence interval= [0.086, 0.210]). For leatherback sea turtles, the Table 2 Curved carapace length (CCL, cm) for loggerhead sea turtles, by trip. Sample Average Standard Trip size CCL deviation Minimum Maximum 1 19 56.9 7.3 46.0 70.0 2 30 57.2 7.5 46.0 68.0 3 5 67.0 5.9 58.0 73.0 Total 54 58.0 7.7 46.0 73.0 difference in CPUE among the trips was significant (chi- square test, x2=9.76, P<0.01), and the proportion of dead leatherback sea turtles was not significantly different among the trips (exact test, P=1.000). The average prob- ability of death at capture for leatherback sea turtles for the three trips was 0.050 ( 95r>i confidence interval= [0.001, 0.249]). The average sea surface temperature (Table 1) was significantly different among the trips (ANOVA, ;? = 34. F=55.37, P<0.001). The average temperature on the first trip was significantly lower than those on the second and third trips, and the average temperature on the second trip was significantly higher than that on the third trip (Tukey's post hoc test). For loggerhead sea turtles, CPUEs were generally higher on the first trip, which had the lowest average temperature (Fig. 2). For leatherback sea turtles, on the contrary, the lowest CPUEs were found on the first trip, on which only one leatherback sea turtle was captured (Table 1). CCLs of captured loggerheads were in the range of 46-73 cm. Detailed loggerhead CCL data are presented in Table 2. There was a significant difference in average log- gerhead CCL among the trips (Table 2); the average CCL on the third trip was greater than those on the first and second trips (ANOVA, n = 54, F=4.209, P=0.020, Tukey's 396 Fishery Bulletin 102(2) D a. O 25 20 5 - Caretta O O o — r- — l— 15 — r~ 25 Temperature (:C) Figure 2 CPUE (number of captured turtles/1000 hooks I by sea surface temperature (°C) in each set, by species. Circles = first trip, triangles = second trip, stars = third trip. In each graph, the dashed vertical line, arbitrarily placed at 16.7 °C, marks a separation between the temperatures in the first trip and those in the second and third trips (except for one set in the first trip). Note that the two graphs have different vertical scales, and that, in the construction of this figure, temperature measurements (but not the CPUEsl have been jittered (see "Materials and methods" section i. post hoc test). Although leatherback sea turtles could not be hauled aboard for measurements, on board observa- tions and video recordings indicated that they were sub- adult or adult animals. Most of the loggerhead turtles were hooked through their mouths or esophagus, but a small number were hooked through their flippers or were found to be simply entangled in the lines. Loggerhead sea turtles taken aboard had their hooks removed, sometimes in a care- less way that caused severe injury, and they were then returned to the sea. Leatherback sea turtles were found entangled in the lines or hooked either through the flippers or carapace or through the mouth. Because no leatherback sea turtle was hauled aboard, we could not tell if any were hooked in the esophagus. Discussion Achaval et al. (2000 ) reported data obtained from nine trips aboard two different longline vessels operating within the Uruguayan EEZ and in international waters in the South Atlantic in different seasons of the year, and employing different longline methods. Those authors reported that 28 loggerhead and 28 leatherback sea turtles were cap- tured in 86 sets with 75.033 hooks in zones I and II, that correspond approximately to the fishing area covered in this study, yielding a CPUE of 0.37/1000 hooks for both loggerhead and leatherback sea turtles. For loggerhead sea turtles, there was a significant difference between our CPUE (Table 1) and that of Achaval et al. (chi-square test, X2=226.4, P<0.001 ); whereas for leatherback sea turtles no significant difference was found i chi-square test, x2=1.97, P=0.161). Although the variations in CPUE observed in our study could be explained by differences in temperatures (Fig. 2), other physical, spatial, or temporal factors (or a combina- tion of these factors) could be involved. The trips were car- ried out at different times of the year (Table 1); the third trip was more to the south and closer to the coast, and the first trip had sets more to the east (Fig. 1). Our estimates of sea turtle mortality at capture may be lower than the actual mortality rates from longlines because our estimates do not consider postrelease deaths derived from 1) wounds caused by hooks removed from turtles on board, 2) embedded hooks and lines, and 3) stress caused by capture itself. Other researchers have also recognized that, because of factors such as these, there is great uncertainty in the estimates of mortality levels for sea turtles captured in longline gear (Balazs and Pooley, 1994; Eckert, 1994). Captured loggerhead sea turtles were smaller (Table 2 1 than loggerhead sea turtles nesting in Brazil (minimum CCL = 83.0 cm, average CCL= 103.0 cm, nesting season 1982-83 through nesting season 1999-2000; Projeto TAMAR3) and in several places in the North Atlantic and the Caribbean (minimum CCL=75.4 cm, average CCL in the range of 94.0-105.1 cm; Dodd, 1988). However, logger- head sea turtles nesting in Cape Verde, in the northeast- ern Atlantic, are smaller than those nesting in those other places: minimum CCL = 68.0 cm, average CCL=82.9 cm. data from 1998 (Cejudo et al., 20001. There is an overlap between the observed CCL range and that of adult Cape Verde loggerhead sea turtles (seven loggerhead turtles out of 54 observed, or 13.0'7f , had a CCL equal or greater than the minimum Cape Verde CCL), but the average CCL of the captured loggerhead sea turtles (Table 2 i was well below that of loggerhead sea turtles nesting in Cape Verde. We estimate that the captured loggerhead sea turtles were generally juveniles, although a small number of them could have been adult turtles. However, size is 3 Projeto TAMAR. 2000. Unpubl. data Salvador, BA 40210-970, Brazil. Caixa Postal 2219. NOTE Kotas et al.: Incidental capture of Caretta caretta and Dermochelys conacea by the pelagic longline fishery 397 not a reliable indicator of maturity or breeding status for sea turtles (Miller, 1997). Along the southern coast of Brazil (between latitudes 23°S and 33°S), loggerhead sea turtles stranded or in- cidentally captured in fishing gear with CCLs as small as 32.5 cm have been observed (Projeto TAMAR4), but usually loggerhead sea turtles found in that region have CCLs greater than 50 cm, most commonly in the range of 60-90 cm (Pinedo et al., 1998; Bugoni et al., 2001; Projeto TAMAR4 ). Loggerhead sea turtles have also been found in Uruguay and Argentina (Frazier, 1984; Fallabrino et al., 2000). Their CCLs in those countries have been reported to be in the approximate range of 50-115 cm (Frazier, 1984). The loggerhead sea turtles reported here have an average CCL smaller than that usually observed for loggerhead sea turtles stranded or captured in southern Brazil, Uruguay, and Argentina, although most of the turtles (45 out of 54, or 83%) had CCLs equal to or greater than 50 cm, that is, they were within the size range for that region. Cumulative evidence obtained from genetic and size- distribution data around oceanic basins, as well as tag returns, shows that the ontogenetic development of log- gerhead sea turtles involves a pelagic juvenile stage (Carr, 1987; Musick and Limpus, 1997; Bolten et al., 1998). Trans- oceanic developmental migrations establishing a link be- tween juveniles in feeding grounds and hatchlings from nesting beaches on opposite sides of the ocean basin have been demonstrated through genetic analysis for the North Atlantic and North Pacific (Bowenet al., 1995; Bolten et al., 1998). It has been suggested that a similar pattern may be expected for the South Atlantic (Bolten et al., 1998), where loggerhead sea turtles nest in Brazil and possibly in Africa (Marcovaldi and Laurent, 1996; Fretey, 2001). The inciden- tal captures reported in our study, indicating the use of the pelagic environment by juvenile loggerhead sea turtles in the South Atlantic, support the hypothesis of transoceanic developmental migrations for those turtles in that ocean. Future genetic analysis of turtles incidentally captured in the South Atlantic would help to clarify their natal origin. For leatherback sea turtles, there are important nesting grounds in the Atlantic, mainly in French Guiana and Su- riname in South America, and Gabon and Congo in Africa (Spotila et al., 1996; Fretey, 2001). Leatherback sea turtles are known to travel long distances from their nesting beaches into pelagic waters (Goff et al., 1994; Morreale et al., 1996; Eckert and Sarti, 1997; Eckert, 1998). Satellite telemetry data indicate that leatherback sea turtles nest- ing in eastern South Africa can enter the South Atlantic (Hughes et al., 1998; Hughes5). In the southwestern Atlan- tic, leatherback sea turtles have been observed or captured in Brazil, Uruguay, and Argentina (Frazier, 1984; Pinedo et al., 1998; Achaval et al., 2000; Fallabrino et al., 2000; Bugoni etal., 2001). Some measure of the significance of the three trips re- ported in the present study in terms of the potential for turtle capture and mortality in the South Atlantic longline fishery can be obtained by looking at information concern- ing the total fishing effort in the study area. In 1999, the Brazilian longline fleet consisted of 70 longliners (42 Bra- zilian and 28 leased foreign vessels); among them, 33 ves- sels were operating out of ports in southern Brazil, in the states of Sao Paulo, Santa Catarina, and Rio Grande do Sul. In that year, the total number of hooks of that long- line fleet (both Brazilian and leased vessels) amounted to 13,598,260 hooks (ICCAT6). However, the southwestern Atlantic is fished not only by Brazil-based longliners, but also by longliners from Uruguay, Chile, Japan, Taiwan, and Spain (Folsom, 1997; Weidner and Arocha, 1999; Weidner et al., 1999). According to ICCAT's (International Commis- sion for the Conservation of Atlantic Tunas) CATDIS data set (ICCAT)7 longliners operating during 1995-97 in the area delineated by the present study (latitudes 25°S and 35°S and longitude 35°W, or eight ICCAT 5x5° statistical blocks. Fig. 1) had an average annual catch of tunas and swordfishes of 6885 metric tons (t) (the total hold capacity of the vessels on the three trips reported in this study was 130 t). However, due to unreported landings by vessels flying flags of convenience (FAO, 2001; FAO8) and other sources, the estimate obtained from ICCAT data should be considered a minimum estimate of the total annual tuna and swordfish catch ( ICCAT9 ). Furthermore, because North Atlantic stocks of swordfishes and some species of tuna are considered overfished (NMFS10), quota or closure regula- tions (or both) in the North Atlantic may be driving longline fleets to the South Atlantic, increasing the risk of incidental capture of sea turtles there. In Brazil, sea turtle capture is prohibited by federal legislation (Marcovaldi and Marcovaldi, 1999), and mea- sures have been taken to address the problem of inci- dental capture by longlines and other kinds of fishing 4 Projeto TAMAR. 2000. Unpubl. data. Rua Antonio Athanasio 273, Ubatuba, SP 11680-000, Brazil. 5 Hughes, G. R. 2002. Personal commun. Ezemvelo KZN Wildlife, P O Box 13053, Cascades 3202, South Africa. 6 ICCAT (International Commission for the Conservation of Atlantic Tunas). 2001. National report of Brazil. Report for biennial period, 2000-2001, part I (2000), vol. 1, English version, p. 312-315. Calle Corazon de Maria, 8, 28002 Madrid, Spain. 7 ICCAT (International Commission for the Conservation of Atlantic Tunas). 2002. CATDIS dataset. Calle Corazon de Maria, 8, 28002 Madrid, Spain. (Available from http://www. iccat.org.] s FAO (Food and Agriculture Organization of the United Nations). 2001. International plan of action to prevent, deter and eliminate illegal, unreported and unregulated fish- ing, 24 p. FAO, Rome. (Available from http://www.fao.org/ docrep/003/yl224e/yl224e00.htm.] 9 ICCAT (International Commission for the Conservation of Atlantic Tunas). 1999. Detailed report for swordfish, ICCAT SCRS swordfish stock assessment session (Madrid, Spain, September 27 to October 4, 1999), 176 p. Calle Corazon de Maria, 8, 28002 Madrid. Spain. 10 NMFS (National Marine Fisheries Service). 2000. 2000 stock assessment and fishery evaluation for Atlantic highly migratory species, 150 p. U.S. Dep. Commer., NOAA, NMFS. Highly Migratory Species Management Division, 1315 East- West Highway, Silver Spring, MD 20910. 398 Fishery Bulletin 102(2) gear. Since 2001, Projeto TAMAR has been developing and implementing (through partnerships with other institu- tions) an action plan whose main objective is to reduce incidental sea turtle capture, including captures occurring in the open sea (Marcovaldi et al., 2002). The action plan includes, among other things, an assessment of fishery- related sea turtle mortality, the development of mitigation methods, and a proposal of adequate conservation and enforcement policies (Marcovaldi et al., 2002). However, because the longline fleet is composed of vessels from many nations, the reduction of incidental capture in the open sea calls for international cooperation ( Eckert and Sarti, 1997; Trono and Salm, 1999; Crowder, 2000). The observations reported in this study and the pres- ence of a sizable longline fleet operating in the southwest- ern Atlantic indicate 1) the need for research to clarify habitat use by sea turtles in that part of the ocean (Eckert and Sarti, 1997; Bolten et al., 1998), 2) the need for contin- ued research to quantify the impact of longline fishing on sea turtles in the pelagic realm of that ocean (Balazs and Pooley, 1994; Eckert, 1994), and 3) the implementation of conservation measures for sea turtles in that environment. We suggest the implementation of an International Ob- servers Program on board longliners operating throughout the South Atlantic ocean. Acknowledgments This note is the result of observations made possible through an agreement between the REVIZEE Program (National Program for the Assessment of the Sustain- able Fishing Potential of the Exclusive Economic Zone Live Resources, a Brazilian Government program) and Projeto TAMAR's station at Ubatuba, State of Sao Paulo. We would like to thank Jose Kowalsky of the Kowalsky fishing company and Marcelino Talavera (Itajai, State of Santa Catarina), owners of the vessels Yamaya III and Basco, respectively, for kindly allowing access to the fish- ing vessels, and the crew of the two longliners, and also the fishing research center Centro de Pesquisa e Extensao Pesqueira do Sudeste-Sul-CEPSUL/IBAMA ( Itajai, State of Santa Catarina), and particularly Jorge Almeida de Albuquerque, for making this research possible. We also thank Larisa Avens and Matthew Godfrey for their gener- ous reviews of the paper, and the two anonymous referees, whose suggestions helped to improve our work. Projeto TAMAR is affiliated with IBAMA (the Brazilian Institute for the Environment and Renewable Natural Resources), is co-managed by Fundacao Pro-TAMAR, and officially sponsored by Petrobras. In Ubatuba. TAMAR is supported by Ubatuba's municipal government ( Prefeitura Municipal de Ubatuba). S.S. and V.G.A. were supported by CNPq (Brazilian National Research Council). Literature cited Achaval, F., Y. H. Marin, and L. C. Barea. 2000. Captura incidental de tortugas con palangre pela- gico oceanico en el Atlantico sudoccidental. In Captura de grandes peces pelagicos (pez espada y atunesi en el Atlan- tico Sudoccidental. y su interaccion con otras poblaciones (G. Arena and M Rey, eds.), p. 83-88. Instituto Nacional de Pesca, Programa de las Naciones Unidas para el Desar- rollo, Montevideo. Uruguay. Balazs, G. H., and S. G Pooley (comps.). 1994. Research plan to assess marine turtle hooking mor- tality: results of an expert workshop held in Honolulu. Hawaii, November 16-18, 1993. NOAA Tech. Memo. NOAA-TM-NMFS-SWFSC-201, 166 p. Bolten, A. B., K. A. Bjorndal. H. R. Martins, T. Dellinger, M. J. Biscoito, S. E. Encalada, and B. W. Bowen. 1998. Transatlantic developmental migrations of log- gerhead sea turtles demonstrated by mtDNA sequence analysis. Ecol. Appl. 8:1-7. Bolten, A. B., J. A. Wetherall, G. H. Balazs. and S. G. Pooley (comps.). 1996. Status of marine turtles in the Pacific Ocean relevant to incidental take in the Hawaii-based pelagic longline fishery. NOAA Tech. Memo. NOAA-TM-NMFS-SWFSC- 230, 167 p. Bowen, B. W.. F. A. Abreu-Grobois, G. H. Balazs, N. Kamezaki. C. J. Limpus, and R. J. Ferl. 1995. Trans-Pacific migrations of the loggerhead turtle [Caretta caretta) demonstrated with mitochondrial DNA markers. Proc. Natl. Acad. Sci. USA 92: 3731-3734. Bugoni, L., L. Krause, and M. V. Petrv. 2001. Marine debris and human impacts on sea turtles in southern Brazil. Mar. Pollut. Bull. 42:1330-1334. Carr, A. 1987. New perspectives on the pelagic stage of sea turtle development. Conserv. Biol. 1: 103-121. Cejudo, D., I. Cabrera, L. F Lopez-Jurado. C. Evora, and P. Alfama. 2000. The reproductive biology of Caretta caretta on the Island of Boavista ( Republic of Cabo Verde. Western Afri- ca). NOAA Tech. Memo. NMFS-SEFSC-443:244-245. Cleveland, W. S. 1993. Visualizing data, 360 p. Hobart Press, Summit, NJ. Crowder. L. 2000. Leatherback's survival will depend on an interna- tional effort. Nature 405:881. Dodd, C. K., Jr. 1988. Synopsis of the biological data on the loggerhead sea turtle Caretta caretta (Linnaeus 1758). U.S. Fish Wildl. Serv. Biol. Rep. 88. 110 p. Eckert, S. A. 1994. Evaluating the post-release mortality of sea turtles incidentally caught in pelagic longline fisheries. NOAA Tech. Memo. NMFS-SWFSC-201:106-110. 1998. Perspectives on the use of satellite telemetry and other electronic technologies for the study of marine turtles, with reference to the first year long tracking of leatherback sea turtles. NOAA Tech. Memo. NMFS- SEFSC-415:44-46. Eckert, S. A., and M. L. Sarti. 1997. Distant fisheries implicated in the loss of the world's largest leatherback nesting population. Mar. Turtle Newsl. 78:2-7. Fallabrino. A., A. Bager. A. Estrades, and F Achaval. 2000. Current status of marine turtles in Uruguay. Mar. Turtle Newsl. 87:4-5. FAO (Food and Agriculture Organization of the United Nations). 2001. Report of the second technical consultation on illegal, NOTE Kotas et al.: Incidental capture of Caretta caretta and Dermochelys conacea by the pelagic longlme fishery 399 unreported and unregulated fishing. Rome, 22-23 Febru- ary 2001. FAO Fisheries Rep. 646, 38 p. FAO, Rome. Folsom, W. B. 1997. World swordfish fisheries: an analysis of swordfish fisheries, market trends and trade patterns, vol. VI: Western Europe. NOAA Tech. Memo. NMFS-F/SPO-29, 324 p. Frazier, J. 1984. Las tortugas marinas en el Atlantico sur occidental. In Serie divulgacidn no. 2, p. 2-21. Associacion Herpeto- logica Argentina, La Plata, Argentina. Fretey, J. 2001. Biogeography and conservation of marine turtles of the Atlantic coast of Africa. CMS Technical Series Publ. no. 6, 429 p. UNEP/CMS Secretariat. Bonn. Germany. Goff, G. P., J. Lien, G. B. Stenson. and J. Fretey. 1994. The migration of a tagged leatherback turtle, Der- mochelys coriacea, from French Guiana, South America, to Newfoundland, Canada, in 128 days. Can. Field Nat. 108:72-73. Hughes, G. R., P. Luschi, R. Mencacci, and F. Papi. 1998. The 7000 km oceanic journey of a leatherback turtle tracked by satellite. J. Exp. Mar. Biol. Ecol. 229: 209-217. IUCN (International Union for Conservation of Nature and Natural Resources! Species Survival Commission. 1995. A global strategy for the conservation of marine turtles, 25 p. IUCN, Gland, Switzerland. Lindgren, B. W. 1993. Statistical theory, 4th ed., 633 p. Chapman & Hall, New York, NY. Lutcavage, M. E., P. Plotkin, B. Witherington, and P. L. Lutz. 1997. Human impacts on sea turtle survival. In The biol- ogy of sea turtles (P. L. Lutz and J. A. Musick, eds.), p. 387-409. CRC Press, Boca Raton, FL. Marcovaldi, M. A., and A. Laurent. 1996. A six season study of marine turtle nesting at Praia do Forte, Bahia, Brazil, with implications for conservation and management. Chelonian Conserv. Biol. 2:55-59. Marcovaldi, M. A., and G. G. dei Marcovaldi. 1999. Marine turtles of Brazil: the history and structure of Projeto TAMAR-IBAMA. Biol. Conserv. 91:35-41. Marcovaldi, M. A., J. C. Thome, G. Sales, A. C. Coelho. B. Gallo. and C. Bellini. 2002. Brazilian plan for reduction of incidental sea turtle capture in fisheries. Mar. Turtle Newsl. 96:24-25. Miller. J. D. 1997. Reproduction in sea turtles. In The biology of sea turtles (P. L. Lutz and J. A. Musick, eds.), p. 51-81. CRC Press, Boca Raton, FL. Morreale, S. J., E. A. Standora, J. R. Spotila, and F. V. Paladino. 1996. Migration corridor for sea turtles. Nature 384: 319-320. Musick, J. A., and C. J. Limpus. 1997. Habitat utilization and migration in juvenile sea turtles. In The biology of sea turtles (P. L. Lutz and J. A. Musick, eds.), p. 137-163. CRC Press, Boca Raton, FL. NRC (National Research Council I. 1990. Decline of the sea turtles: causes and prevention, 259 p. National Academy Press, Washington, D.C. Nishemura, W„ and S. Nakahigashi. 1990. Incidental capture of sea turtles by Japanese research and training vessels: results of a questionaire. Mar. Turtle Newsl. 51:1-4. Oravetz. C. A. 1999. Reducing incidental catch in fisheries. In Research and management techniques for the conservation of sea turtles (K. L. Eckert, K. A. Bjorndal, F. A. Abreu-Grobois. and M. Donnelly, eds.). p. 189-193. IUCN SSC Marine Turtle Specialist Group publication no. 4. Pinedo, M. C, R. Capitoli, A. S. Barreto, and A. L. V. Andrade. 1998. Occurrence and feeding of sea turtles in southern Brazil. NOAA Tech. Memo. NMFS-SEFSC-412:117- 118. Spotila, J. R.. A. E. Dunham, A. J. Leslie. A. C. Steyermark, P. T. Plotkin, and F.V. Paladino. 1996. Worldwide decline of Dermochelys coriacea: are leatherback turtles going extinct? Chelonian Conserv. Biol. 2:209-222. Teas, W. G. 1993. Species composition and size class distribution of marine turtle strandings on the Gulf of Mexico and southeast United States coasts, 1985-1991. NOAA Tech. Memo. NMFS-SEFSC-315, 43 p. Tobias, W. 1991. Turtles caught in Caribbean swordfish net fishery. Mar. Turtle Newsl. 53:10-12. Trono, R. B., and R. V. Salm. 1999. Regional collaboration. In Research and manage- ment techniques for the conservation of sea turtles (K. L. Eckert, K. A. Bjorndal, F. A. Abreu-Grobois, and M. Donnelly, eds.), p. 224-227. IUCN SSC Marine Turtle Specialist Group publication no. 4. Weidner, D. M„ and F. Arocha. 1999. World swordfish fisheries: an analysis of swordfish fisheries, market trends and trade patterns. Vol. IV, Part A2b: Brazil. NOAA Tech. Memo. NMFS-F/SPO- 35:237-628. Weidner, D. M.. F. J. Fontes, and J. Serrano. 1999. World swordfish fisheries: an analysis of swordfish fisheries, market trends and trade patterns. Vol. IV part A2c: Uruguay, Paraguay and Argentina. NOAA Tech. Memo. NMFS-F/SPO-36:631-916. Williams, P., P. J. Anninos, P. T. Plotkin, and K. L. Salvini (comps.l. 1996. Pelagic longline fishery — sea turtle interactions. Proceedings of an industry, academic and government experts, and stakeholders workshop held in Silver Spring, Maryland. 24-25 May 1994. NOAA Tech. Memo. NMFS- OPR-7, 77 p. Zar, J. H. 1996. Biostatistical analysis, 3rd ed., 662 p. Prentice Hall, LTpper Saddle River, NJ. 400 Diet changes of Pacific cod (Gadus macrocephalus) in Pavlof Bay associated with climate changes in the Gulf of Alaska between 1980 and 1995 Mei-Sun Yang Alaska Fisheries Science Center National Marine Fisheries Service, NOAA 7600 Sand Point Way NE Seattle. Washington 98115 E mail address mei-sunyangffinoaa gov The diet of Pacific cod (Gadus mac- rocephalus) in the area of Pavlof Bay, Alaska, was studied in the early 1980s by Albers and Anderson (1985). They found that the dominant prey spe- cies were forage species like pandalid shrimp, capelin iMallotus villosus), and walleye pollock [Theragra chal- cogramma). The shrimp fishery in Pavlof Bay began in 1968 and closed in 1980 because of low shrimp abun- dance (Ruccio and Worton1). Survey data indicate that, during the period between 1972 and 1997, the abun- dance of forage species such as pan- dalid shrimp and capelin declined and higher trophic-level groundfish such as Pacific cod increased. There is a general recognition that a long- term ocean climate shift in the Gulf of Alaska has been partially responsible for the observed reorganization of the community structure (Anderson and Piatt, 1999). Because there has been an appar- ent shift in the abundance of both predators and prey in Pavlof Bay, it is important to understand how trophic relationships may also have changed. 1 Ruccio, M. P.. and C. L. Worton. 1999. Annual management report for the shell- fish fisheries of the Alaska peninsula area, 1998. In Annual management report for the shellfish fisheries of the westward region. 1998. Regional Infor- mation Report 4K99-49, 312 p. Alaska Department of Fish and Game, Division of Commercial Fisheries, 211 Mission Road, Kodiak, Alaska 99615. In order to partially address this ques- tion, stomach samples of Pacific cod and other groundfishes were taken in 1995. By performing a comparison of the diet of Pacific cod right after the climate shift with Pacific cod and other groundfishes well after the shift, this analysis may demonstrate how the relative abundance of prey in the Gulf of Alaska may have changed. Methods Stomachs of Pacific cod, walleye pol- lock, and arrowtooth flounder (Atheres- thes stomias) were collected by National Marine Fisheries Service (NMFS) sci- entists on board the chartered vessel FV Arcturus conducting a trawl survey in Pavlof Bay, Alaska. (Fig. 1) from 5 August to 7 August 1995. The survey targeted shrimp and used a high- opening net with small mesh (32-mm stretched mesh). Each tow was about 1.2 km in length. The average depth of the 13 hauls where stomachs were col- lected was 108.9 (±9.5) m with a range from 90 to 123 m. When a sampled stomach was retained, it was put in a cloth stomach bag. A field tag with the species name, fork length (FL in cm) of the fish, and haul data (vessel, cruise, haul number, specimen number) was also put in the bag. All the samples col- lected were then preserved in buckets containing a 10% formalin solution. When the samples arrived at the labo- ratory, they were transferred into 70% ethanol before the stomach contents were analyzed. In the laboratory, the stomach was cut open, the contents were removed and blotted with a paper towel. Wet weight was then recorded to the nearest 0.1 g. After obtaining the total weight for a stomach's contents, the contents were placed in a Petri dish and examined under a microscope. Each prey item was classified to the lowest practical taxonomic level. Prey weights and numbers of commercially important fish were recorded. Stan- dard lengths of prey fish and carapace width of crabs were also recorded. The diet of Pacific cod was summarized to show the percent frequency of occur- rence, the percentage by number, and the percentage of the total weight of each prey item found in the stomachs. Stomach contents of walleye pollock and arrowtooth flounder were ana- lyzed for comparisons. Results Of 130 Pacific cod stomachs analyzed. 129 contained food. Pacific cod sizes ranged from 40 to 80 cm FL (fork length); a mean size was 55.4 (SD ±7.2) cm. Polychaetes, crangonid shrimp, pea crab, and clams were the most fre- quently found prey items in Pacific cod stomachs (Table 1). However, in terms of weight, eelpouts (zoarcids). Tanner crab (Chionoecetes bairdi), crangonid shrimp, hermit crab, and polychaetes were the most important prey of Pa- cific cod. Pandalid shrimp, spinyhead sculpin (Dasycottus setiger), prickle- backs (stichaeid). Pacific sandlance (Ammodytes hexapterus), arrowtooth flounder lAtheresthes stomias), and flathead sole (Hippoglossoides elasso- don ) were minor prey. Invertebrates (mainly crangonid shrimp, polychaetes, and crabs) were the principal prey of Pacific cod smaller than 60 cm (Fig. 2). There were nine prey categories as shown in Figure 2. The miscellaneous prey included Si- puncula, Echiura. fish offal (processed Manuscript approved for publication 2 I I Vcember 2003 by Scientific Editor. Manuscript received 20 January 2004 at NMFS Scientific Publications Office. Fish. Bull. 102:400-405(2004). NOTE Yang: Diet changes of Gadus macrocephalus associated with climate changes in Pavlof Bay 401 Figure 1 Location of study area in 1980, 1981, and 1995. 100 fish parts like head, tail, pyloric caeca, etc.), and all other prey organisms not included in the other eight prey catego- ries. The importance of fish in the diet of Pacific cod increased after 60 cm FL. Walleye pollock were consumed only by Pacific cod >60 cm FL. In general, Pacific cod ate prey of small individual size (Table 2). Tanner crabs iChionoecetes bairdi) ranged from 4.5 to 42.3 mm carapace width. Eelpouts ranged in length from 36.2 to 256.6 mm standard length. Other fish prey ranged in length from 32.7 to 81.5 mm. Walleye pollock were consumed by Pacific cod but were not measurable. In 1995, when Pacific cod stomachs were collected in Pavlof Bay, 218 wall- eye pollock and 80 arrowtooth flounder stomachs were also collected. Similar to the results for Pacific cod, pandalid shrimp and capelin were not important food of walleye pollock and arrowtooth flounder either (Fig. 3). These prey each comprised less than 39c of the total stomach content weight of walleye pol- lock and arrowtooth flounder. Instead, eelpouts, pricklebacks, euphausiids, and walleye pollock were important food of arrowtooth flounder, and euph- ausiids (83% by weight) were the main food of walleye pollock. N=26 40-49 □ Polychaete □ Pollock □ Misc. fish □ Tanner crab S Pagurid □ Other crab El Pandalid O Crangonid □ Misc. prey 50-59 Predator fork length (cm) 60-80 Figure 2 Variations in the main food items of Pacific cod, by predator size, in Pavlof Bay in 1995. /i=sample size. Discussion This study shows that eelpouts hermit crabs, polychaetes, and , Tanner crabs, crangonids, echiuroids were the princi- 402 Fishery Bulletin 102(2) Table 1 Percent frequency of occurrence (%F), percentage by number (7cN), and percentage by weight C7rW) of prey items of Pacific cod collected in Pavlof Bay, Alaska, 1995. Prey name 9c F %N %W Polychaeta (worm) 79.8 11.4 9.2 Gastropoda (snail) 14.0 0.8 0.4 Bivalvia (clam) 55.0 6.1 2.1 Cephalopoda (squid and octopus) 10.1 0.5 2.1 Copepoda 0.8 0.0 0.0 Peracarida Mysidacea (mysid) 31.8 11.5 0.2 Cumacea (cumaceanl 13.2 0.9 0.0 Amphipoda (amphipod) 17.1 1.2 0.0 Euphausiacea leuphausiid) 15.5 10.0 0.7 Natantia (unidentified shrimp) 12.4 0.7 0.1 Caridea (shrimp) 12.4 1.2 1.2 Hippolytidae (shrimp) 17.8 1.2 0.2 Pandalidae (shrimp) 41.1 5.7 2.3 Crangonidae (shrimp) 76.0 18.9 13.3 Reptantia (unidentified crab) 11.6 0.5 1.9 Paguridae (hermit crab) 22.5 1.3 9.5 Decapoda Brachyura (crab) 0.8 0.0 0.1 Hyas sp. (lyre crab) 0.8 0.0 0.9 Hyas lyratus (lyre crab) 1.6 0.1 0.6 Chionoecetes sp. (snow and Tanner crab) 40.3 3.5 13.9 Pinnotheridae (pea crab) 1.6 0.1 0.1 Pinnixa sp. (pea crab) 68.2 8.4 3.2 Sipuncula I marine worm) 0.8 0.0 0.6 Echiura (marine worm) 24.0 1.4 6.6 Ophiuroidea (basket and brittle star) 9.3 0.7 0.1 Chaetognatha (arrow worm) 1.6 0.2 0.0 Rajidae (skate) 2.3 0.1 0.4 Osteichthyes Teleostei (fish) 12.4 1.1 0.6 Nongadoid fish remains 47.3 6.5 2.3 Gadidae (unidentified) 1.6 0.1 0.4 Theragra chalcogramma (walleye pollock) 2.3 0.1 1.4 Zoarcidae (eelpout) 16.3 0.9 14.0 Cottoidei (Sculpim 2.3 0.1 0.2 Dasycottus setiger (spinyhead sculpin) 0.8 0.0 0.2 Stichaeidae (prickleback) 8.5 1.5 0.6 Lumpenus sp. (prickleback) 0.8 0.0 0.0 Ammodytes hexapterus (Pacific sand lance) 0.8 0.0 0.0 Pleuronectidae (flatfish) 2.3 0.2 0.2 Atheresthes stomias (arrowtooth flounder) 1.6 0.1 0.0 Hippoglossoides elassodon (flathead sole) 3.9 0.2 0.5 Unidentified organic material 10.1 0.5 0.6 Unidentified worm-like organism 5.4 0.3 0.5 Fish offal (processed fish parts, e.g., head, tail) 0.8 0.0 8.1 Total prey weight 2715 g Total stomachs 1.30 Total empty stomachs 1 NOTE Yang: Diet changes of Gadus macrocephalus associated with climate changes in Pavlof Bay 403 D Pacific cod G Walleye pollock ■ Arrowtooth flounder Main prey items Figure 3 Percentage by weight of the main prey in the diet of Pacific cod (n=129), walleye pollock (n= and arrowtooth flounder 0.1 kg/km ). The data from this study corroborates Anderson's (2000) results. Anderson (2000) also reported that during the period of the decline of pandalid shrimp in inshore waters of the Gulf of Alaska, the abundance of some pleuronectids, Pacific cod, and walleye pollock increased. These species are predators of pandalid shrimp (Yang and Nelson, 2000). One hypothesis is that predators keep pandalid shrimp populations low. Albers and Anderson (1985) suggested that cod predation was one reason for the failure of the pink shrimp stock to rebuild in Pavlof Bay. In the Northwest Atlantic. Lilly et al. (2000) showed that the large in- crease in shrimp biomass seen in the 1990s was related to the collapse of cod \Gctdus morhua) populations during the late 1980s and 1990s in the northeast Newfoundland shelf. The impact of cod on Barents Sea shrimp (P. borealis) was also reported by Berenboim et al. (2000). They found that when cod biomass is high, the shrimp frequency of occurrence in cod stomachs declines; there is a significant inverse correla- tion between the abundance of cod and shrimp. Tanner crabs consumed by Pacific cod in this study ranged from 5 to 42 mm carapace width (CW). In general, the size of Tanner crabs consumed in- creases as Pacific cod size increases. The size range of Tanner crabs con- sumed by Pacific cod in this study is similar to that (5-45 mm) found in Pacific cod stomachs in Albers and An- derson's ( 1985 ) study and is also similar to that (1-40 mm) found in Hunter's (1979) study near Kodiak Island. Jewett's ( 1978 ) Pacific cod diet study around Kodiak Island from 1973 to 1976 showed that Tanner crabs were the most frequent (37%) prey of Pa- cific cod; pandalid shrimp occurred in 8-10% of the stomachs examined from 1973 to 1975; and walleye pollock were found in 49r of the stomachs examined. The importance of Tanner crabs as food of Pacific cod in Jewett's ( 1978 1 study is coincident with our study. This study suggests that there were substantial differences between the diets of Pacific cod in Pavlof Bay be- tween the early 1980s and 1995. In the 1980s, pandalid shrimp and cap- elin were the main food of Pacific cod, whereas benthic species (polychaetes, NOTE Yang: Diet changes of Gadus macrocephalus associated with climate changes in Pavlof Bay 405 80 Figure 6 Pandalid shrimp consumed by Pacific cod sampled at different bottom depths (m) in Pavlof Bay in 1995, and in the Gulf of Alaska in 1990, 1993, and 1996. hermit crabs, Tanner crabs, and eelpouts) were the domi- nant food in 1995. This change was probably due to the climate shift from cold to warm in the Gulf of Alaska. Acknowledgments I would like to thank Paul Anderson, Troy Buckley, and Patricia Livingston for reviewing the manuscript and for their very helpful suggestions. I also want to thank the two anonymous reviewers for their comments and suggestions. Literature cited Albers W. D., and P. J. Anderson. 1985. Diet of the Pacific cod. Gadus macrocephalus. and predation on the northern pink shrimp, Pandalus borealis, in Pavlof Bay, Alaska. Fish. Bull. 83:601-610. Anderson, P. J . 2000. Pandalid shrimp as indicators of ecosystem regime shift. J. Northw. Atl. Fish. Sci. 27:1-10. Anderson, P. J„ and J. F. Piatt. 1999. Community reorganization in the Gulf of Alaska fol- lowing ocean climate regime shift. Mar. Ecol. Prog. Ser. 189:117-123. Berenboim, B. I., A. V. Dolgov, V. A. Korzhev, and N. A. Yaragina. 2000. The impact of cod on the dynamics of Barents Sea shrimp (Pandalus borealis) as determined by multispecies models. J. Northw. Atl. Fish. Sci. 27:69-75. Hunter, M. A. 1979. Food resource partitioning among demersal fishes in the vicinity of Kodiak Island, Alaska. M.S. thesis, 120 p. Univ. Washington, Seattle, WA. Jewett, S. C. 1978. Summer food of the Pacific cod, Gadus macrocepha- lus, near Kodiak Island, Alaska. Fish. Bull. 76:700-706. Lilly, G. R., D. G. Parsons, and D. W. Kulka. 2000. Was the increase in shrimp biomass on the North- east Newfoundland shelf a consequence of a release in predation pressure from cod? J. Northw. Atl. Fish. Sci. 27:45-61. Yang, M-S., and M. W. Nelson. 2000. Food habits of the commercially important ground- fishes in the Gulf of Alaska in 1990, 1993, and 1996. NOAA Tech. Memo. NMFS-AFSC-112, 174 p. 406 Fishery Bulletin 102(2) Superintendent of Documents Publications Order Form *5178 I | YES, please send me the following publications: Subscriptions to Fishery Bulletin for $55.00 per year ($68.75 foreign) The total cost of my order is $ Prices include regular domestic postage and handling and are subject to change. (Company or Personal Name) (Please type or print! 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DC 20402. Ii i- also available free in limited numbers to hi" arch institutions, Slate and Federal agencies, and in exchange lor other scientific publications U.S. Department of Commerce Seattle, Washington Volume 102 Number 3 July 2004 Fishery Bulletin Contents Articles 407—417 Abascal, Francisco J., Cesar Megina, and Antonio Medina Testicular development in migrant and spawning bluefin tuna (Thunnus thynnus (U) from the eastern Atlantic and Mediterranean 418-429 Bobko, Stephen J., and Steven A. Berkeley Maturity, ovarian cycle, fecundity, and age-specific parturition of black rockfish (Sebastes melanops) 430-440 Brock, Daniel J., and Timothy M. Ward Maori octopus (Octopus maorum) bycatch and southern rock lobster (Jasus edwardsii) mortality in the South Australian lobster fishery The conclusions and opinions expressed in Fishery Bulletin are solely those of the authors and do not represent the official position of the National Marine Fisher- ies Service INOAA) or any other agency or institution. The National Marine Fisheries Service (NMFSi does not approve, recommend, or endorse any proprietary product or pro- prietary material mentioned in this pub- lication. No reference shall be made to NMFS. or to this publication furnished by NMFS, in any advertising or sales pro- motion which would indicate or imply that NMFS approves, recommends, or endorses any proprietary product or pro- prietary material mentioned herein, or which has as its purpose an intent to cause directly or indirectly the advertised product to be used or purchased because of this NMFS publication. 441—451 Dawson, Stephen, Elisabeth Slooten, Sam DuFresne, Paul Wade, and Deanna Clement Small-boat surveys for coastal dolphins; line-transect surveys for Hector's dolphins (Cephalorhynchus hector/) 452—463 Laidig, Thomas E., Keith M. Sakuma, and Jason A. Stannard Description and growth of larval and pelagic juvenile pygmy rockfish (Sebastes wilsoni) (family Sebastidae) 464—472 McGarvey, Richard Estimating the emigration rate of fish stocks from marine sanctuaries using tag-recovery data 473-487 Roumillat, William A., and Myra C. Brouwer Reproductive dynamics of female spotted seatrout (Cynoscion nebutosus) in South Carolina Fishery Bulletin 102(3) 488-497 Taggart, S. James, Charles E. O'Clair, Thomas C. Shirley, and Jennifer Mondragon Estimating Dungeness crab (Cancer magister) abundance: crab pots and dive transects compared Companion papers 498-508 Tollit, Dominic J., Susan G. Heaslip, Tonya K. Zeppelin, Ruth Joy, Katherine A. Call, and Andrew W. Trites A method to improve size estimates of walleye pollock (Theragra chalcogramma) and Atka mackerel (Pleurogrommus monopterygius) consumed by pinnipeds: digestion correction factors applied to bones and otoliths recovered in scats 509-521 Zeppelin, Tonya K., Dominic J. Tollit, Katherine A. Call, Trevor J. Orchard, and Carolyn J. Gudmundson Sizes of walleye pollock (Theragra chalcogramma) and Atka mackerel (Pleurogrommus monopterygius) consumed by the western stock of Steller sea lions (Eumetopias /ubatus) in Alaska from 1999 to 2000 522-532 Tollit, Dominic J., Susan G. Heaslip, and Andrew W. Trites Sizes of walleye pollock (Theragra chalcogramma) consumed by the eastern stock of Steller sea lions (Eumetopias /ubatus) in Southeast Alaska from 1994 to 1999 533-544 Tremain, Derek M., Christopher W. Harnden, and Douglas H. Adams Multidirectional movements of sportfish species between an estuanne no-take zone and surrounding waters of the Indian River Lagoon, Florida 545-554 Wells, R. J. David, and Jay R. Rooker Distribution, age, and growth of young-of-the year greater amberjack (Seriola dumerili) associated with pelagic Sargassum Note 555-560 Hiroishi, Shingo, Yasutaka Yuki, Eriko Yuruzume, Yosuke Onishi, Tomoji Ikeda, Hironobu Komaki, and Muneo Okiyama Identification of formalin-preserved eggs of red sea bream (Pagrus ma/or) (Pisces: Spandae) using monoclonal antibodies 561 Subscription form 407 Abstract— Testis histological structure was studied in bluefin tuna {.Thunnus thynnus I from the eastern Atlantic and Mediterranean during the reproductive season (from late April to early Junei. Testicular maturation was investi- gated by comparing samples from bluefin tuna caught on their eastward reproductive migration off Barbate i Strait of Gibraltar area) with samples of bluefin tuna fished in spawning grounds around the Balearic Islands. Histological evaluations of cross sec- tions showed that the testis consists of two structurally different regions, an outer proliferative region where germ cells develop synchronously in cysts, and a central region made up of a well- developed system of ducts that convey the spermatozoa produced in the prolif- erative region to the main sperm duct. Ultrastructural features of the differ- ent stages of the male germ cell line are very similar to those described in other teleost species. The bluefin tuna testis is of the unrestricted spermatogonial testicular type, where primary sper- matogonia are present all along the germinative portion of the lobules. All stages of spermatogenesis were pres- ent in the gonad tissue of migrant and spawning bluefin tuna, although sper- matids were more abundant in spawn- ing fish. The testis size was found to increase by a factor of four ( on average ) during migration to the Mediterranean spawning grounds, whereas the fat bodies (mesenteric lipid stores associ- ated with the gonads) became reduced to half their weight, and the liver mass did not change significantly with sexual maturation. Linear regression analy- sis of the pooled data of migrant and spawning bluefin tuna revealed a sig- nificant negative correlation between the gonad index (IG) and the fat tissue index (IF), and a weaker positive cor- relation between the gonad index (IG) and the liver index (IL). Our analyses indicate that the liver does not play a significant role in the storage of lipids and that mesenteric lipid reserves con- stitute an important energy source for gametogenesis in bluefin tuna. Testicular development In migrant and spawning bluefin tuna {Thunnus thynnus (L.)) from the eastern Atlantic and Mediterranean Francisco J. Abascal Cesar Megina Antonio Medina Departamento de Biologia Facultad de Ciencias del Mar y Ambientales Universidad de Cadiz Av Republica Saharaui 11510 Puerto Real Cadiz, Spain E-mail address (for A Medina, contact author): antonio.medina@uca.es Manuscript submitted 27 April 2003 to Scientific Editor's Office. Manuscript approved for publication 25 March 2004 by the Scientific Editor. Fish. Bull. 102:407-417 (2004). The Atlantic northern bluefin tuna (Thunnus thynnus thynnus (L.)), is one of the most commercially valu- able wild animals in the world. In the last two decades this species has been subject to intense over-fishing, which has caused a decline in both the east- ern and western populations because of lowered recruitment (Mather et al., 1995; Sissenwine et al., 1998). The bluefin tunas (7! thynnus and T. maccoyii) are unique among tuna spe- cies in that they live mainly in cold waters and move into warmer waters to spawn (Olson, 1980; Lee, 1998; Schaefer, 2001); therefore the migra- tory pattern of these species depends substantially on reproduction. The eastern stock of Atlantic bluefin tuna spawns from June through August in the Mediterranean Sea. where natural conditions are apparently optimal for the survival of offspring. From late April to mid June, bluefin tuna breed- ing stocks migrate from the North Atlantic to spawning grounds in the Mediterranean (Mather et al., 1995; Ravier and Fromentin, 2001). A good understanding of the reproductive parameters (especially sexual matu- ration, fecundity, and spawning) of tunas is of paramount importance for population dynamics studies and the management of fisheries that target tunas. Nevertheless, "a very limited amount of scientifically useful infor- mation is available on the reproduc- tive biology for most tunas" ( Schaefer, 2001). Recent work has increased our knowledge on the reproductive biol- ogy of female Thunnus thynnus in the eastern Atlantic and the Medi- terranean (Susca et al„ 2000, 2001a, 2001b; Hattour and Macias, 2002; Medina et al., 2002; Mourente et al., 2002), but many questions remain still to be answered regarding male reproductive activity in this and other tuna species. Histological examination of gonads is a useful tool for assessing the ma- turity state offish. However, very few light-microscopy studies have been published on bluefin tuna and no ul- trastructural studies of reproductive organs are yet available. The male reproductive cycle of T thynnus has been characterized histologically by Santamaria et al. (2003), and Ratty et al. ( 1990 ) and Schaefer ( 1996, 1998 ) have reported valuable histological descriptions on male and female go- nads of the Pacific albacore (Thun- nus alalunga) and the yellowfin tuna (Thunnus albacares), respectively. In this article we report biometric and histological data on male T. thynnus caught during their reproductive mi- gration and spawning period in order to provide further information on the biological aspects of reproduction for this species. 408 Fishery Bulletin 102(3) Materials and methods Statistical analysis Samples and condition indices During the eastward migration, 62 adult male bluefin tuna weighing between 71 and 273 kg (mean 195.17 kg) were obtained from the trap fishery in the area of the Strait of Gibraltar (Barbate. Cadiz, southwestern Spain) from late April to early June 1999. 2000, and 2001. Thirty-four mature males, weighing between 19 and 349 kg (mean 115.11 kg), were sampled in June-July 1999-2001 from the purse-seine fleet operating in the Mediterranean spawning grounds of bluefin tuna off the Balearic Islands. Whenever possible, the total body weight (W) was recorded to the nearest kg. When indi- vidual body weights were not available, W was estimated from the fork length (LF) measurements (recorded to the nearest cm), according to the formula: W = 0.000019 x LF3 (Table VIII in Rodriguez-Roda, 1964). Following dissection, the liver, testes, and the fat bodies associ- ated with the gonads were removed and weighed to the nearest g. The condition of the fish was assessed by three different indices. The gonad index (gonadosomatic index) (IG) is indicative of the maturation state and was calcu- lated as: IG = (WG I W) x 100, where WG = gonad weight. The liver index (hepatosomatic index) (IL) and fat-body index (IF) were calculated as IL = (WL I W) x 100, and Ip- = (WF I W) x 100 (where WL and WF represent liver and fat-body weights), respectively, and are considered as good indicators of the metabolic condition and energy reserves of the fish. All measurements are expressed as means +SD. The bluefin tuna specimens used in this study showed considerable variability in size, especially those caught by purse seine in Balearic waters, where weight ranged between 12 and 349 kg. The purse-seine fishery is, in fact, much less size-selective than are traps, which seldom catch small bluefin tuna (Rodriguez-Roda, 1964: Mather et al., 1995). Analysis of covariance (ANCOVA), with body weight as covariate, was used as the most suitable method (Garcia-Berthou, 2001) to test interan- nual differences in the weight of the organs within the two sampling sites. ANCOVA was likewise applied to compare the weights of the three organs between both areas. All data were previously log-transformed to meet the prerequisites of normality and homoscedasticity (Zar, 1996). Linear least-squares regression analyses were performed to test possible correlations between IG and the two other indices (IL and IF) by using the pooled data of Barbate and the Balearic Islands. In the regression between IG and IF, the Balearic samples corresponding to year 2001 were excluded because the reduced fat-body size (adipose tissue was almost non- existent in the mesentery) of these small bluefin tuna did not permit an accurate weight measurements on board. The values of the indices were arcsine-trans- formed prior to the statistical analysis (Zar, 1996). A P-value <0.05 was considered statistically significant for all tests. Results Histology For light microscopy, tissue samples from the central part of the testes were fixed for 48-96 hours in 10% formalin in phosphate buffer, 0.1 M, pH 7.2. After dehy- dration in ascending concentrations of ethanol, a part of each sample was embedded in paraffin wax and the remainder was embedded in plastic medium (2-hydroxy- ethyl-methacrylate). Paraffin sections (6 /im thick) were stained with haematoxylin-eosin, and plastic sections (3 /jm thick) were stained with toluidine blue. These were examined and photographed on a Leitz DMR BE light microscope. For electron microscopy, small fragments of testis were fixed for 3-4 hours in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate buffer (pH 7.2). Following two 30-min washes in cacodylate buffer, they were postfixed for 1 hour at 4C in cacodylate-buffered 1% osmium tetroxide, rinsed several times in buffer, dehydrated in ascending concentrations of acetone, and embedded in epoxy resin (either Epon 812 or Spurr). Thin sections (-80 nm thick) were picked up on copper grids, stained with uranyl acetate and lead citrate, and examined in a Jeol 1200 EX transmission electron microscope. Ap- proximate dimensions provided for germ cells are mea- surements (means ±SD) of the largest cell diameters on electron micrographs. Condition indices ANCOVA did not reveal significant interannual differ- ences in gonad, liver, and fat-body weight in the samples of Barbate as well as in those of the Balearic Islands. In contrast, a strongly significant difference in testicular size (P<0.0001) was found in comparing data of matur- ing bluefin tuna from Barbate (migrant tuna) with fully mature fish from the Balearic Islands (spawning fish). In fact, as shown in Figure 1, the average I0 was more than fourfold higher in the Balearic Islands than it was in Barbate (4.81 ±1.77 vs. 1.12 ±0.57). This finding may indicate a noticeable increase in sperm production during reproductive migration to the Mediterranean spawning grounds. Significant differences between maturing and spawning tuna were also found in fat-body weight, the volume of which dropped to about half by spawning time. Thus, IF fell from 0.36 ±0.24 in migrating fish to 0.16 ±0.12 in spawning fish isee Fig. 1). The liver mass, however, did not differ significantly (P=0.31> between the two samples. Figure 2 illustrates linear regression analysis be- tween IG and I, , and between IG and IF. A significant negative correlation (;-'- = 0.34; P<0.0001) was found be- tween Iq and Ip, indicating that the amount of mesenteric fat tissue decreases as the gonad matures. In contrast, there was a positive, though somewhat weak, correlation Abascal et al.: Testicular development in Thunnus thynnus 409 V? ™i 'G V////A l|_ c=i If j&A Barbate Balearic Islands Figure 1 Differences in gonad index (IG) liver index 1 1, ), bluefin tuna [Thunnus thynnus) from Barbate and male tuna from the Balearic Islands. (r2=0.21; P<0.0001) between IG and IL, which suggests a slight growth of the liver with sexual maturation. Histology The testes of Thunnus thynnus are paired, elongate organs that appear attached to the dorsal body wall by a mesentery. The fat body, which is closely associated with the gonad, consists of a variable amount of adipose tissue. The testis is composed of a dense array of lobules converging on the main sperm duct (vas deferens) and terminating blindly beneath the tunica albuginea at the periphery ( Fig. 3, A and B). Two distinct zones can be distinguished in cross sections of the testes (Fig. 3A). At the outer region, the seminiferous lobules have a thick wall formed by the germinal epithelium, where germ cells develop in association with Sertoli cells; the lumina of the lobules are filled with spermatozoa that have been released after completion of the spermiogenetic process (Fig. 3, B and C). As a result of the release of mature sperm from spermatocysts into the lobule lumina, the germinal epithelium becomes discontinuous (Fig. 3B). The transition from the outer to the central region of the testis is marked by an abrupt change in the configuration of the testicular lobules, which lose the germinal epithe- lium and become ducts where lobule function has shifted from sperm production to sperm storage (Fig. 3C). Thus, the only sex cells that are found in the central part of the testis are mature spermatozoa, which fill the swollen lumina of the lobules. In this zone the testis ducts con- stitute an intricate network of channels that convey the spermatozoa produced in the proliferative region to the main sperm duct (Fig. 3, A and D), which is thick walled and located in the center of the testis (Fig. 3D). 0,25 A 0,20 o ^ 0,10 o 0,05 • 0,00 0,00 0.05 0.10 0,15 0,20 0.25 0,30 0,35 arcsin[(lG/100)1'2] 0,12 • B • 0.10 • c\j 5- 0.08 o o jL 0,06 - o 0.04 - 03 • > • • • .^>^» • • ^"~\^^ 0.02 •\. • ^--^ 0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 arcsin[(lG/100)"2] Figure 2 Linear regression between gonad index (IG) and liver index (ILl I Al, and between gonad index (I,-. ) and fat body index (IFI (Bi (data were pooled from the two areas). In B, samples of bluefin tuna (Thunnus thynnus) collected off the Balearic Islands in 2001 were excluded. The gametes develop in groups of isogenic cells called germinal cysts or spermatocysts, where the process of differentiation is synchronous (Fig. 4). Primary sper- matogonia are large, single cells (Fig. 4A) that are dis- tributed all along the germinal epithelium, as is char- acteristic of the teleost unrestricted testicular type. Spermatogonia B resulting from successive mitoses of spermatogonia A are found in small groups, whereas spermatocytes and spermatids are grouped within larger spermatocysts (Figs. 3, B and C, 4). The cysts contain- ing late spermatids and spermatozoa, prior to spermia- tion, display a particular alveolar appearance due to the orientation of the spermatid heads facing the lobule walls and the bundles of fiagella directed toward the seminiferous lobule lumen (Fig. 4, A, C, and D). Active spermatogenesis was observed to occur both in migrant bluefin tuna from the Strait of Gibraltar (Fig. 4, A and B) and spawning fish from the Mediterranean (Fig. 4, C and D). In both cases, all stages of the male 410 Fishery Bulletin 102(3) Figure 3 Light micrographs depicting the histological organization of the bluefin tuna (Thunnus thyn- nus) testis. iAi Transverse section showing the outer proliferative region (PR) of the testis and the inner region, which includes the testis duct system (D) and the main sperm duct (MSD). (B) Peripheral zone of the testis where the distal ends of some tubules i dotted lines! terminate beneath the tunica albuginea (TA). (C) Transition (dotted line) between the outer region (PR) of the testis, where the lobules contain developing germinal cysts, and the inner region (Dl, whose lobule walls enclose only mature spermatozoa. (Dl Main sperm duct iMSDi filled with a compact mass of spermatozoa that are incorporated (arrowhead) from the testis ducts (D). Arrows = discontinuities in the germinal epithelium; sc = spermatocytes; sd = spermatids; sg = spermatogonia; sz = spermatozoa. All samples are from Barbate (A and D, paraffin-embedded sections stained with haematoxylin-eosin; B and ('. toluidine-blue stained plastic-embedded sections). Abascal et al.: Testicular development in Thunnus thynnus 411 L , tf K *.T<£W*. »5r * .'50 ufk Figure 4 Spermatocysts in the testis proliferative region of bluefin tuna (Thunnus thynnus) from Barbate (A and B) and the Balearic Islands (C and D). All stages of spermatogenesis are present in both cases, although spermatid cysts containing late spermatids and spermatozoa (asterisks) are somewhat more abundant in specimens from the Balearic Islands. Arrow = dividing sperma- tocytes; sc = spermatocytes; sd = spermatids; sg = primary spermatogonion; sz = spermatozoa. Plastic-embedded sections (A-D) were stained with toluidine blue. germ cell line were present in the gonads. In addition, large amounts of spermatozoa had accumulated in the central system of ducts and in the main sperm duct, both of which appear to function as reservoirs of sperm. In specimens from Barbate, spermatocytes and sper- matids were abundant (Fig. 4B), whereas in most tuna collected in the Balearic area spermatids predominated over spermatocytes. Cysts containing late spermatids and spermatozoa were particularly common (Fig. 4, C and D). 412 Fishery Bulletin 102(3) sgA sd2 sgA B^'-V- ^ 4/r Se (.. A !."»" , ■■■':'.'^' m /*-■» • I sd3 mc sgA ^ // 'I cf Se 2 urn Figure 5 Electron micrographs of testicular tissue from bluefin tuna (Thunnus thynnus). i A and Bl over- views displaying several spermatocyst types, including single primary spermatogonia (sgA). and clusters of spermatogonia B IsgB), primary spermatocytes iscl I, mid spermatids ( sd2 1. late spermatids (sd.3), and spermatozoa (sz). The germ cells are surrounded by Sertoli cells (Se). Arrowheads = perinuclear bodies ("image"); N = nucleus; n = nucleolus; cf = collagen fibers; mc = myoid cell. infrastructure Primary spermatogonia are large, ovoid cells (8.55 ±1.07 jim) whose nucleus (>5 Jim in its largest diameter) shows diffuse chromatin and a single central nucleolus. The cytoplasm contains free ribosomes, a few mitochondria, endoplasmic reticulum cisternae, and several masses Abascal et al.: Testicular development in Thunnus thynnus 413 of electron-dense perinuclear material ("nuages") that indicate nucleocytoplasmic transport (Fig. 5, A and B). Such chromatoid bodies persist throughout spermatogen- esis until the spermatid stage, but their size and number is far higher in primary spermatogonia. Spermatogonia B are grouped in clusters of a few cells. They are per- ceptibly smaller (6.75 ±0.37 um) than spermatogonia A and their nucleus contains patchy chromatin (Fig. 5, A and B). Spermatocytes form clusters in which the cells are interconnected by cytoplasmic bridges. Primary sperma- tocytes (4.84 ±0.45 Jim) show a heterochromatic nucleus (-3.5 um in diameter) that varies in appearance depend- ing on the prophase-I stage. The cytoplasm contains free ribosomes (mostly polysomes), mitochondria, clear vesicles, and the diplosome (Figs. 5A, 6A). Synapton- emal complexes are clearly recognizable at pachytene (Fig. 6A). Secondary spermatocytes are apparently short-lived cells because they are rare in histological samples — a finding that suggests that the second meiotic division is triggered shortly after completion of the first division. Spermatocytes II are difficult to distinguish morphologically from early spermatids, although they are slightly larger (3.31 ±0.47 /jm). The cytoplasm is more reduced than in spermatocytes I and the nucleus shows diffuse chromatin forming moderately electron- dense patches (Fig. 6B). During spermiogenesis, the spermatid nucleus changes in shape and decreases in volume as the chromatin con- denses. In early spermatids (2.39 ±0.28 /Jm) the spheri- cal nucleus shows a dense chromatin with some elec- tron-lucent areas (Fig. 6C). Then the chromatin becomes more homogeneous in mid spermatids (2.56 ±0.21 fim) (Figs. 5A, 6D), and eventually in late spermatids (1.81 ±0.32 /iml condenses into a coarse granular pattern, whereas the nucleus assumes an ovoid shape and forms a basal indentation over the proximal segment of the axoneme (Fig. 6E). Cytoplasmic changes involve elonga- tion of the flagellum, reduction of the cytoplasmic mass, and coalescence of the mitochondria into a few large spherical units located around the proximal portion of the axoneme. Rotation of the nucleus does not take place during spermiogenesis, therefore the flagellum axis remains parallel to the base of the nucleus and the spermatozoon shows the typical ultrastructure of teleostean type-II sperm (Fig. 6F). Discussion Histologically, the bluefin tuna testis is of the unre- stricted spermatogonial testicular type found in most teleosts, where spermatogonia occur along the greater part of the testicular tubules. In the restricted sper- matogonial testicular type of the atheriniforms, on the other hand, the spermatogonia are confined to the distal end of the tubules, and spermatogenesis proceeds as the germ cells approach the efferent ducts (Grier et al., 1980; Grier, 1981). Efferent ducts are generally absent in unre- stricted spermatogonial testes, so that germinal cysts form along the testicular tubule length (Grier et al., 1980; Grier, 1981; Lahnsteiner et al., 1994). However, in maturing and spawning bluefin tuna a well-developed network of ducts collects the sperm produced by the ger- minal epithelium and voids them into the main sperm duct. The central ducts of the testis are continuous with the proliferative segment of the testicular lobules, which lose the germinal epithelium in the innermost region of the testis and function as sperm storage structures. This process has been documented in the common snook iCentropomus undecimalis) (Grier and Taylor, 1998), the cobia (Rachycentrum canadum) (Brown-Peterson et al.. 2002), and the swamp eel (Synbranchus marmoratus) (Lo Nostro et al., 2003). Grier et al. (1980) showed that in the atheriniform Fundulus grandis the efferent duct wall cells derive from Sertoli cells. A system of efferent ducts has been described in other species of teleosts pos- sessing testes of the unrestricted spermatogonial type (Rasotto and Sadovy, 1995; Manni and Rasotto, 1997). As has been shown in other species of the genus (Ratty et al., 1990; Schaefer, 1996; 1998), the main sperm duct of T. thynnus has a thick wall and is located near the center of the testis, whereas in many other teleosts the main duct is dorsal (Grier et al., 1980). Ultrastructural features of bluefin tuna spermatogen- esis are comparable to those described extensively in teleosts (for examples of recent literature see Gwo and Gwo, 1993; Stoumboudi and Abraham, 1996; Quagio- Grassiotto et al., 2001; Huang et al., 2002; Koulish et al., 2002; Fishelson, 2003). The primary spermatogonia are the largest male germ cells and exhibit several conspicuous perinuclear ("nuage") bodies. After several divisions they give rise to cysts of secondary spermato- gonia that enter meiosis to produce successively primary and secondary spermatocytes. Primary spermatocytes are abundant, particularly at the pachytene phase, and are therefore thought to be of long duration. In contrast, the spermatocyte-II stage is thought to be the shortest spermatogenetic step, because, as occurs in teleosts in general, it is the least frequent in histological samples. Spermiogenesis develops without the occurrence of rotation of the spermatid nucleus, resulting in a teleostean type-II spermatozoon (Mattei, 1970), in which the flagellar axis lies tangential to the nucleus instead of being inserted perpendicular to its base (Abascal et al., 2002). Santamaria et al. (2003) divided the testicular cycle of T. thynnus caught in Mediterranean waters from Febru- ary to September into five periods. Those developmental stages are similar to stages 2-6 classified by Grier (1981) for a generalized teleost annual reproductive cycle. Most probably, stage 1 (spermatogonial proliferation) occurs in Mediterranean bluefin tuna between October and January. More recently, annual histological changes in the germinal epithelium have been used to identify five distinct reproductive classes in males of several teleost species (Grier and Taylor, 1998; Taylor et al., 1998: Brown-Peterson et al., 2002; Lo Nostro et al., 2003). It is assumed that the most advanced maturation classes in males are characterized by the presence of a discontinu- ous germinal epithelium. According to this criterion, all 414 Fishery Bulletin 102(3) Figure 6 Electron micrographs of spermatocytes I (A), spermatocytes II (B), early spermatids (C), mid spermatids (D), late spermatids (E), and spermatozoon (F) from bluefin tuna {Thunnus thynnus). Arrows = synaptonemal complexes; arrowheads = cytoplasmic bridges between spermatids; ax = axoneme; c = centriole; cc = cytoplasmic canal; d = diplosome; dc = distal centriole; f = flagellum; Gc = Golgi complex; m = mitochondria; N = nucleus; pc = proximal centriole. of the samples examined in the present study correspond to the mid- and late-maturation stages proposed by Grier and Taylor (1998), and Taylor et al. (1998). Testes at these stages become storage organs that are filled with sperm. The present study encompasses only a short period of the reproductive cycle, which comprises final gonad maturation. However, descriptions of the testicu- lar histology throughout the annual cycle (Santamaria et al., 2003) appear to indicate that different maturation classes might be defined in the bluefin tuna based on histological examination of the germinal epithelium (see Taylor et al, 1998; Brown-Peterson et al., 2002). Final sexual maturation involves a considerable in- crease in testis size, but no apparent remarkable histo- logical changes, with the exception of a slightly higher frequency of the most advanced stages of spermatogen- Abascal et al.: Testicular development in Thunnus thynnus 415 esis in fully mature bluefin tuna. The different testicular development of maturing and spawning tuna is reflected by their respective average IG, which was fourfold higher in spawning fish. An equivalent gonad growth was found in the females collected in the same samplings (Medina et al., 2002), indicating a spatiotemporal parallelism in the gonad maturation cycle and a good synchronization of the reproductive peak in the two sexes. The matura- tion schedule differs between the two sexes, however, in that males are capable of generating mature spermatozoa while still on migration, whereas females do not appear to develop fully mature oocytes until they have reached the spawning grounds (Medina et al., 2002). Therefore, even though mature spermatozoa can be found in tes- ticular ducts during prolonged periods throughout the reproductive cycle, it is unlikely that males are actually capable of spawning out of reproductive season. The seasonal IG profile of the bluefin tuna appears to be similar to that of the pelagic, highly migratory perciform Rachycentron canadum (Brown-Peterson et al., 2002), and the swamp eel (Synbranchus marmora- tus) (Lo Nostro et al., 2003), in which peak Ic; values occur when the reproductive activity is at a maximum. A different situation has been reported in the common snook (Taylor et al., 1998), where the highest IG levels correspond with the mid maturation class and decrease during the latter part of the reproductive season. The biological significance of these different IG profiles in terms of reproductive strategies is yet unknown because a very limited number of species have been examined so far. Because spermatozoa are by far the most abundant cells in mature testes, the gonad weight becomes a good indicator of the quantity of sperm produced by a fish (Billard, 1986). Therefore, the significant increase in IG that occurred between samplings off Barbate and the Balearic Islands would indicate that, during migra- tion, bluefin tuna can raise several times the volume of sperm accumulated in the testes. The apparently high spermatogenetic activity observed in bluefin tuna caught on the spawning grounds suggests that bluefin tuna have the ability to regenerate testicular sperm stores. Continuous sperm production could be important be- cause external fertilization requires the release of large amounts of sperm to ensure successful fertilization of eggs, especially when egg size is small. In addition, it should be noted that tunas spawn multiple times (June, 1953; Yuen, 1955; Buriag, 1956; Otsu and Uchida, 1959; Baglin, 1982; Stequert and Ramcharrun. 1995) and can spawn almost daily throughout the reproductive season (Hunter et al.. 1986; McPherson, 1991; Schaefer, 1996, 1998, 2001; Farley and Davis, 1998; Medina et al., 2002). From histological examination of the sperm ducts, and based on the amount of sperm present and the staining of the epithelium, Schaefer (1998) proposed a spawning interval of 1.03 days for spawning male Thun- nus albacares throughout the eastern Pacific Ocean. The spawning rate estimated for reproductively active females with the postovulatory-follicle method was 1.19 days (Schaefer, 1998), which coincides with the spawn- ing interval estimated for female T. thynnus around the Balearic Islands (Medina et al., 2002). Unfortunately, we could not make a reliable estimation of the male spawning interval in our samples. Two possible reasons may account for this failure. One reason is that many of the samples of gonadal tissue did not include the main sperm duct. On the other hand, no clear evidence of spawning was identified by histological examination of those specimens processed that had sperm ducts. A plausible explanation for this fact is that recent sperm release can be detected only within 12 hours after the spawning event (Schaefer, 1996); hence for male spawn- ing to be detected the fish would have to be sampled in a narrow range of times following spawning, which Schaefer (1996) established between 00.01 and 12.00 hours after spawning for Thunnus albacares. It would be worth conducting further research on bluefin tuna at their spawning grounds, by attempting to cover a broad range of sampling times in order to ensure collection of specimens shortly after gamete release. In this way, use- ful information would be obtained on such reproductive parameters as spawning schedules, fecundity, and the energy cost of spawning, which are essential for ecologi- cal assessments of the reproductive potential. It is noteworthy that male tuna, as small as 20 kg in weight (-100 cm LF), were caught on the spawning grounds in our study. They had gonad indices over 5% and histological features indicative of full maturity. These observations indicate that the eastern stock of Atlantic northern bluefin tuna can reach maturity at age 3 years and thus support conclusions of previous studies (Rodriguez-Roda, 1967; Hattour and Macias, 2002; Susca et al., 2001a, 2001b; Medina et al., 2002); western bluefin tuna, on the other hand, mature at an older age, which has been estimated at 6 years (Baglin, 1982). Prior to sexual maturation, marine fish generally accumulate large lipid deposits, primarily triacylgly- cerols, which are subsequently mobilized to support gonad development and spawning migration (Bell, 1998). The major lipid storage sites are the mesenteric tissue, muscle, liver, and subdermal fat layers (Ackman, 1980). In bluefin tuna the liver does not appear to play an important role in lipid storage but is mainly involved in processing fatty acids mobilized from other bodily sources (Mourente et al„ 2002). This metabolic pattern is consistent with our observations of weight modifica- tions for liver and fat body from maturation through the spawning period. Although IL increases only slightly with sexual maturation, IF undergoes a marked decrease at the time of maximum gonad development. Thus, the regression analysis of the relationship between IG and IF shows a significant negative correlation, which reveals a depletion of mesenteric fat stores as the testes grow. The occurrence of a similar situation in females (Medina et al., 2002; Mourente et al., 2002) and in male and female Thunnus alalunga (Ratty et al., 1990) has led to the con- clusion that fat-body lipid reserves provide an important energy source for gametogenesis in tunas. 416 Fishery Bulletin 102(3) Acknowledgments This study has been funded by the Spanish government and the European Union (projects 1FD1997-0880-C05- 04 and Q5RS-2002-01355). The authors wish to thank two anonymous reviewers for helpful recommendations. 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U.S. Fish and Wildlife Service Special Scientific Report: Fisheries 150, 30 p. Washington, DC. Zar, J. H. 1996. Biostatistical analysis. 662 p. Prentice-Hall. Upper Saddle River, NJ. 418 Abstract-From 1995 to 1998. we col- lected female black rockfish (Sebastes mt'lanops) off Oregon in order to describe their basic reproductive life history and determine age-specific fecundity and temporal patterns in parturition. Female black rockfish had a 50^ probability of being mature at 394 mm fork length and 7.5 years-of- age. The proportion of mature fish age 10 or older significantly decreased each year of this study, from 0.511 in 1996 to 0.145 in 1998. Parturition occurred between mid-January and mid-March, and peaked in February. We observed a trend of older females extruding larvae earlier in the spawning season and of younger fish primarily responsible for larval production during the later part of the season. There were dif- ferences in absolute fecundity at age between female black rockfish with prefertilization oocytes and female black rockfish with fertilized eggs; fertilized-egg fecundity estimates were considered superior. The likelihood of yolked oocytes reaching the devel- oping embryo stage increased with maternal age. Absolute fecundity esti- mates ( based on fertilized eggs) ranged from 299,302 embryos for a 6-year-old female to 948,152 embryos for a 16- year-old female. Relative fecundity (based on fertilized eggs I increased with age from 374 eggs/g for fish age 6 to 549 eggs/g for fish age 16. Maturity, ovarian cycle, fecundity, and age-specific parturition of black rockfish (Sebastes melanops) Stephen J. Bobko Steven A. Berkeley Department of Fisheries and Wildlife Hatfield Marine Science Center 2030 SE Marine Science Drive Oregon State University Newport, Oregon 97365 Present address (for S. A. Berkeley, contact author): Long Marine Laboratory University of California, Santa Cruz 100 Shaffer Rd Santa Cruz, California 95060 E-mail address (for S A Berkeley, contact author): stevenab@cats.ucsc.edu Manuscript submitted 13 March 2003 to Scientific Editor's Office. Manuscript approved for publication 30 March 2004 bj the Scientific Editor. Fish Bull. 102:418-429(2004). Many fish species in the North Pacific have a long reproductively active life span, which increases the likelihood of producing offspring during peri- ods of favorable environmental condi- tions. This bet hedging reproductive strategy reduces the impact of envi- ronmental variation on reproductive success (Goodman, 1984; Leaman and Beamish, 1984; Schultz, 1989). In species with age-structured spawn- ing schedules, a broad age distribu- tion will maximize the length of the spawning season. The more protracted the reproductive period, the greater the likelihood that some spawning will occur during conditions favorable for larval survival (Lambert, 1990). Age-related differences in the timing of spawning have been observed in many fishes; usually larger, older fish spawn earlier (Simpson, 1959; Bage- nal, 1971; Berkeley and Houde, 1978; Shepherd and Grimes, 1984; Lambert, 1987), but in some cases younger fish spawn earlier in the season (Hutch- ings and Myers, 1993). Age truncation, an inevitable result of fishing, can increase recruitment variability by reducing the length of the spawning season or by selectively- removing older, more fit individuals from the population. Factors that might affect individual reproductive success include the number of eggs produced, the quality of eggs (e.g., yolk or oil globule volume), and the size or health of eggs and larvae. Off the coast of Oregon, widow rockfish (Sebastes entomelas) have exhibited increased absolute fecundity, and more importantly have increased rela- tive fecundity, with age (Boehlert et al., 1982). Individual populations of shortbelly rockfish (Sebastes jordani), have been found to produce larvae with differing lipid and protein com- positions and consequently potentially differing rates of survival (MacFar- lane and Norton. 1999). Zastrow et al. (1989) reported that striped bass eggs stripped from wild fish increase in quality with maternal age due to increased amounts of proteins and lipids, although relative concentra- tions remain unchanged. Black rockfish ( Sebastes melanops), like most other rockfish, are long- lived, moderately fecund livebearers with long reproductive life spans. Al- though their longevity and low rate of natural mortality is presumed to be an adaptation to allow success- ful reproduction over their lifespan despite long periods between favor- able environmental conditions, it al- so makes them more susceptible to overexploitation. The objective of our research presented in the present article is twofold. First, we describe the basic reproductive life history of black rockfish. with an emphasis on the ovarian developmental cycle and maturity schedule. Second, we investigate age-specific fecundity and temporal patterns in parturition and Bobko and Berkeley: Maturity, ovarian cycle, fecundity, and parturition of Sebastes melanops 419 discuss their effect on reproductive success in a population undergoing truncation of the upper end of its age distribution. Materials and methods We collected female black rockfish during the months of peak female reproductive development, November through March, for three successive years from 1995-96 through 1997-98. Female black rockfish were primarily obtained from recreational charter boat landings in Newport, Depoe Bay, and Charleston, Oregon, in addition to some fish from commercial landings from Port Orford, Oregon (Fig. 1). We also collected fish by rod and reel and spearfishing. When possible, the sex of all avail- able black rockfish was determined, and females were staged as immature or mature. Immature females were measured (FL), and mature females were returned to the laboratory. On extremely busy days when numerous charter boats were fishing, all mature females were collected, but immature fish were not measured. In total we collected 1643 female black rockfish. Immediately upon return to the laboratory, we recorded fork length, total weight when possible (most samples from charter boats were carcasses only), liver weight, and ovary weight. Ovaries were assigned a maturity stage based on macroscopic appearance and preserved in 10% buffered formalin. We initially followed the gross maturity stage scheme of Nichol and Pikitch (19941 for darkblotched rockfish ( Sebastes crameri) but ultimately- abandoned their classification of maturity stages in favor of the simplified maturity stages reported by Gunderson et al. (1980) (Table 1). Sagittal otoliths were removed and stored dry for age determination. All aging was done by an expert age reader from the Oregon Department of Fish and Wildlife (ODFW), who used the break-and-burn technique (Beamish and Chilton, 1982). Ten percent of the otoliths were randomly selected for a second read- ing to ensure consistency in interpretation of annuli. It should be noted that black rockfish ages have not been validated. However, ages have been validated for yellowtail rockfish (Sebastes flavidus), a closely related species (Leaman and Nagtegaal, 1987), by using otoliths; moreover, the break-and-burn aging method is widely accepted as valid for aging rockfish (MacLellan, 1997), and ages thus derived are routinely used in rockfish stock assessments. Because our sample included all mature females that we encountered, we used these data to estimate the age distribution of mature females in each time period, and the age distribution of parturition during each time interval. Histological preparations were made from the ova- ries of 175 females collected monthly from March 1996 through March 1997 to track seasonal ovarian devel- opment. Females collected in March 1996 and Novem- ber 1996 through March 1997 were from our regular sampling program, whereas fish collected from April 45° 00'N " 42° 50' ' 124° 30' 120°00'W Figure 1 Map of the Oregon coast showing the study area where black rockfish (Sebastes melanops) were collected. through October 1996 were obtained from Newport recreational charter boat landings. Females were ran- domly selected from each maturity stage observed each month and from as wide a range of ages as available (Table 2). Ovaries were embedded in paraffin, sectioned at 4-5 jjm, and stained with gill-3 haematoxylin and eosin y solution. We determined stage-specific fecundity in black rock- fish for females with unfertilized yolked oocytes (?i=184) and fertilized eggs (n = 85). Postfertilization ovaries were very fragile and tended to rupture easily and release embryos under the slightest pressure. Consequently, for estimating fecundity for these stages, we used only fish collected by ourselves so that we were certain that no eggs or larvae had been released during capture. To ensure that no eggs were lost after capture, these fish were immediately placed into plastic bags in order to retain any eggs that might be extruded before the ovary could be processed. Ovaries were processed fol- lowing procedures modified from Lowerre-Barbieri and Barbieri (1993) to separate eggs and embryos from connective tissue. Briefly, fixed ovaries were manually manipulated and rinsed with water through a 1-mm square mesh sieve, which retained most of the con- nective tissue, into another sieve with 0.75-mm mesh. Ovary connective tissue was retained in the coarse sieve, and freed eggs were collected in the fine-mesh sieve. Freed eggs were patted dry, weighed (nearest 0.1 g), and three subsamples were collected, weighed (nearest 0.001 g), and placed in 10% buffered formalin. 420 Fishery Bulletin 102(3) Table 1 Macroscopic and histological descriptions of stages used to describe female black rockfish maturity. Maturity stage Macroscopic description Histological description 1 Immature Small and translucent ovary, pink during months without sexual activity and yellowish (except for very small fish) during months with reproductive activity. 2 Vitellogenesis Ovary firm and yellow or occasionally cream in color. Large range of size, but all with visible opaque eggs. 3 Fertilization 4 Eyed larvae 5 Spent 6 Resting Eggs are golden and translucent. Ovary extremely large in relation to body cavity. Ovary wall thin and easily torn. Eyes of developing embryos visible, giving ovary an overall greyish color. Ovary fills a large portion of the body cavity. Ovary flaccid, purplish-red in color. Eyed larvae may still be visible. Ovary again firm and pink in color. Black spots may be visible. Oocyte cytoplasm intensely basophilic. Densely packed oogonial nests and developing oocytes, with larger oocytes containing small clear vesicles. Oogonia and developing oocytes still visible, but ovary dominated by large oocytes with numerous small red-staining yolk globules. Fertilized eggs ovulated and found within the ovarian cavity. Eggs have a single pink-staining yolk mass and clear oil droplet. Presence of developing larvae with black pigmented eyes. Yolk mass absorbed in late-stage larvae, but oil droplet usually present. Early-stage oocytes loosely associated. Extensive network of blood vessels. Possibility of encountering residual larvae. Similar appearance to immature fish. Ovary wall slightly thicker in early summer. Table 2 Monthly ranges for age, length, and maturity stage of black rockfish collected off Oregon from March 1996 through March 1997 for histological analysis. Month Age (yr range FL(mm) range Maturity stage range n March 7-25 375-510 1.4-6 10 April 7-18 364-447 1.5-6 12 May 7-13 340-465 1 and 6 15 June 5-13 349-432 1 and 6 15 July 5-13 360-475 1 and 6 14 August 5-11 357-493 1-2,6 15 September 6-16 366-488 1 and 2 12 October 5-16 357-420 1 and 2 11 November 5-11 355-434 1 and 2 16 December 5-14 365-439 1 and 2 10 January 6-17 369-473 1-4 16 February 7-17 378-464 1-5 17 March 6-13 380-467 1,5-6 12 AF = EW The number of ova in the subsamples were counted and absolute fecundity was estimated by using the following algorithm: issc ssw where AF = absolute fecundity, or the total number of eggs per female; EW = weight of rinsed eggs (or larvae); SSCt = subsample count i, where j=l to 3; and SSWt = subsample weight i, where £=1 to 3. Relative fecundity (RF), based on gonad-free somatic weight was estimated by RF = — —. TW-GW where AF = absolute fecundity, or the total number of eggs per female; TW = total weight: and GW = gonad weight. For our analyses of fecundity, we used only fish in which the number of eggs or larvae estimated from the three subsamples had coefficients of variation less than or equal to 59;, and for prefertilization eggs we used only females with average egg diameters of at least 450 jj to ensure inclusion of all developing oocytes. Only one cohort of developing oocytes is present in the ovary of Bobko and Berkeley: Maturity, ovarian cycle, fecundity, and parturition of Sebastes melanops 421 5n -i Maturity stage 2 • Vitellogenic 40 - Age 8 Collected February 1996 30 - 10 - o - i i ' 1 L 1 1 — i — L i i i i i 50 40 30 20 10 0 0.50 0 55 0 60 0 65 0 70 0 75 0.80 0.85 0.90 0.95 1.00 Maturity stage 3 - Fertilized Age 8 Collected February 1996 -[ — ' — i — ' — i 1 1 — — ' — — i — — 0 50 0 55 0 60 0.65 0.70 0 75 0 80 0 85 0 90 0 95 100 Oocyte or egg diameter (mm) Figure 2 Prefertilization- and fertilized-egg-diameter frequency distributions show- ing a single mode of developing oocytes at both developmental stages. black rockfish, either during development (stage 2) or after fertilization (stage 3) (Fig. 2). Analysis of covari- ance (ANCOVA) was used to test for annual effects in the relationship between prefertilization fecundity and age and a maturity-stage effect (prefertilization vs. fertilized-egg development stages) on both absolute and relative fecundity at age. We also used ANCOVA to test for a maturity stage effect in the relationship between absolute fecundity and fork length. All ANCOVA analy- ses were conducted by using multiple linear regression with the function lm in S-PLUS 2000 (MathSoft. Inc.. Seattle, WA). To predict the probability of a female black rockfish being mature based on its fork length, we fitted our maturity-at-length data to a logistic regression. Dur- ing those months without reproductive activity, late spring through early fall, it was difficult to distinguish between immature and mature-resting ovaries. Conse- quently, only those females collected during the peak months of reproductive development and from sampling events where all fish, mature and immature, were col- lected were included in our analysis. Binary maturity observations (0=immature, l=mature) and fork length were fitted to a logistic model by using the function glm, family = binomial of S-PLUS (S-PLUS 2000). The model used was S0+/Jl«. mFL)=P(Y = \\FL)-- \ + e 00+010. ■ where P(Y=1\FL) probability of female black rockfish being mature at size FL; and /30 and P{ = regression coefficients for the inter- cept and fork length, respectively. For functional purposes, the response variable was interpreted as the percentage of female black rockfish mature at length. Assuming this relationship of fork length to maturity had not changed over time, we ap- plied our logistic model to fork-length data from random sampling conducted by ODFW during the summers of 1992-2000 to calculate the percentage of female black rockfish caught by the recreational fishery off Newport that were mature in each year. Fork length-at-age data for female black rockfish were fitted with the von Bertalanffy growth function (VB- GF) by using the nonlinear function nisi ) in S-PLUS 2000. Age at 50% maturity was calculated by using our estimate of length at 509i maturity and a VBGF rearranged to the form ^U', '() ~*~ ' where t L |9j = age at 50% maturity; ,,= = asymptotic length; k = Brody growth parameter; t0 = age at zero length; and ._ = length at 50% maturity. Timing of parturition was estimated by microscopi- cally determining embryo development stages for all females with fertilized eggs following Yamada and 422 Fishery Bulletin 102(3) Kusakari's (1991) stages of embryonic development for kurosoi (Sebastes schlegeli) modified to reflect the gesta- tion period of 37 days for black rockfish (Boehlert and Yoklavich, 1984). Gestation period is likely to vary with water temperature. In determining gestation period, Boehlert and Yoklavich (1984) held black rockfish in the laboratory at 9-11 :,C. Mean water temperatures in our study area during the period of egg and larval development (December-April) were 10.9°, 10.1°, and 11.4°C in 1995-96, 1996-97, and 1997-98, respectively (http://co-ops.nos.noaa.gov/data). Even in the strong El Nino year of 1997-98, nearshore water temperature during the winter larval development period was only slightly outside this range. Therefore, we assumed a 37-day gestation period for all years of our study. Us- ing the Boehlert and Yoklavich (1984) equation; (stage duration = 0.0452 xstage1 090) we solved for duration at each stage by adding 5 days to account for the time between hatching (stage 32) and parturition (also from Boehlert and Yoklavich, 1984). To calculate the time until parturition for each stage, we subtracted the pre- vious stage durations from the total gestation period of 37. For example, at stage 1, parturition would occur in 37 days. At stage 2, parturition would take place in 37 days - stage-1 duration (-2 days) = 35 days. For each year of our study, estimated parturition dates for all females in our sample were grouped into one-week time intervals and further subdivided into age categories: 6-8: 9-11; 12-14; and >15. These num- bers were then multiplied by the appropriate value for age-class-specific fecundity based on fertilized eggs (Table 3) to estimate relative spawning output by week for each age class. Results Ovarian development Black rockfish off Oregon exhibited group-synchronous oocyte development; and females extruded only one brood of larvae per year (Fig. 2). Based on our observa- tions of ovarian development from all three years of this study, parturition took place from mid-January through mid-March and peaked in February. Following parturition, unextruded larvae were quickly resorbed and the ovary lost much of its vascularization. From April through early August ovaries were in a resting state and contained oogonial nests and slightly larger oocytes with a basophilic cytoplasm and a maximum diameter of 50 jjl. Also present at this time were develop- ing oocytes ranging from 50 to 150 n in diameter with small lipid vacuoles surrounding the nuclear membrane. Yolk deposition (vitellogenesis) began in late August and was observed through the third week of February. In the final stages of vitellogenesis. the largest oocytes were approximately 700 n in diameter and had numer- ous oil vacuoles and yolk globules throughout the cyto- plasm. The first female with fertilized eggs (stage 3) was observed during the second week of January, and stage-3 Table 3 Age group-specific absolute fecundity (based on fertilized eggs l and age distribution of mature females as a percent- age of all mature females, used to estimate larval pro- duction. Calculated from data pooled from 1996 through 1998. Age group (yr) Absolute fecundity' Percentage of all mature females represented by each age group 6-8 9-11 12-14 15 and older 364,183.5 558,837.1 753,490.7 948,144.3 42.19 38.48 13.94 5.39 ' Absolute fecundity for each age group is the estimated fecundity (based on fertilized eggs) for ages 7. 10, 13, and 16. respectively. females were observed until the third week of February. Recently fertilized eggs were approximately 850 ,u in diameter. The period of parturition as indicated by the occurrence of ovaries containing eyed larvae extended from the second week in January through the second week of March. Spent females were first collected during the last week of January and were most frequently col- lected in late February and early March. Sexual maturity Parameter values for the length-maturity logistic model were ft, = -26.73 and ^ = 0.068. The smallest mature female black rockfish we observed was 345 mm; all individuals were mature by 450 mm. Fifty percent of females were estimated to be mature at 394 mm fork length (Fig. 3). As reflected in our length-maturity logis- tic model, there was a decreasing trend in the percent maturity for female black rockfish in recreational land- ings from ODFW collections from 1992 through 2000 (Fig. 4). The von Bertalanffy parameter estimates for female black rockfish were Lre = 442 mm, k = 0.33, t0 = 0.75 (Fig. 5). Using these estimates, along with the fork length at 509c maturity, we estimated the age at 50% maturity for female black rockfish to be 7.5 years. The median age of mature females decreased in each col- lection year from 10 years in 1996 to 9 in 1997 and to 7 years in 1998. In addition, we observed a significant decrease in the proportion of mature fish age 10 or older over the three years of our study (Pearson's ^2 = 52.4, df=2, P<0.001). The proportions decreased from 0.511 in 1996, to 0.318 in 1997, and 0.145 in 1998. Fecundity Absolute fecundity for prefertilization female black rockfish ranged from 482,528 oocytes for a 5-year-old female to 998,050 oocytes for a 19-year-old female. The results of ANCOVA (Table 4) over a common age Bobko and Berkeley: Maturity, ovarian cycle, fecundity, and parturition of Sebastes melanops 423 100 e-26 731+0 068-FL •^ — • — • — • P(Y= UFL) = • / 1 + g-26 731+0 068-FL 80 • / • / • 60 - •/ 40 - 20 - *^% U I I I I 250 300 350 400 Fork length (mm) 450 500 Figure 3 Logistic regression model for the estimated percentage of sexually mature female black rockfish as a function of fork length, with associated observed percent mature at 10-mm length intervals. range showed no evidence of differences in slopes among the years 1996-98 (P=0.161). ANCOVA also showed no significant difference in elevations (P= 0.632), indicating no annual effect and allowing one model to be fitted to the pooled data (Fig. 6). Absolute fecundity for females with fertilized eggs ranged from 299,302 embryos for a 6-year-old to 948.152 embryos for a 16-year-old. Because of the low number of females with developing embryos collected in 1996 and 1998, 19 and 4 females, respectively, and based on the results of prefer- tilization females, all data were pooled and fitted with one model (Fig. 7). Although we were able to pool the data for all years for fecundity-age regressions for both prefertiliza- tion females and fertilized females, there was evidence of interaction (i.e., unequal slopes) between stage-specific absolute fecundity and age (2-tailed t-test, P=0.020) requiring sepa- rate linear regressions to be fitted to the data (Fig. 8). Similar to the ANCOVA results for abso- lute fecundity, there were no differences in slopes or elevations for relative fecundity for prefertilization females for the years 1996-98 (Table 4). Again, based on the results of the ANCOVA for prefertilization females and due to the low number of fertilized females collected and 1998, all relative fecundity data for femal fertilized eggs were pooled. Unlike the results • 50" • • 40" • • • • 30" 1 1 • • 1 1992 1994 1996 Year 1998 2000 Figure 4 Estimated percent maturity for recreationally landed female black rockfish from Newport, Oregon, based on our logistic regression model of fork length on maturity. Data were from regular random summer port sampling conducted by the Oregon Department of Fish and Wild- life from 1992 through 2000. in 1996 relation between absolute fecundity and age there was es with no evidence of interaction (i.e., unequal slopes) between for the stage-specific relative fecundity and age (2-sided f-test 424 Fishery Bulletin 102(3) Table 4 Results of analyses of covariance testing for differences in slopes and elevations of annua 1 absolute fecundity-age relati on and annual relative fecundity-age relation. Response variables = AF and RF. treatment factors = year, and covariate = age. Source of variation df Sum of squares Mean square F P Absolute fecundity i based on prefertilization oocytes) Equality of slopes 2 105.347 52,674 1.85 0.161 Error 160 4,564.320 28,527 Equality of elevation 2 168,544 84,272 0.29 0.747 Error 162 3,459,048 288,254 Relative fecundity i based on prefertilization oocytes) Equality of slopes 2 259.04 129.52 0.58 0.559 Error 160 35,452.22 221.58 Equality of elevation 2 799.52 399.76 1.81 0.166 Error 162 35.711.26 220.44 300 200 " 100 L = 442.02 •( 1 -e1" ° 33 '(a9e"( ° 75 1") 10 15 20 Age (years) Figure 5 Fork length at age fitted to the von Bertalanffy growth model for female black rockfish. (2-tailed i-test, P<0.0001,) of a stage effect (i.e., unequal elevations) which necessitated that the data be fitted with a parallel-line multiple linear regression model (Fig. 10 1. Temporal patterns in parturition From 1996 through 1998 we estimated rela- tive larval production for four age groups: 6-8; 9-11; 12-14: and 15 years and older (Fig. 11). In each year parturition took place from mid-January until mid-March, and older, larger fish extruded larvae earlier than younger fish. In 1996 and 1997, the 9-11 year-old fish dominated larval pro- duction, responsible for 60. lQ and 49. 6^ of all larvae extruded, respectively (Table 5). In 1998 age 6-8 fish produced the largest percentage of larvae (65.3%). In all years, relative larval production was lowest for the oldest age group (15+), declining to near 0 by 1998. Discussion P= 0.096). There was, however, strong evidence (2-tailed /-test, P<0.001,) of a stage effect li.e., unequal elevations) which necessitated that the data be fitted with a paral- lel-lines multiple linear regression model (Fig. 9). Absolute fecundity for prefertilization female black rockfish ranged from 443.671 oocytes for a 381-mm-FL female to 1,135,457 oocytes for a 495-mm-FL female. For fertilized females, absolute fecundity ranged from 283,618 oocytes for a 381-mm-FL female to 1,073,356 oocytes for a 510-mm-FL female. The results of AN- COVA over a common size range showed no evidence of differences in slopes between maturity stages (2-sided /-test P=0.206). There was, however, strong evidence Ovarian development for black rockfish in Oregon was similar to the developmental cycles reported for other rockfish species (Moser, 1967; Bowers, 1992; Nichol and Pikitch, 1994) with the exception of seasonal timing and stage duration. Females underwent vitellogenesis for up to six months before fertilization, which occurred from December through February. In all three years, parturition off the Oregon coast occurred between mid- January and mid-March and peaked in February. Wyllie Echeverria (1987) observed similar timing for parturi- tion of black rockfish off north-central California, with a peak in February but with parturition occurring through May. Bobko and Berkeley: Maturity, ovarian cycle, fecundity, and parturition of Sebastes melanops 425 All female black rockfish. except the smallest immature females, followed a sea- sonal cycle in which their ovaries developed an orange coloring during the months of reproductive activity — a pattern observed in olive rockfish (Love and Westphal, 1981). Similarly, Nichol and Pikitch (1994) ob- served darkblotched rockfish undergoing an "immature cycling" and even assigned these fish a maturity stage. After the re- productive season, the ovaries of immature black rockfish once again became pale pink in color. Because these fish were function- ally immature and there was no way to project when they would become sexually mature, they were combined with those small, young females undergoing no sea- sonal ovarian development and were staged as immature. Our estimate of fork length at 50% ma- turity for female black rockfish off Oregon was similar to the 400 mm estimate re- ported for north-central California females (Wyllie Echeverria, 1987), but lower than the estimate of 422 mm from Washington (Wallace and Tagart, 1994). Our estimated age at 50% maturity of 7.5 years was simi- lar to the estimates of 7.9 and 7 years from Washington and north-central California, respectively. McClure (1982) reported that over 50% of examined female black rockfish collected off Depoe Bay, Oregon, were ma- ture by age six. The difference between our estimate and McClure's was most likely due to using whole otoliths to age fish, which resulted in underestimates of age, and to assigning maturity stages only during summer months, which we have already de- scribed as problematic. Both absolute and relative fecundity increased with age for female black rockfish in Oregon waters, although there was a great deal of varia- tion not accounted for by age. The low r2 values for absolute fecundity regressions for pre- and postfertilization females (0.25 and 0.45 respectively) are due largely to the relatively poor correspondence between age and size (Fig. 5). Black rockfish, like many slow growing, long-lived fish grow slowly after sexual maturity. The rate of growth during their first few years can be quite variable depending on oceanographic conditions and food availability. As a re- sult, young fish can be as large or larger than much older fish (Fig. 5). Length is a better predictor of fecundity than age as judged by the goodness-of-fit of the multiple linear regression model (Fig. 10; /-2=0.70). An increase in absolute fecundity with age was ob- served in both prefertilization and postfertilization Absolute fecuodity (-10,000) ro -p* en cd o ro £* o o o o o o o 1996 • 1997 o 1998 A • A A % % • 0 o • . : • ' » ^- ° • * 1 . 1 ° • o • - . ^S^ « o i 1 * • ' i * 4F(prefertilization) = 298.413 + 36.823 age P<0.001 r2 = 0.23 U I I I I I I 1 1 I 4 6 8 10 12 14 16 18 20 Age (years) Figure 6 Scatter plot of black rockfish absolute fecundity (AFl (based on pre- fertilization eggs) on age by year (1996-98) with a fitted regression line from pooled data. 140" _ 120" o o o ° 100- 1 80- CJ § 60- o _o < 40- 20" 1996 • 1997 o 1998 • • • ! i s : • • • • : • • : y^ • / • 8 i i : t /^(fertilized) = -90,008 + 64,885 age P< 0.001 r2 = 0,45 0 ' ll ii 4 6 8 10 12 14 16 18 20 Age (years) Figure 7 Scatter plot of black rockfish absolute fecundity (AF) (based on fertilized eggs) on age by year (1996-98) with a fitted regression line from pooled data. females, but they occurred at different rates. As il- lustrated in Figure 8, the absolute fecundity for a post- fertilization 6-year-old black rockfish was only 58% of the estimated absolute fecundity for a prefertiliza- tion fish of the same age. By age 15 absolute fecundity 426 Fishery Bulletin 102(3) 140 120 o o °. 100 o ~ 80 C 13 O •2 60 £ 3 o £ 40 < 20 -* — prefertllization -o— fertilized 10 12 14 Age (years) 16 20 Figure 8 Separate-lines regressions fitted to absolute fecundity (based on pre- fertilization and fertilized eggs) on age for black rockfish in Oregon. 800" — ■ — prefertllization ■-o— fertilized • • ! i • • • • 5 600" O) cn 03 ••Is • • 0 >. o § 400- • f \ : s o _Q) . ' • i ro • 8 ° £ 200- o o 0 RF = 375.7 + 17.5-age - 106.5'stage o P< 0.001 ^ = 0.27 0 I i i 6 8 10 12 14 16 Age (years) Figure 9 Parallel-lines model fitted to relative fecundity I based on prefertiliza- tion and fertilized eggs) on age for black rockfish in Oregon. estimates for fertilized and prefertilization females were approximately equal. Yolked oocytes from older females were more successful in reaching the developing embryo stage. This may be attributed to higher rates of fertil- ization, greater viability of embryos, or a combination of both in older female black rockfish. Regardless of the mechanism there should have been signs of greater atresia in the ovaries of young fish, which we did not observe in our histological preparations. This may have been due to rapid resorption of unfertilized oocytes or an artifact of the fragile nature of fertilized ovaries, which made it difficult to obtain representative histo- logical preparations. Nevertheless, these results sug- gest that fecundity in black rockfish is best described after fertilization, but care must be taken to minimize embryo loss. These results also suggest that current Bobko and Berkeley: Maturity, ovarian cycle, fecundity, and parturition of Sebastes melanops 427 estimates of reproductive potential, in which fe- cundity for prefertilization females is used, may overestimate actual larval production because an increasing proportion of the stock consists of young fish. We observed a recurring trend of older, larger fish extruding larvae earlier in the reproductive season and larval output being increasingly domi- nated by younger and younger fish. Eldridge et al. (1991) reported that larger (and most likely older) yellowtail rockfish {Sebastes flavidus) spawned earlier in the season than smaller fish — a pat- tern also reported for darkblotched rockfish (S. crameri) by Nichol and Pikitch (1994). Reduced food availability has been suggested as a poten- tial cause for delayed reproduction in Sebastes for smaller, younger individuals with high metabolic requirements for somatic growth (Larson, 1991). We feel that limiting the amount of energy that can be spent on reproductive development would cause lower fecundity or reduced yolk content, but not necessarily a delay in reproductive develop- ment that would result in suboptimal timing of parturition. Stock assessments rarely consider changes in population age composition resulting from the removal of older age classes except to the extent that total egg and larval production is reduced. The decreasing representation of mature female black rockfish age 10 and older in the three years of our study indicates that age truncation is oc- curring in black rockfish in Oregon. This trun- cation not only removes biomass and potential larval production, but truncation of the upper end of the age distribution eliminates mature females with higher fecundity per individual, a greater success in carrying eggs through to the larval stage, and an age group that extends the overall parturition season. Further research is necessary to explore the controlling mechanisms of differential reproductive success with age and to determine how best to incorporate these find- ings into stock assessment models. Acknowledgments Many individuals contributed to the completion of this study. Tom Rippetoe provided many hours of expert assistance in all aspects of this research. Bob Mikus aged all of the adult black rockfish for this study. We thank him and the Oregon Department of Fisheries and Wildlife for all their support. We thank Dan Detman for his help and time in collecting black rockfish. We also thank Brock McLeod, Jason Castillo, David Stewart, and Michael Hogansen for all their help. We are espe- cially grateful for all those unpaid volunteers who helped with fieldwork: Joe O'Malley, Mark Amend, Bill Pinnix, Wolfe Wagman, and Pat McDonald. 160" /1F = -1 .888,811 +6.122FL -160,053 stage Absolute fecundity (xio.000) o o o r2 = 0 70 . . .*> o • • 1 ^f .. . : '••"tt: .• °. ° fi*.\C? ,"8 . = ° • ° o ° • • •• °^ • . • o o o — • — prefertilization o fertilized 365 390 415 440 465 490 515 Fork length (mm) Figure 10 Parallel-lines model fitted to absolute fecundity (based on prefer- tilization and fertilized eggs) on age for black rockfish in Oregon. I I Age 6 - 8 ^M Age 9 - 1 1 I I Age 12-14 ^mm Age 15 + ^ ^ ' & K*' ^' catch rates and rock lobster (Jasus edwardsii) mortality rates and examines factors that are associated with within-trap lobster mortality in the South Australian rock lobster fishery (SARLF). Since 1983, between 38,000 and 119,000 octopuses per annum have been taken in SARLF traps. Catch rates have fluctuated between 2.2 and 6.2 octopus/100 trap-lifts each day. There is no evidence to suggest that catch rates have declined or that this level of bycatch is unsustainable. Over the last five years, approximately 240,000 lobsters per annum have been killed in traps, representing ~4% of the total catch. Field studies show that over 98% of within-trap lobster mortal- ity is attributable to octopus pre- dation. Lobster mortality rates are positively correlated with the catch rates of octopus. The highest octo- pus catch rates and lobster mortality rates are recorded during summer and in the more productive southern zone of the fishery. In the southern zone, within-trap lobster mortality rates have increased in recent years, apparently in response to the increase in the number of lobsters in traps and the resultant increase in the probability of octopus encountering traps containing one or more lobsters. Lobster mortality rates are also posi- tively correlated with soak-times in the southern zone fishery and with lobster size. Minimizing trap soak- times is one method currently avail- able for reducing lobster mortality rates. More significant reductions in the rates of within-trap lobster mor- tality may require a change in the design of lobster traps. Maori octopus (Octopus maorum) bycatch and southern rock lobster (Jasus edwardsii) mortality in the South Australian rock lobster fishery Daniel J. Brock South Australian Research and Development Institute (Aquatic Sciences) 2 Hamra Ave. West Beach, South Australia 5024, Australia Present address: Department of Soil and Water Adelaide University Adelaide, South Australia 5005, Australia E-mail address: Brock. Daniel a1 saugov.sa.gov au Timothy M. Ward South Australian Research and Development Institute (Aquatic Sciences) 2 Hamra Ave. West Beach, South Australia 5024, Australia Manuscript submitted 28 April 2003 to Scientific Editor's Office. Manuscript approved for publication 2 March 2004 by the Scientific Editor. Fish. Bull. 102:430-440 (2004). Fishing traps are used throughout the world to target a wide range of crusta- ceans, fishes, and cephalopods. Com- mercial trap fisheries, especially those for decapod crustaceans, are often the most valuable fisheries within a region (Phillips et al., 1994). Traps are gen- erally considered to be an efficient and benign form of fishing because they appear to cause relatively minor damage to benthic habitats, can be designed to target particular species and size ranges, and produce live catches in good condition while mini- mizing bycatch (Miller, 1990). There are 49 species of spiny lob- sters (Decapoda: Palinuridae) world- wide, 33 of which support commercial trap fisheries. The largest of these are in Cuba, South Africa, Mexico, Australia, and New Zealand (Wil- liams, 1988). The main trap fisher- ies in Australia are for western rock lobster {Panulirus cygnus) in Western Australia and southern rock lobster (Jasus edwardsii) along the southern coastline. Octopuses constitute a sig- nificant component of the bycatch in both fisheries (Joll1; Knight et al.2) . In South Australia, J. edwardsii supports the State's most valuable commercial fishery. Octopus maorum is a significant bycatch species and is thought to be the major cause of lobster mortality in traps (Prescott et al.3). Although the octopus bycatch of the South Australian rock lobster fishery (SARLF) is saleable, the commercial value of this product does not offset the value of the large number of lob- sters that are killed in traps by octo- pus. Many fishermen are convinced that incidental mortality of octopus resulting from lobster fishing acts to control octopus numbers and that if these rates were reduced, octopus abundance and levels of within trap predation would increase. Despite the prevalence of octopus bycatch in lobster fisheries, there have been only a few studies on the interaction between octopus and lob- 1 Joll, L. 1977. The predation of trap- caught western rock lobster {Panulirus Longipes cygnus) by octopus. Depart- ment of Fisheries and Wildlife, Western Australia, Report 29, 58 p. (Available from Department of Fisheries, 168-170 St George's Terrace. Perth, Western Aus- tralia, 6000.] - Knight, M. A., A. Tsolos, and A. M. Doonan. 2000. South Australian fisheries and aquaculture information and statistics report. Research Report Series 49, 69 p. [Available from SARD] Aquatic Science, 2 Hamra Avenue, West Beach, South Australia 5022.] :! Prescott, J.. R. McGarvey, Y. Xiao, and D. Casement. 1999. Rock lobster. South Australian Fisheries Assessment Series 99/04, 35 p. [Available from SARDI Aquatic Science, 2 Hamra Avenue, West Beach, South Australia 5022.1 Brock and Ward: Octopus bycatch and lobster mortality in the South Australian rock lobster fishery 431 Figure 1 Map of the marine fishing areas (MFAs) of the South Australian rock lobster fishery. Shading shows the MFAs that were considered in this study and where most fishing effort is concentrated. sters in traps (Joll1). Furthermore, there is a paucity of quantitative data on the impact of fishing on octopus populations, the proportion of lobster mortality that is attributable to octopus predation, or the long-term economic and ecological effects that octopus-induced mortality may have on lobster fisheries. In this study, we examined the interaction between O. maorum and J. edwardsii in the South Australian rock lobster fishery (SARLF). The objectives were 1) to determine the number of lobsters and octopus caught and the number of lobsters killed in traps each year in the fishery; 2) to describe the interannual and seasonal patterns in lobster catch rate (CPUEL), octopus catch rate (CPUE0), and lobster mortality rate (ML); 3) to examine some factors that may affect lobster mortal- ity rates; 4) to estimate what proportion of the lobster mortality is attributable to octopus predation; and 5) to determine whether the rate of lobster mortality through octopus predation in traps is size dependent. Materials and methods South Australian rock lobster fishery The SARLF is divided into a northern zone (NZ) and a southern zone (SZ), each of which is further divided into marine fishing areas (MFAs) for statistical purposes (Fig.l). There are 68 and 183 fishermen licensed to oper- ate in the NZ and SZ respectively. The fishing season extends from November to May in the NZ and October to April in the SZ. A quota management system was introduced in the SZ in 1993, whereas the NZ is man- aged by gear restrictions and temporal closures. Total annual catch and effort for the SARLF Catch and effort data are recorded on a daily basis by all individual fishermen. Since 1983. a standardized logbook for recording catch and effort has been used across the fishery. Data provided by fishermen include MFA fished, average depth fished, number of trap-lifts, number and total weight of live lobsters, number of dead lobsters, and number and total weight of octopus. This information is stored in a South Australian rock lobster database that is managed by the South Australian Research and Development Institute, Aquatic Sciences. Interannual and seasonal patterns in CPUEL, CPUEQ, and ML Although commercial fishing for lobsters occurs along most of the South Australian coastline, the majority of effort is concentrated in only a few MFAs. In the NZ over the last 5 years about 72% of total trap-lifts were made in MFAs 15, 28, 39, 40, and 49. In the SZ over the same period 95% of trap-lifts were made in MFAs 51, 55, 56, and 58 (Fig. 1). 432 Fishery Bulletin 102(3) Data from the database were used to calculate catch rates of lobsters (CPUEL), octopus (CPUE0), and MLon an annual and monthly basis for the nine major MFAs listed above. Catch rates from these MFAs for each fisherman were calculated according to the formula: catch rate = catch number/itrap-liftslday). Annual and seasonal trends in CPUEj , CPUE0, and ML were cal- culated for each zone and MFA. Factors that affect within-trap lobster mortality Potential factors that affect within-trap lobster mortality were analysed by using a general linear model (type-3 sums of squares) under the assumption that the number of dead lobsters follows a log-normal distribution. The number of dead lobsters/trap-lift/day/license (with a ln+1 transformation) was used as the measure of lobster mortality. A model of the following structure was used to examine factors that affect the numbers of dead lobster: Dead lobster = License + MFA + Month + Year + Effort + Depth + Octopus + Lobster catch + Soak-time + I License xYear) + I License xMonth) + (YearxMonth) + (YearxMFA) + iSoak-timexYear) + < Soak-time xMonth). In the model. License represents an individual fisher- man, MFA is the marine fishing area. Month accounts for seasonal variation and Year accounts for interannual variation. Effort is the number of trap-lifts/license each day, Depth is the average depth fished by each License on a particular day. Octopus and Lobster are the respec- tive daily catches/license, and Soak-time is the number of days that the traps remained in the water since the previous trap-lift. The interaction terms License xYear and License x Month account for variations in the catch characteris- tics of the individual licenses over time that result from changes in fishing practises and efficiency associated with different boats, license holders, and skippers. The interaction terms YearxMonth and YearxMFA account for variation in the population dynamics of octopus and lobster over time in different locations that could result in differential trends in lobster mortality. The inter- action terms Soak-time xYear and Soak-time xMonth reflects the change in general fishing strategies over time. In quota-managed fisheries the average soak-time will be affected by a number of factors, for example, that may include price, weather, and the fishermen's perceived ability to catch their quota. The analysis was run separately for the SZ (/? = 493,629 traps) and NZ (ra=155,628 traps) because the respective zones have different fishing seasons and management structures. The relationship between the number of dead lobsters and the factors depth, soak-time, and num- ber of octopuses and lobsters were presented graphically by the equation: Lobsters killed in traps <*. factor a, where a = the parameter estimated by use of the model. Source of lobster mortality and size-dependent mortality A sampling program was conducted on three commer- cial vessels from the SZ during the 2001-02 fishing season. Five days were spent on each vessel. All lobsters caught were measured (carapace length, mm), and the sex (male or female), maturity (mature or immature), status (dead or alive), and cause of death (octopus or other) were recorded. The method used to distinguish between lobsters killed by octopus or other means followed that of Joll.1 This suitability of this approach was confirmed through examination of the carcasses of over one hundred lob- sters killed by octopus in aquarium trials (Brock et al.4). Lobsters with shells that were partly or completely separated at the juncture between abdomen and cepha- lothorax but were otherwise undamaged were deemed to have been killed by octopuses, whereas lobsters with shells without this separation and with evidence of bite marks were deemed to have been eaten by other preda- tors (fish or cuttlefish). Anecdotal evidence from fishermen suggests that larger lobsters are more susceptible to predation than smaller ones. The effect of CL on the probability of mortality was examined separately for males and fe- males by generalized linear modeling. The probability of mortality at a given size was modeled with a logistic equation of the form: P{sex, CL) = l/(l+e-,a+6CL,), where P(sex, CL) = the probability of a lobster of a given sex at carapace length CL being dead; and a and b are parameters to be estimated. Results Estimation of total lobster catch, octopus bycatch, and lobster mortality In 1999, there were 1.6 million trap-lifts in the SARLF, and 70% of this total effort was in the SZ (Fig. 2). The number of traps-lifts in the SZ declined from 2.2 mil- lion in 1983 to 1.2 million in 1999 (Fig. 2Ai. In contrast, fishing effort in the NZ remained relatively consistent with 406,000 trap-lifts in 1983 and 480, 000 trap-lifts in 1999 (Fig 2B). The total annual lobster catch has generally increased in each fishing zone since 1983 (Fig. 2, A and B). ' Brock, D. J., T. M. Saunders, and T. M. Ward. In review. A two-chambered trap with potential for reducing within-trap predation by octopus on rock lobster. Can. J. Fish. Aquat. Sci., 19 p. lAvailable from SARDI Aquatic Science, 2 Hamra Avenue. West Beach, South Australia 5022.1 Brock and Ward: Octopus bycatch and lobster mortality in the South Australian rock lobster fishery 433 A 7 -r 6 |< 5 4 3 2 1 0 Southern zone I lobster — ♦ — trap lifts \ T 2'5 -- 2 1 05 0 B 7 6 5 + 4 3 2 1 0 Northern zone I lobster — ♦ — trap lifts 2.5 ■■ 2 -- 1.5 1 uxi -♦-♦-♦ iinlniiilli i"i"i"i"i"i"i"i"i"i"i"i"i' 0.5 0 1983 1987 1991 1995 1999 1983 1987 1991 1995 1999 1983 1987 1991 1995 1999 1983 1987 1991 1995 1999 Season Figure 2 Annua] total catch and effort for each zone of the South Australian rock lobster fishery for number of live lobsters caught (A l, number of trap-lifts used (B). number of dead lobsters caught (C), and number of octopuses caught (D) (Note: change of scale in D). In the SZ, the annual lobster catch rose from 3.8 mil- lion lobsters to a peak of 6.4 million lobsters in 1991 and was 5.4 million lobsters in 1999 (Fig. 2A). In the NZ, 560,000 lobsters were taken in 1983 compared to 850,000 in 1991 (Fig. 2B). The total annual octopus catch varied among years in both zones, but between 70% and 907c of the total octo- pus catch were landed in the SZ (Fig. 2). The total num- ber of octopus ranged from 36,000 in 1986 to 109,000 in 1992 (Fig. 2C) in the SZ, and from 4700 octopuses in 1985 to 11,200 in 1998 in the NZ (Fig. 2D). In 1999, over 226,000 lobsters were killed in traps in the SARLF (Fig. 2). Since 1983, the mean proportion of dead lobsters out of the total catch has been approxi- mately 4%. In the SZ, the number of lobsters killed in traps has generally increased from 118,000 in 1983 to 196,000 in 1999; a peak of 274,000 dead lobsters oc- curred in 1992 (Fig. 2C). In the NZ, there has also been a general increase in the number of lobster killed in traps each year; 24,000 dead lobsters were recorded in 1983, compared to 31,000 in 1999 and a peak of 39,000 dead lobsters recorded in 1998 (Fig. 2D). Interannual and seasonal patterns in CPUEL CPUE0,and ML Southern zone Mean annual CPUEL in the SZ increased from 175 to 466 lobsters/I 100 trap-lifts/day) between 1983 and 1999, and the largest increase occurred between 1997 and 1999 (Fig. 3A). Mean annual CPUE0 ranged from 1.8 to 6.2 octopus/dOO trap-lifts/day) in 1987 and 1992, respectively (Fig. 3C). Mean annual ML rose from 5 to 17 dead lobster/(100 trap-lifts/day) between 1983 and 1999 (Fig. 3E). Peaks in both CPUE0 and ML occurred in 1985, 1992, and 1995. Mean monthly CPUEL declined during the fishing sea- son from 310 to 164 lobster/(100 trap-lifts/day) between October and April (Fig. 4A). In contrast, mean monthly CPUE0 increased from 2.6 to 3.7 octopus/dOO trap-lifts/ day) between October and December and declined to 1.8 octopus/dOO trap-lifts/day) in April (Fig. 4C). Similarly mean monthly ML increased from 10.7 to 12.8 dead lob- ster/(100 trap-lifts/day) between October and November and declined to 6.7 dead lobster/) 100 trap-lifts/day) in April (Fig. 4E). 434 Fishery Bulletin 102(3) A lobster 700 , Southern zone Northern zone maiahmiiii -i — I — I — I — I — I — I — I — I — I — I — I — i — I — r- -1 — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I 1983 1987 1991 1995 1999 1983 1987 1991 1995 1999 (_ octopus 10 -, L) octopus 10 — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i 1983 1987 1991 1995 1999 6 -I 4 2 0 H#fe^%ftft t. dead lobster 30 Q- 25 8 20 £ 15 V> 2 10 •o TO . The mean monthly M, declined from 7.5 to 3.4 dead lobster/100 trap-lifts/day between November and May (Fig. 4Fi. Since 1983, mean annual CPUEL has been relatively low and stable in MFAs 15, 28, and 40 but has been Brock and Ward: Octopus bycatch and lobster mortality in the South Australian rock lobster fishery 435 A lobster Southern zone 600 500 - 400 o o s; 300 w CD « 200 Oct Nov Dec Jan Feb Mar Apr octopus in % 6 CL ra 5 - i *■ ~S 3 CD en o =3 ^ a. 2 1 o o 0 Oct Nov Dec Jan Feb Mar Apr k dead lobster 20 15 1 1 1 1 1 1 1 Oct Nov Dec Jan Feb Mar Apr B lobster Northern zone 600 -, 500 400 300 200 - 100 0 D — i 1 1 1 1 1 1 Nov Dec Jan Feb Mar Apr May octopus 6 5 4 3 - 2 1 0 1 1 1 1 1 1 1 Nov Dec Jan Feb Mar Apr May 15 - 10 ■ 5 - 0 - — i 1 1 1 Nov Dec Jan Feb Mar Apr May Month Figure 4 Mean monthly catch rates in each fishing zone for lobsters lCPUEL) (A and Bl, octopus (CPUE0) (C and Di, and dead lobsters (ML)(E and F). Error bars are ±SD of mean. higher and more variable in MFAs 39 and 49 (Fig. 5B). There were large interannual fluctuations in CPUE(1 in each MFA, and these trends were similar among MFAs (Fig. 5D). ML was highest in MFA 40, where a maximum of 12.5 dead lobsters/l 100 traps lifts) was re- corded 1998 and lowest in MFA 15 where the maximum was 5.2 dead lobsters/100 trap-lifts in 1997 (Fig. 5F). No clear long-term trends in ML were apparent in any MFA. Factors that affect within-trap lobster mortality Based on the mean square values, the number of octo- pus had the greatest effect on lobster mortality in both zones (Table 1, A and B). The number of dead lobsters increased with both octopus and lobster catches and with soak-time and decreased as depth increased (Figs. 6 and 7). Based on the relative size of the mean square values, the factor with the greatest effect on the number of dead lobsters in the SZ was the number of octopus caught, followed by soak-time, number of lobsters caught, and depth. In the NZ, the number of octopus caught was also the most important factor, followed by the number of lobsters caught, depth, and soak-time. Source of lobster mortality and size-dependent mortality A total of 3627 lobsters from 635 trap-lifts were mea- sured. In the sample there were 212 lobsters killed in traps of which 207 (98%) were killed by octopus and 5 436 Fishery Bulletin 102(3) Southern zone -i — i — i — i — i — i 1995 1999 octopus 1983 li, dead lobster - 1 — i — i — i — i — i — 1987 1991 — i — r — i — i — r- 1983 1987 1991 -MFA 51 -MFA 56 1995 -MFA 55 MFA 58 1999 B lobster Northern zone 600 500 400 - 300 200 100 0 1983 1987 D octopus -1 — I — I — 1991 1995 -T 1 1 1 1 1 1 1 T 1983 1987 1991 1995 >_MFA 15 g MFA 28 a MFA 39 MFA 40 _*_MFA 49 Figure 5 Annual catch rates of the major MFAs in each fishing zone for lobsters (CPUEL) (A and Bi. octopus (CPUE0i i C and D), and dead lobsters (MLl (E andF). by other predators. The mean CL of dead male lobsters was greater than live males (120 ±21.1(SD) vs. 110 ±18.3 (SD) mm, P<0.001). There was no significant difference in the mean size of live and dead female lobsters. For both sexes the probability of mortality increased with size according to the following relationships: P (ML, males) = i/i+e-(-5.04+o.02CL) P F Model Error Corrected total 5319 483.961 489,280 314,536 194,140 508,677 59.13 0.401 147.41 ;0.0001 Source df Type-3 SS Mean square F- value P>F License MFA Year MFAxYear Month Effort Lobster catch Depth Octopus Soak-time Soak-time x Year License x Year License x Month Year x Month Soak-time * Month 245 3 17 51 6 1 1 1 1 1 17 3415 1460 94 6 49158.1 9.6 2494.6 233.0 2830.7 229.6 6728.7 1335.0 35930.5 6842.1 286.9 53019.8 5900.2 3760.4 310.2 200.6 3.2 146.7 4.7 471.8 229.6 6728.7 1335.0 35930.5 6842.1 16.9 15.5 4.0 40.0 51.7 500.2 8.0 365.8 11.6 1176.1 572.4 16773.4 3327.9 89568.8 17056.1 42.1 38.7 10.1 99.7 128.9 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 H Source df Squares Mean square F-value P>F Model Error Corrected total 2159 148,731 150,890 39,217 64,713 103,931 18.17 0.435 41.75 <0.0001 Source df Type-3 SS Mean square F-value P>F License MFA Year MFAxYear Month Effort Lobster catch Depth Octopus Soak-time Soak-time x Year License x Year License x Month Year x Month Soak-time x Month 95 4 17 68 7 1 1 1 1 1 17 1287 553 95 6 3361.5 174.8 241.2 175.4 317.3 27.1 1299.7 391.3 6305.1 210.8 117.7 7275.6 1170.7 275.9 2.8 35.4 43.7 14.2 2.6 45.3 27.1 1299.7 391.3 6305.1 210.8 6.9 5.7 2.1 2.9 0.5 81.3 100.4 32.6 5.9 104.2 62.3 2987.2 899.3 14491.0 484.4 15.9 13.0 4.9 6.7 1.1 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.3743 438 Fishery Bulletin 102(3) A depth D soak time 1 ♦ 41 0 8 ■ 0.6 - 3 - * 2- * • » 0 4- ***■" '''»»• | M | [J, 1 " ♦ 0) 3 0.2 - ■DO 50 100 150 0 5 10 15 -g Depth (m) Soak time (days) o o O octopus D lobster a 5 -, 10 i im CO a> 4 - „♦»♦♦* 8. ^^^^ QC 3 - ♦ ♦** yS^ 2 - ♦ *# f ♦ ♦■ 1 - ♦ 2 -, 0 5 10 15 20 0 200 400 600 800 No. of octopuses No. of lobsters Figure 6 The relative number of dead lobster as a function of lAl depth, iBi soak-time, (C) number of octopus, and (D) no. of lobsters for the southern zone. Error bars are ±SD of mean. . 1 0 15- t: o 0 1- 0 05 - male j$ J2jMlsfrm*F*F^^ female 60 80 100 120 140 160 180 200 Carapace length (mm) Figure 8 Size-dependent mortality of lobsters with respect to sex. J. edwardsii (O. maorum), (Gardener5), and a localized study in the New Zealand fisheries for J. edwardsii (O. maorum) found the proportion of the lobster catch killed by octopus to be as high as 10% (Ritchie6). The estimates of lobster mortality from these other studies should be treated with caution however because the cur- rent study is the only one that documents within-trap lobster mortality from a fishery-wide data set. The general linear modeling approach that we used to determine the factors associated with ML has some limi- tations. For example, the logbook data for the SARLF, like the monitoring data for most other fisheries, are not completely independent, and interdependence among observations can bias estimates of parameters. Simi- larly, some of the factors in the model, notably CPUEL and CPUE0 are partially correlated. In addition, the large number of observations and degrees of freedom tend to make most factors significant. We considered all of these issues when interpreting the results of the analyses and used the mean square (MS) values to rank the importance of factors. In both zones, inter- and intra-annual fluctuations in ML largely reflect the effects of CPUE0 and CPUEL. The broad trends in annual CPUE0 have largely cor- responded to those for M, with peaks in both generally synchronous in both fishing zones. In the SZ, the gen- eral increase in ML since 1983 appears to result from the increase in CPUE, which has more than doubled over this period. This assessment is supported by catch- rate data from individual MFAs. The two MFAs in the SZ that have had the greatest increases in CPUEL over the last 5 years (56 and 58) have also had the highest 5 Gardener, C. 2002. Personal commun. Tasmanian Aqua- culture and Fisheries Institute, Private Bag 49, Hobart, Tasmania 7001. 6 Ritchie. L. D. 1972. Octopus predation on trap-caught rock lobster — Hokianga area, N.Z. September-October 1972. New Zealand Marine Department. Fisheries Technical Report 81, 40 p. (Available from Ministry of Fisheries, 101-103 The Terrace, Wellington, New Zealand, 1020.] corresponding increase in ML. Increases in CPUEL are likely to elevate ML by both increasing the probability of octopus encountering traps containing lobsters and the number of lobsters in traps entered by octopus. However, ML is also positively correlated with soak- time, especially in the SZ. This finding is consistent with patterns observed in the New Zealand fishery for J. edwardsii6 and reflects the increased opportunities for octopus predation when pots containing lobsters remain in the water for longer periods. In the SZ, fish- ermen return to port each day and choose to fish or not to fish each day according to factors such as weather and price; therefore, although a 24-h soak period is still most common, soak times can range from one to five days. In the NZ, fishermen remain at sea for extended periods and consequently soak times longer than 24 hours are rare. There was considerable variation in the fishery data, especially in the southern zone. It is likely that much of this variation is related to the large geographical extent of the fishery as opposed to fishing practises. Across the fishery lobster growth rates and subsequent catch rates vary greatly (McGarvey et al., 1999a). For example, since 1991, the CPUEL in MFAs 56 and 58 have been twice those of MFAs 51 and 55 in the SZ. Although the variation in CPUE0 between the zones has been similar, the higher variability in CPUEL in the SZ is reflected in the variation in ML. Data spanning 17 years and covering about 50.000 km2 represent one of the few long-term and large-scale data sets on the distribution and abundance of an octo- pus species (Hernandez-Garcia et al., 1998: Quetglas et al., 1998). The paucity of octopus studies on these scales reflects the logistical constraints of fishery-independent surveys of octopus populations and the poor and incon- sistent methods generally used to record fishery catch and effort data (Boyle and Boletsky, 1996). The few data that are available on the distribution patterns of octo- pus have been obtained mainly from small commercial fisheries and CPUE(, has been included as a measure of relative abundance (Defeo and Castilla, 1998; Her- nandez-Garcia et al., 1998). This approach has proven useful, but several potential biases must be considered when CPUE0 data are being interpreted: these include 1) changes in fishing methods and efficiency over time; 2) the distribution pattern (e.g., random or aggregated) of the species under consideration; and 3) spatiotempo- ral fluctuations in catchability (Richards and Schnute, 1986; Rose and Kulka, 1999). There are several reasons why the data from the SARLF may provide a useful measure of the relative abundance of octopus over these spatial and temporal scales. Most importantly, the ba- sic unit of effort in the fishery, the trap, has remained unchanged since 1983. Furthermore, although O. mao- rum is retained as bycatch and kills J. edwardsii in traps, it is neither targeted nor avoided by fishermen, and fishing effort is thus relatively independent of its distribution patterns because the economic effects of both the sale of octopus bycatch and the costs of lobster predation are relatively small compared to the primary 440 Fishery Bulletin 102(3) economic driving force for the fishery, the lobster catch rates, and because the catch rates of octopus are dif- ficult to predict. In addition, O. maorum is a solitary animal that tends to be dispersed randomly throughout areas of suitable habitat (Mather et al., 1985). The higher total catches and catch rates of both lob- ster and octopus in the SZ, compared to the NZ, prob- ably reflect the more extensive reef habitat and more intense nutrient-enrichment upwelling in this portion of the SARLF (Lewis, 1981). There have been large interannual fluctuations in CPUE0 in both zones since 1983. Such fluctuations in population size are common among other cephalopods, especially squid, and may result from life history strategies that are characterized by rapid growth, short lifespan (50 m) vessels (e.g. Barlow, 1988) and hence are extremely expensive ($US 10,000/ day). Such costs usually put high-qual- ity surveys such as those conducted for harbor porpoise in the U.S. (e.g., Carretta et al„ 2001) beyond the reach of less affluent nations. The need for abundance estimates is especially great for the coastal and riverine spe- cies found in Asia, Africa, Australasia, and South America (Table 1). Several of these species have apparently small populations and restricted distribu- tions, and all suffer from being taken as bycatch in fishing gear, principally in gill nets (IWC, 1994). In addition, it is difficult or impossible for large ves- sels to work close to shore, in shallow waters, where some of these species are most common. The work described in this contri- bution had two aims: 1) to adapt ship- based line-transect methods (e.g., Barlow, 1988) to a 15-m catamaran, and 2 ) to provide an updated estimate of the abundance of Hector's dolphin (Cephalorhynchus hectori). Hector's dolphin, a small delphinid found only in the inshore waters of New Zealand, is subject to bycatch in gill nets throughout its range (Dawson et al., 2001). At least in the Canterbury region, and off the North Island west coast, recent catch levels are clearly unsustainable (Dawson and Slooten, 1993; Martien et al., 1999; Slooten et al., 2000; Dawson et al.. 2001). Stud- ies of mt-DNA indicate that the very small North Island population is dis- tinct and that there are at least three separate populations in South Island waters (Pichler et al., 1998; Pichler and Baker, 2000; see also Baker et al., 2002). At the time of the present study the only quantitative population estimate was from a strip-transect survey conducted in 1984-85 (Daw- son and Slooten, 1988), in which the offshore distribution, as well as the 442 Fishery Bulletin 102(3) proportion of dolphins detected within the strip, was estimated. A current, more robust estimate is needed for management. This study describes line-transect boat surveys conducted to estimate Hector's dolphin abundance on the north, east, and south coasts of the South Island of New Zealand. Figure 1 Photograph of the observer platform on the catamaran Cat Materials and methods Vessel choice and field methods Displacement catamarans are inherently suitable for inshore surveys because of their resistance to rolling and their ability to sustain reasonably high cruising speeds with modest power. We based our surveys from a 15.3-m sailing catama- ran (RV Catalyst), which is powered by two 50-hp diesel engines, and cruises at 9-10 knots while using <10 liters of fuel per hour. We fitted a collapsible aluminum sighting platform (~6 m eye height; Fig. 1) to increase the resolution with which observers could measure the downward angles to sightings (see Lerc- zak and Hobbs, 1998. for details) and to allow the observers to see animals far- ther away. The surveys were conducted with a crew of six (five observers, one skipper). Three people stood on the platform at any given time; one scanned the surface waters to the right of the platform, and the other scanned to the left, and a third person (the recorder) recorded sightings dictated by the observers. Sightings made by the recorder were not used in our analyses because his or her sight- \Jj ^ ing effort was unavoidably uneven (the recorder could not make sightings while recording another sighting). The record- er did not point out sightings to observ- ers. Observers and data recorder rotated alyst Table 1 Examples of coastal and riverine species of special conservation concern. Common name Scientific name Habitat Vaquita Phoeoena sinus Northern Gulf of California Chilean dolphin Cephalorhynchus eutropia Inshore coastal Chile Hector's dolphin < 'ephalorhynchus hectori Inshore coastal New Zealand Commerson's dolphin Cephalorhynchus commersoni Inshore coastal Chile, Argentina, Falkland Is, Kerguelen Is. Heaviside's dolphin ( 'ephalorhynchus heavisidii Inshore coastal South Africa and Namibia Peale's dolphin Lagenorhynchus australis Coastal Chile. Argentina, Falkland Is. Finless porpoise Neoph ocoena phocaenoides Coastal and riverine Asia and Indonesia Indo-Pacific humpbacked Sousa chinensis Inshore tropical and estuarine habitats in western Pacific dolphins and Indo Pacific Burmeister's porpoise Phoeoena spinipinnis Coastal Chile. Argentina, Uruguay. Brazil Franciscana Pontoporia blainvillei Coastal Brazil and Argentina Indus river dolphin Platanista minor Indus River Ganges river dolphin Platanista gangetica Ganges, tiramaputra. Karnphuli, Meghna rivers Boto Inia geoffrensis Amazon River Tucuxi Solatia fluviatilus Coastal and estuarine Atlantic Central and South America Dawson et al.: Line-transect surveys of Cepha/orhynchus hectori 443 every 30 minutes to avoid fatigue. Although Hector's dolphins are easily identified from other species, and group size is typically small (usually 2-8; Dawson and Slooten, 1988), in order to maintain even sighting effort on both sides of the trackline, observers did not confer during a sighting. Sighting information was entered into a custom-written program on a Hewlett-Packard 200LX palmtop computer on the sighting platform. Data record- ed included horizontal sighting angle, downward angle to sighting (in reticles), species, group size, orientation of the animals when first sighted, depth, Beaufort sea state, swell height, glare, GPS fix, date, and time. The program also recorded survey effort by storing a GPS fix every 60 seconds. Weather conditions were recorded at the start of field effort, and whenever they changed. Observers used reticle- and compass-equipped Fujinon 7x50 (WPC-XL) binoculars to make sightings and to measure the downward angle from the land, or horizon, to the sighting. If the former, the corresponding dis- tance to land was measured with RADAR (Furuno 1720 model), or, if within a few hundred meters of shore, with a Bushnell lightspeed laser rangefinder (tested accuracy ±1 m from 12 to 800 m). We calibrated the ac- curacy of the RADAR by comparison with transit fixes and laser rangefinder measurements. Sighting angles were recorded by using angle boards (see Buckland et al., 1993) in the first season, and thereafter with the compasses in the binoculars. There were no ferrous metals or significant electrical fields within 6 m of the sighting platform. Navigation was facilitated by the use of a Cetrek 343 GPS chartplotter with digitized C-MAP charts onto which transect waypoints were plotted. Depths were measured with a JRC JFV-850 echosounder (at 200 kHz). At the start of each survey, several days were spent training observers at Banks Peninsula, where sighting rates are high. Training continued until we gained about 100 sightings (data gathered in this period were not used in the analyses). An observer manual (avail- able from authors) specified scanning behavior and recording methods. To ensure a wide shoulder on the histograms of perpendicular sighting distances, observ- ers were instructed to concentrate their effort within 45° of the trackline and to spend less time searching out to 90°. Observers spent about 85% of the time scan- ning with binoculars. Regular scans with the naked eye minimized fatigue and reduced the chance of missing groups close to the boat. To promote consistency, observ- ers were asked to re-read the manual at least once a week throughout the survey. While the survey was underway, exploratory data analyses were undertaken to assess data quality. These analyses showed that in the early stages of the first sur- vey, observers were rounding angles of sightings close to the trackline to zero. The use of the angle boards was modified to minimize this problem, and they were not used in subsequent surveys. The data from these early lines were discarded and the survey lines repeated. Survey effort was restricted to sea conditions of Beau- fort 3 or less and swell heights of <2 meters. Transect lines were run down-swell and down-sun to minimize pitching and effects of glare. Deviations of up to 10° from the intended course were made if needed to further re- duce pitching or glare. The inshore end of each line was surveyed to just outside the surf zone on open coasts, or until a 2 m depth was reached, or to within 50 m of rocky shores. All surveys were conducted in passing mode to minimize the extent of vessel attraction. Line-transect data were collected in three surveys in three consecutive summer seasons, each focussing on a particular coastal area (Fig 2; Motunau to Timaru, 5 January-21 February 1998; Timaru to Long Point, 9 December 1998-16 February 1999; Farewell Spit to Motunau, 17 December 1999-28 January 2000). Survey design In order to obtain a clear picture of density and to mini- mize variance in encounter rate, Buckland et al. (1993) recommend placing transects across known density gradients. Because short-distance, alongshore move- ments are well-known for Hector's dolphins (Slooten and Dawson, 1994: Brager et al., 2002) and the dolphins' density declines sharply with distance offshore (Dawson and Slooten, 1988), transects were placed at 45° to the coast. On curved coastlines (within strata) we divided the coastline into blocks, drew an imaginary baseline along the coast, and placed lines at 45° to that baseline. The starting point of the first line along the baseline was decided randomly; thereafter lines were spaced at regular intervals according to the sampling intensity required in that stratum (Fig. 2). Within harbors we placed lines at 45° to an imaginary line down the center of the harbor (Fig. 3). The aim of this scheme was to ensure that, within a stratum, any one point had the same chance of being sampled as any other. Survey effort was stratified according to existing data on distribution, obvious habitat differences, and areas of intrinsic management interest. In summer, very few Hector's dolphins are seen beyond four nmi from shore (Dawson and Slooten, 1988); therefore most sampling effort was placed in this inshore zone (i.e. 45° lines at 2-, 4-, or 8-nmi spacings, approximately proportional to density as determined from previous surveys). Within harbors, transect spacings were either one or two nau- tical miles. In the offshore zone (from 4 to 10 nmi) we expected very low densities, and therefore used sparse transect spacing (-30 nmi apart). It was not our inten- tion to estimate density in this offshore zone. A subse- quent aerial survey was found to be better suited for this purpose (Rayment et al.1). Our goal was to estimate effective half strip width (ESW) separately for strata with different exposure to wind and swell. Hence, in each survey we aimed to gain sufficient sightings to estimate ESW separately for harbors or protected waters, and open coasts. To reach 1 Rayment, W., E. Slooten, and S. M. Dawson. 2003. Unpubl. data. Department of Marine Science, Univ. Otago, P.O. Box 56, Dunedin, New Zealand. 444 Fishery Bulletin 102(3) 172 55'S N / /, t X// -' • "' > '^\\ / ^ -M-- ^ (\\ o '>&°x£ W „ov- * Akaroa (^ yp%%y\ lx^fv>^ ^ *#3? (^NS. o^Jy J3_5P!§ _>_ -\febO - .2*C - x/^ /\\ / /A ^ J^-^j, 6 It it \J\l\ - wrr -_T- ; ■ 1 km Figure 2 Map of New Zealand's South Island, showing transect lines and sightings of Hector's dophins (dots) 1997-2000. Figure 3 Example of transect layout in harbors 1 1997- 98 Akaroa Harbor transect lines and sight- ings, showing three replicates I. Buckland et al.'s (1993) target of 60-80 detections for robust ESW estimation, in the 1997-98 survey we con- ducted replicate surveys (with a new set of lines each time) in the harbors and bays stratum (e.g., Fig. 3). Low sighting rates in the area surveyed in 1999-2000 would have required unrealistic effort levels to reach this target; therefore we gained extra sightings from areas with the same exposure but higher sighting rates (e.g., data used to calculate ESW for the Marlborough Sounds were supplemented by data gathered in Akaroa Harbour by the same observers, in the same summer). Hence different sample sizes were available to estimate density and ESW (Table 2). Because observers changed between surveys, we did not pool sightings across years for estimating ESW. Strata areas were measured from nautical charts with a digital planimeter. Data analysis Within each stratum, Hector's dolphin abundance (Ns) was estimated as (Buckland et al., 1993): Ns=^^. (1) 2LESW where A = size of the study area; n = number of groups seen; S = expected group size; L = length of transect line surveyed, and ESW = the effective half strip width. Because there was no significant relationship between group size and detection distance, expected group size was estimated as a simple mean group size. Dawson et al.: Line-transect surveys of Cephalorhynchus hecton 445 Table 2 Survev effort by stratum. Number of sightings is the total number made before truncation and quality auditing see "Vessel choice and field methods"). Survev effort No. of Sightings Survey zone Stratum (km) sightings per km Motunau to Timaru Banks Peninsula harbors and bavs 223 89 0.399 (1997-98) Banks Peninsula Marine Mammal Sanctuary (BPMMSi 265 66 0.249 (excluding open coasts) <4 nmi offshore, to the north and south of BPMMS 174 21 0.121 Offshore (4-10 nmi ) 89 4 0.045 Timaru to Long Point Timaru-Long Point (excluding Te Waewae Bay) 336 13 0.04 (1998-99) Te Waewae Bay 101 14 0.14 Offshore (4-10 nmi) 106 0 0 Motunau to Farewell Spit Farewell Spit-Stephens Island 120 0 0 (1999-20001 Marlborough Sounds (including Queen Charlotte Sound i 205 3 0.015 Cape Koamaru-Port Underwood 68 0 0 Cloudy Bay and Clifford Bay 89 13 0.146 Cape Campbell-Motunau 192 0 0.026 Offshore (4-10 nmi I 93 2 0.022 Using the program Distance 3.5 (Research Unit for Wildlife Population Assessment, University of St. An- drews, UK), we fitted detection functions to perpendicu- lar distance data to estimate ESW (note that this value is derived directly from f(0)). Akaike's information crite- rion (AIC) was used to select among models fitted to the data. Models and adjustments were the following: haz- ard/cosine, hazard/polynomial, half-normal/hermite, half- normal/cosine, uniform/cosine (Buckland et al., 1993). Following Buckland et al. (1993), perpendicular sighting distances were truncated to eliminate the farthest 5% of sightings and binned manually for /10) estimation. The coefficient of variation (CV) for the abundance es- timate was calculated from the coefficients of variation of each variable element in Equation 1 above (Buckland et al., 1993): CV(N5)=JcV-{)]) + CV2(S) + CV2[ESW). (2) The CV(n) was estimated empirically as recommended by Buckland et al. (1993): CV{n) = vardi) (3) (4) where var(«) = /.£/,<«, II,-n I L): I ik-\). lj = the length of transect line i; nj = the number of sightings on transect i; and k - number of transect lines. CV(S) was estimated from the standard error of the mean group size. CVlESW) was estimated with the bootstrapping option in Distance 3.5 software. This process incorporates uncertainty in model fitting and model selection (Buckland et al., 1993). Measuring the effect of attraction Conventional line-transect estimates can be biased as a result of responsive movement of the target species and animals on or near the trackline being missed by observers (Buckland et al., 1993). Buckland and Turnock (1992) presented a method using co-ordinated boat and helicopter surveys to quantify and adjust for the com- bined effects of responsive movements of dolphins to the boat and to eliminate the bias from observers failing to see animals on or near the trackline. Their approach is better suited to the restricted space available on small boats than to a dual-platform approach (Palka and Ham- mond, 2001). Additionally, sightings can be made much farther ahead (reducing the possibility that the animals have already responded), and the two sighting teams are totally isolated from each other. For these reasons we adapted Buckland and Turnock's (1992) approach in our trials of 1998-99. Simultaneous boat-and-helicopter surveys were car- ried out to the south of Banks Peninsula, predominantly between Birdlings Flat and the mouth of the Rakaia River. This area was chosen because it displayed rep- resentative and varying densities. A Robinson R22 helicopter with pilot and one observer (ES) followed a zig-zag flight path approximately 1.5 km in front of the boat, traveling out to 1000 m on either side of the vessel's trackline at a height of 500 ft ( 152 m) (Fig. 4). To aid the process of tracking sightings from the air, sighting positions were marked with Rhodamine dye bombs.2 The position of the helicopter in relation Dye bombs consisted of a tablespoon of Rhodamine dye in a paper cup 2/3 filled with sand. An additional (empty) paper cup was taped upside down on top of the first cup with paper-based masking tape. On impact the two cups broke apart, releasing the sand+dye mix into the water. 446 Fishery Bulletin 102(3) Figure 4 Schematic diagram of simultaneous helicopter-and-boat surveys for Hector's dolphins south of Bank Peninsula, South Island, New Zealand. to the boat was determined with the boat's RADAR. The absolute position of the boat was determined to an accuracy of 2-5 m by differential GPS (Trimble GeoExplorer; postprocessed). Distances to land were obtained at the time of sighting with RADAR or during analysis by using GIS coastline data and the computer program "SDR Map" (Trimble Navigation, Christchurch, New Zealand). Boat observers followed our standard sighting pro- cedures (see above). On most occasions the helicopter was outside the field of view of the observers' binoculars because the observers were scanning the water surface, and the helicopter was well above what the observers could sec through the binoculars. When it was within their view, observers made a conscious effort to remain unbiased by the movements of the helicopter. On mak- ing a sighting, the helicopter observer informed an independent observer located in the cabin (observers on the platform could not hear communications from the helicopter observer and vice versa). The helicopter then hovered briefly above the sighting while a range and bearing in relation to the boat was taken by RA- DAR. The helicopter then ceased hovering but tracked the group of dolphins either until the boat observers had sighted the group, or the group had passed abeam of the boat. A second range and bearing were then taken. Sightings lost by the helicopter observer during tracking were discarded in our analyses. The indepen- dent observer, in liaison with the helicopter observer and boat observers, determined whether the sighting was a duplicate (i.e., made by both helicopter and boat observers) by using information on location and group size. These decisions were checked again in analysis by inspection of plotted locations of sightings made from either platform or both platforms. Following the approach of Buckland and Turnock (1992), let gjy) = the probability that a group detected from the helicopter at perpendicular distance y from the trackline of the ship is subsequently detected from the ship; fs(y) = SsW'H' with ,» = |M "< y)2 were removed before f(0) estimation. The error for the correction factor (c) was estimated by bootstrapping on transect lines and applying the estimation procedure to each of 199 bootstrap data sets. The standard deviation of the bootstrap estimates was used as the standard error of c. Ideally, the correction factor would be estimated sepa- rately for each survey from separate sets of boat-and-he- licopter trials conducted in areas of representative den- sity. Financial and logistical constraints prevented this; therefore the correction factor estimated in 1998-99 was applied to each of the line-transect surveys reported in the present study. We note that this is not uncommon (e.g., Carretta et al., 2001). Unbiased abundance estimates were calculated by N, "■N, (10) The CVs of the corrected abundance estimates (NL,) were calculated with the following equation (Turnock et al„ 1995): CV(NU) = JCV2(£) + CV2(NS), where CV(c) 5£(r) (11) (12) Upper (Nuc) and lower (NLC) 95% confidence inter- vals for Nv were calculated by using the Satterthwaite degrees of freedom procedure outlined in Buckland et al. (1993). This procedure assumes a log-normal distribu- tion of Nc, using NLC = NL. I C, and Nuc = NUC, where C = exp \ r, ( 0.025 ) log, 1 + -[CV(N, >]" (13) (14) The Satterthwaite degrees of freedom (df) for corrected abundance estimate confidence intervals were calcu- lated by Table 3 Summary of variables required for corr ection factor (boat-and-helicopter trials) Parameter Estimate Length of transect. L (km) 308 Truncation distance, w (km) 1.0 Number of helicopter detections, nh 58 Number of ship detections, ns 126 Number of duplicate detections, nhs 33 ESW of helicopter (km) 0.532 ESW for duplicates ( km I 0.342 Apparent ESW of boat (kmi 0.268 Apparent density estimate ( groups/km2 1 0.7631 Corrected density estimate (groups/km2) 0.3839 Boat detection probability "near" trackline 0.8861 Correction factor (c) 0.5032 Standard error, SE(c) 0.0912 df= CV\N, ) Cl'V) CV\NS) (15) tf-1 dfs where B is the number of bootstrap samples, and dfs is the Satterthwaite degrees of freedom for the uncorrected abundance estimate, Ns (see Buckland et al., 1993). The CV of combined abundance estimates (Nai) was computed by SEUouih J{SE\Nm ) + SE-(N,,) + ... + SE-iN. «)}• and SEiloiah CV{total) = -^- N, (total) (16) (17) Results The three line-transect surveys covered 2061 km of tran- sect, and 231 sightings were used to estimate density (Table 2). Sighting rates were highest around Banks Peninsula (Table 3). The simultaneous boat-and-helicopter surveys indi- cated that boat observers missed 11.4% of the dolphins on the trackline, but that strong responsive movement towards the boat resulted in apparent densities twice as high as they normally would be (Table 3). If the observ- ers' attention was drawn to dolphin groups by the posi- tion of the helicopter, the results of these trials would be biased. This is unlikely, however, because several groups sighted by the helicopter observer subsequently passed within 200 m of the boat and were not seen by observers. We saw no evidence that the dolphins were affected by the helicopter. Detection functions for boat-and-helicopter sightings (Fig. 5, C and D) are relatively smooth in comparison 448 Fishery Bulletin 102(3) A 0 50 100 150 200 250 300 350 400 450 500 550 600 650 1 0 08 06 04 02 n a' a -v^^ 0 100 200 300 400 500 600 700 800 900 1000 0 50 100 150 200 250 300 350 400 450 500 550 600 0 50 100 150 200 250 300 350 400 450 500 550 600 E 100 700 300 400 S00 600 700 SO 100 ISO 200 2S0 300 3S0 400 4S0 500 Perpendicular distance (m) Figure 5 Histograms of perpendicular sighting distances, and their fitted detection functions as used to estimate effective strip width. /; = number of sightings. The fitted model (hazard, cosine, uniform, or half normal) and any adjustments to it (cosine or none) are given in brackets. (A) 1997-98 har- bors and bays (n = 71; hazard/cosine); (B) 1997-98 open coasts U?=75; uniform/cosine); (C) 1998-99 open coasts (re=121; half-normal/cosine); (D) 1998-99 helicopter sightings (/i = 58; half-normal i; (Ei 1998-99 duplicate sightings (n = 33; uniform/cosine); (F> 1999-2000 harbors and sounds (ra=70; hazard/cosinei; (G) 1999-2000 open coasts (n = 89; uniform/cosine). Dawson et al.: Line-transect surveys of Cepha/orhynchus hecton 449 with those presented in Turnock et al. (1995). The de- tection function for the duplicate sightings (Fig. 5E) was more difficult to fit. Given the restricted sample size of duplicates (n=33), this result is not unexpected. In the 1998-99 Timaru to Long Point and 1999-2000 Motunau to Farewell Spit surveys, robust estimation of ESW was facilitated by addition of extra sightings gained under similar sighting conditions at Banks Peninsula (Fig. 5, C, F, G). None of the three surveys showed significant evidence of larger groups being seen farther away. A broad pattern of abundance declining to the north and south of the Timaru-Banks Peninsula area is evident (Fig 2, Table 2). We made six sightings on 288 km of offshore lines (4-10 nmi offshore), con- firming that densities in this zone are low. Information on sea state is usually collected dur- ing boat line-transect surveys and sometimes used to poststratify data (e.g.. Barlow, 1995). In our study this was not advantageous, for three reasons. 1) We avoided collecting data in conditions with whitecaps; therefore only a few sightings were collected in Beaufort 3. Hence variance estimates for this Beaufort state are large. 2) Differences among Beaufort states for key parameters such as sighting rate, average group size, and effective strip width were small and showed overlapping confi- dence intervals (we concede that statistical power is low because of reason 1 stated above). Note that data were pooled in the same way as for ESW estimation. 3) Stratification by Beaufort state does not produce abundance estimates that match the zones of intrinsic management interest (e.g., Banks Peninsula Marine Mammal Sanctuary; Dawson and Slooten, 1993). Discussion The catamaran survey platform was a near-ideal vessel for close inshore surveys. The sighting platform (Fig. 1) was a relatively inexpensive modification (-US$2000) that could be dismantled in about 10 minutes to allow sailing. The vessel's minimal draught allowed coverage of very shallow areas, which are an important part of the distribution of Hector's dolphin and many other inshore cetaceans. Although catamarans are inherently resistant to rolling, pitching can be a problem when motoring into a head sea or swell. We minimized this pitching by arranging lines so they could be run down-swell. The 45° placement of lines facilitated this reduction in pitching because it provided two alternative sets of lines (at 90° to one another). Further, these could be run inshore or offshore, allowing a choice of four options. A significant advantage of vessels with low running costs is that the cost of training is low. We could af- ford to spend 7-10 days training before each survey. Further, waiting for weather to improve is inexpensive; therefore one does not need to gather data in marginal sighting conditions. Estimated abundances (Table 4) were not significantly different from those estimated in the 1984-85 strip transect survey. Recent mark-recapture estimates of dolphin abundance at Banks Peninsula in 1996, based on photo-ID data, differed from the line-transect es- timate for this area by less than 6% (Gormley, 2002; Jolly-Seber model allowing different capture probabili- ties between first and subsequent captures). Our surveys confirmed previous work showing the patchy nature of Hector's dolphin distribution (Dawson and Slooten, 1988). Research at Banks Peninsula on the alongshore range of individually identified dolphins has shown a mean alongshore range of about 31 km (SE = 2.43; Brager et al., 2002). Despite wide-ranging surveys over 13 years, the most extreme sightings of any individual were 106 km apart. These data indicate very high site fidelity and indicate that even small-scale discontinuities in distribution may be long lasting. Lack of extensive movement along-shore, and hence limited contact with neighboring populations, is likely to be the mechanism by which Hector's dolphin has become segregated into genetically distinct populations (Pichler et al., 1998; Pichler and Baker, 2000). The new abundance data, in combination with the genetic data indicating segregation of Hector's dolphin into four populations (Pichler and Baker, 2000) and modeling work indicating that the species is in decline in most of its range owing to bycatch in gill nets (Mar- tien et al., 1999; Slooten et al., 2000), underscore the urgent need for better information on bycatch rates. Despite strong evidence of bycatch throughout the species' range, observer coverage sufficient to estimate bycatch has been achieved only in one area (Canter- bury) for one fishing season (1997-98; Baird and Brad- ford, 2000). During this season six Hector's dolphins were observed entangled in commercial gill nets (a further two were caught but released alive), resulting in a bycatch estimate of 17 individuals (Starr3). One mortality was observed in a trawl net, but very low observer coverage prevented any calculations of overall trawl bycatch (Baird and Bradford, 2000). No attempt was made to assess bycatch in recreational gillnetting during this period, but during a more recent summer (2000-01) five Hector's dolphin mortalities occurred in gill nets that were probably set by recreational fish- ermen (Department of Conservation and Ministry of Fisheries, 2001). It is not reasonable to assume that all mortalities in recreational gillnets are detected. In our opinion it is likely that combined commercial and recreational gillnet bycatch off Canterbury is about 15-30 individuals per year. Hector's dolphin abundance on the north, east, and south coasts of the South Island estimated from the sur- veys reported in the present study is 1880 individuals (CV=15.7%). Hector's dolphins are more common on the Starr, P. 2000. Comments on "Estimation of the total bycatch of Hector's dolphins (Cephalorhynchus hectori) from the inshore trawl and setnet fisheries off the east coast of the South Island in the 1997-98 fishing year." Unpublished paper presented to Conservation Services Levy Working Group, 28 p. Department of Conservation, P.O. Box 10-420 Wellington. New Zealand. 450 Fishery Bulletin 102(3) Table 4 Corrected abundance estimates (only strata with sightings are listed I . Number of sighti ngs represents only those made in that stratum and used for density estimation. The number rsed for estimating effective half strip width. (ESW) differs because it includes sightings from extra transects in areas of similar exposure ar d transects repeal ed on the same day ( rnd hence not true replicates for the purposes of estimating density). No. of ESWim) N(c) Lower Upper Survey zone Stratum sightings (n,CV%) rv. 95% CI 95% CI Motunau to Timaru Akaroa harbor 56 275 62 32 121 (1997-981 (71,22.6) (33.9) Other harbors and bays 8 275 171,22.6) 14 (67.5) 3 79 Banks Peninsula Marine M ammal Sanctuary 62 261 821 535 1258 iBPMMS) (excluding harbors and bays) (75, 10.3) (22.1) <4 nmi offshore, to the north and south of BPMMS 19 261 300 133 679 175, 10.3) (36.5) Timaru to Long Point Timaru-Long Point (exclud ng Te Waewae Bay 13 268 310 201 478 (1998-99) (121, 10.5) (28.4) Te Waewae Bay 14 268 (121, 10.5) 89 (32.4) 36 218 Motunau to Farewell Spit Queen Charlotte Sound 3 214 20 4 111) (1999-2000) (70,20.2) (100.5) Cloudy and Clifford Bay 13 277 (89,6.1) 162 (55.4) 56 474 Cape Campbell-Motunau 5 277 (89,6.1) 102 (55.2) 34 305 Total 1880 (21.3) 1246 2843 South Island west coast, where an aerial survey of simi- lar design resulted in an estimate of 5388 (CV=20.6<*; Slooten et al., in press). Thus Hector's dolphin abun- dance in South Island waters is estimated at 7268 in- dividuals (CV=15.8'7r ). The North Island subspecies of Hector's dolphin, now considered critically endangered (IUCN4) remains to be surveyed quantitatively. The new abundance estimates provide an empirical basis from which to calculate levels of take that would still allow the currently depleted populations to recover (e.g., Wade, 1998). These levels of take should be seen as short-term targets for bycatch reduction in gill and trawl nets. For the management of Hector's dolphin to be put on a rational basis, a more comprehensive and wide-ranging assessment of bycatch, including statisti- cally robust observer programs in coastal fisheries, is urgently needed. nificant contributions to equipment used in the survey- were made by the New Zealand Whale and Dolphin Trust and the University of Otago. We are very grateful for the hard work put in by the other observers: Laszlo Kiss, Nadja Schneyer, Gail Dickie, Niki Alcock, Lesley Douglas, James Holborow, Ellie Dickson, Guen Jones, Will Rayment, and Dan Cairney. Jay Barlow, Jeff Laake, Anne York, and Debbie Palka shared their thoughts on survey design and field methods. David Fletcher helped with aspects of variance estimation. Akaroa Harbour Cruises provided much appreciated field support. Daryl Coup wrote the sightings program we used to collect data in the field. Otago University's Department of Surveying very helpfully provided the GPS base-station data for postprocessing our GPS fixes. Literature cited Acknowledgments These surveys were funded principally by Department of Conservation Contracts SCO 3072, 3074, and 3075. Sig- 4 IUCN. 2000. IUCN red list of threatened species. In- tel national Union for Conservation of Nature and Natural Resources. Species Survival Commission. [Available at www.redlist.oru I Baird. S. J., and E. Bradford. 2000. Estimation of the total bycatch of Hector's dolphins Wcphalurhynchiis hectori) from the inshore trawl and setnel fisheries off the east coast of the South Island in the L997-98 fishing year. 28 p. Published client report on contract 3024. funded by Conservation Services Levy, Department of Conservation. Wellington. Web site: http://csl.doc.govt.nz/CSL3024.pdf. [Accessed 8 March 2001.1 Dawson et al.: Line-transect surveys of Cepholorhynchus hectori 451 Baker, A. N.. A. N. H. Smith, and F. B. Pichler. 2002. Geographical variation in Hector's dolphin: recogni- tion of a new subspecies of Cephalorhynchus hectori. J Roy. Soc. N.Z. 32:713-727. Barlow, J. 1988. Harbor porpoise, Phocoena phocoena, abundance estimation for California, Oregon, and Washington: I. Ship surveys. Fish. Bull. 86:417-432. 1995. The abundance of cetaceans in California waters. Part 1: Ship surveys in summer and fall of 1991. Fish. Bull. 93:1-14. Brager, S., S. M. Dawson, E. Slooten, S. Smith, G. S. Stone, and A. Yoshinaga. 2002. Site fidelity and along-shore range in Hector's dol- phin, an endangered marine dolphin from New Zea- land. Biol. Conserv. 108:-287. Buckland, S. T.. D. R. Anderson, K. P. Burnham, and J. L. Laake 1993. Distance sampling: estimating abundance of bio- logical populations, 446 p. Chapman and Hall, New York, NY. Buckland, S. T., and B. J. Turnock. 1992. A robust line transect method. Biometrics 48: 901-909. Carretta, J. V., B. L. Taylor, and S. J. Chivers. 2001. Abundance and depth distribution of harbor por- poise i Phocoena phocoena) in northern California de- termined from a 1995 ship survey. Fish. Bull. 99:29-39. Dawson, S. M., and E. Slooten. 1988. Hector's Dolphin Cephalorhynchus hectori: distri- bution and abundance. Rep. Int. Whal. Comm. (special issue) 9:315-324. 1993. Conservation of Hector's dolphins: the case and process which led to establishment of the Banks Pen- insula Marine Mammal Sanctuary. Aquat. Cons. 3:207-221. Dawson, S. M., E.. Slooten, F Pichler, K. Russell, and C. S. Baker. 2001. North Island Hector's dolphins are threatened with extinction. Mar. Mamm. Sci. 17:366-371. Department of Conservation and Ministry of Fisheries. 2001. Hector's dolphin and setnetting Canterbury Area: a paper for public comment, 9 p. [Available from Min- istry of Fisheries, Private Bag 1926, Dunedin, New Zealand. I Gormley, A. 2002. Mark-recapture abundance estimation in marine mammals. M.S. thesis., 105 p. Univ. Otago, Dunedin, New Zealand. IWC (International Whaling Commission). 1994. Report of the International Whaling Commission workshop and symposium on the mortality of cetaceans in passive fishing nets and traps. Rep. Int. Whal. Comm. (special issueil5. 629 p. Lerczak, J. A., and R. O. Hobbs. 1998. Calculating sighting distances from angular read- ings during shipboard aerial, and shore-based marine mammal surveys. Mar. Mamm. Sci. 14:590-599. Martien. K. K., B. L. Taylor, E. Slooten, and S. M. Dawson. 1999. A sensitivity analysis to guide research and man- agement for Hector's dolphin. Biol. Conserv. 90:183- 191. Palka, D. L., and P. S. Hammond. 2001. Accounting for responsive movement in line transect estimates of abundance. Can. J. Fish. Aquat. Sci. 58(41:777-787. Pichler, F, and C. S. Baker. 2000. Loss of genetic diversity in the endemic Hector's dolphin due to fisheries-related mortality. Proc. Roy. Soc. (Londi, Series B. 267:97-102. Pichler, F. B., S. M. Dawson, E. Slooten, and C. S. Baker. 1998. Geographic isolation of Hector's dolphin popula- tions described by mitochondrial DNA sequences. Cons. Biol. 12:676-682. Slooten, E.. and S. M. Dawson. 1994. Hector's Dolphin. In Handbook of marine mam- mals, vol V ( Delphinidae and Phocoenidae M S. H Ridgway and R. Harrison, eds.), p. 311-333. Academic Press. New York, NY. Slooten. E.. S. M. Dawson, and W. J. Rayment. In press. Aerial surveys for coastal dolphins: Abundance of Hector's dolphins off the South Island west coast, New Zealand. Mar. Mamm. Sci. 20(3 I. Slooten. E.. D. Fletcher, and B. L. Taylor. 2000. Accounting for uncertainty in risk assessment: Case study of Hector's dolphin mortality due to gillnet entanglement. Cons. Biol. 14:1264-1270. Turnock, B. J.. S. T. Buckland. and G. C. Boucher. 1995. Population abundance of Dall's porpoise iPhocoe- noides dalli) in the Western North Pacific Ocean. Rep. Int. Whal. Comm. (special issue) 16:381-397. Wade, P. 1998. Calculating limits to the allowable human-caused mortality of cetaceans and pinnipeds. Mar. Mamm. Sci. 14:1-37. 452 Abstract— A developmental series of larval and pelagic juvenile pygmy rockfish (Sebastes wilsoni) from cen- tral California is illustrated and described. Sebastes wilsoni is a non- commercially. but ecologically, impor- tant rockfish, and the ability to dif- ferentiate its young stages will aid researchers in population abundance studies. Pigment patterns, meristic characters, morphometric measure- ments, and head spination were recorded from specimens that ranged from 8.1 to 34.4 mm in standard length. Larvae were identified ini- tially by meristic characters and the absence of ventral and lateral midline pigment. Pelagic juveniles developed a prominent pigment pattern of three body bars that did not extend to the ventral surface. Species identifica- tion was confirmed subsequently by using mitochondrial sequence data of four representative specimens of various sizes. As determined from the examination of otoliths, the growth rate of larval and pelagic juvenile pygmy rockfish was 0.28 mm/day, which is relatively slow in compari- son to the growth rate of other spe- cies of Sebastes. These data will aid researchers in determining species abundance. Description and growth of larval and pelagic juvenile pygmy rockfish (Sebastes wilsoni) (family Sebastidae) Thomas E. Laidig Keith M. Sakuma Santa Cruz Laboratory Southwest Fisheries Science Center National Marine Fisheries Service, NOAA 110 Shaffer Rd Santa Cruz, California 95060 E-mail address: torn laidigiS'noaa. gov Jason A. Stannard La Jolla Laboratory Southwest Fisheries Science Center National Marine Fisheries Service, NOAA P. O. Box 271 La Jolla, California 92038 Manuscript submitted 9 -June 2003 to Scientific Editor's Office. Manuscript approved for publication 25 February 2004 by the Scientific Editor. Fish. Bull. 102:452-463(20041. Rockfishes (genus Sebastes) form a diverse group comprising at least 72 species occurring in the northeast- ern Pacific (Love et al., 2002). Many of these species represent a substan- tial portion of the groundfish fishery off the west coast of North America, accounting for 20% of the groundfish landings in California in 2000 (Pacific Fishery Management Council, 2000). A few species are relatively abun- dant but are not harvested because of their small size. These species play vital roles in the community ecology, including providing prey for the larger, commercially important species. The pygmy rockfish (Sebastes wilsoni) having a maximum size of 23 cm total length, is among these small species (Love et al., 2002). Pygmy rockfish are common over sediment and rocky sea- floor habitats at a depth of 30-274 m (Stein et al., 1992; Yoklavich et al., 2000). Stein et al. (1992) observed that pygmy rockfish were by far the most abundant fish species off Heceta Bank, Oregon, and Love et al. (1996) reported "clouds" of pygmy rockfish mixed with two other small species, squarespot rockfish (S. hopkinsi ) and halfbanded rockfish (S. semicinctus) off southern California. In Soquel Canyon in central California, pygmy rockfish dominated fish assemblages in rock-boulder habitat at 75-175 m (Yoklavich et al., 2000). Accurate identification of larval stages is critical. Biomass of rock- fish populations can be estimated from larval production (Ralston et al., 2003) and larval and juvenile abundance studies (Moser and But- ler, 1987; Hunter and Lo, 1993 I. If the larval and juvenile rockfish ana- lyzed in these studies are not correct- ly identified, it could lead to either over- or underestimates of biomass or recruitment potential of a popula- tion. Identification of young stages of Sebastes has been accomplished through rearing studies and through descriptions based on developmental series of field-caught specimens of various sizes (Matarese et al., 1989; Moser, 1996). Otolith morphologies have been useful in discerning some Sebastes species (Laidig and Ralston, 1995; Stransky, 2001). Recently, mo- lecular methods have proven to be an effective tool for the identification of Sebastes larvae (Seeb and Kendall, 1991; Rocha-Olivares, 1998; Rocha- Olivares et al„ 2000). In this study, we identify and de- scribe the larvae and pelagic juveniles of pygmy rockfish based on morpho- metries and pigmentation patterns, and estimate age and growth at two Laidig et al.: Descriptions and growth of larval and juvenile Sebastes wilsoni 453 developmental stages. Further, we examine otolith ra- dius at time of larval extrusion to separate pygmy rock- fish from other similarly pigmented Sebastes specimens. We also use mitochondrial DNA (mtDNA) sequence data to identify four putative pygmy rockfish specimens representing a continuum of late-larval through pelagic juvenile stages. The molecular results are used to con- firm identifications based on morphological, meristic, and pigmentation characters and to assure that the assembled developmental series is monospecific. Methods Specimen collection Specimens of larval and pelagic juvenile pygmy rockfish were obtained from research cruises conducted aboard the NOAA RV David Starr Jordan off central California. Specimens were collected in midwater (5-30 m) from mid-May to mid-June, 1990-92, between Bodega Bay (north of San Francisco) and Cypress Point (south of Monterey Bay) by using a 26 mx26 m modified Cobb midwater trawl (12.7-mm stretched-mesh codend liner). Specimens also were collected during early March, 1992-93, between Salt Point (north of San Francisco) and Cypress Point with a 5 mx5 m modified Isaacs-Kidd (MIK) frame trawl with 2-mm net mesh and 0.505-mm mesh codend. Specimens from the Cobb trawl were frozen and specimens from the MIK frame trawl were preserved in 95% ethanol for later analysis. Meristics, morphometries, and body pigmentation We examined pigmentation patterns and physical char- acteristics of 122 pygmy rockfish larvae and pelagic juveniles. Standard length (SL) was measured for each individual and sizes ranged from 8.1 to 34.4 mm. Speci- mens greater than 19.9 mm were identified by using meristic characters (Chen. 1986; Matarese et al.. 1989; Moreland and Reilly, 1991; and Laroche1), and pigment patterns were recorded. Specimens less than 20 mm were identified initially from pigment patterns from a series starting with the smallest (8.1 mm SL) identifi- able individuals with complete fin-ray counts. Counts of dorsal-, anal-, and pectoral-fin rays, and the number of gill rakers on the first arch were recorded whenever possible and subsequently used in identifications. Gill raker counts were obtained only from fish larger than 15 mm SL. We measured snout-to-anus length, head length, snout length, eye diameter, body depth at the pectoral fin base, body depth at anus, and pectoral-fin length on 16 specimens ranging from 8.1 to 29.6 mm SL, follow- ing Richardson and Laroche (1979). Head spination was examined on thirty-three specimens (8.1 to 29.6 mm 1 Laroche, W. A. 1987. Guide to larval and juvenile rock- fishes (Sebastes) of North America. Unpubl. manuscript, 311 p. Box 216, Enosburg Falls, VT 05450. SL) that were stained with alizarin red-s. Terminol- ogy for head spination follows Richardson and Laroche (1979). In the following descriptions, larval and juvenile lengths always refer to SL and pigmentation always refers to melanin. Otolith examination Sagittal otoliths were removed from 61 larval and pelagic juvenile pygmy rockfish (8.1-34.4 mm SL), and growth increments were counted beginning at the first incre- ment after the extrusion check (the mark in the otolith formed when the larvae are released from their mother) by using a compound microscope at lOOOx magnifica- tion (see Laidig et al., 1991). No validation of the these growth increments was performed during the present study, and none has been conducted by other research- ers. However, we assumed that these growth increment counts corresponded to daily ages based on validation of daily growth increments in other co-occurring rock- fishes, namely shortbelly rockfish, S. jordani (Laidig et al., 1991), black rockfish, S. melanops (Yoklavich and Boehlert, 1987), bocaccio, S. paucispinis, chilipepper, S. goodei, widow rockfish, S. entomelas, and yellowtail rockfish, S. flavidus (Woodbury and Ralston, 1991). The radius of the otolith was measured from the primordium to the postrostral edge of the extrusion check for com- parison with similar measurements from other Sebastes spp. (as reported in Laidig and Ralston, 1995). Transfor- mation from the larval stage to the pelagic juvenile stage was ascertained by the presence of accessory primordia (Laidig et al., 1991; Lee and Kim, 2000). Molecular confirmation Total genomic DNA was isolated from skeletal muscle tissue of four larval and juvenile putative pygmy rock- fish specimens by using a CTAB and phenol-chloro- form-isoamyl alcohol protocol ( Winnepenninckx et al., 1993; Hillis et al., 1996). These four specimens ranged in length from 15 to 27 mm and had pigment patterns similar to the fish identified as pygmy rockfish in the present tudy. Polymerase chain reaction (PCR) ampli- fications and sequencing of partial mitochondrial DNA regions (cytochrome b [cyt-6] and control region [CR]) followed the methods of Rocha-Olivares et al. (1999a, 1999b). PCR products were verified on 29c agarose gels and purified by using a QIAquick™ PCR Cleanup Kit (Qiagen, Inc., Valencia, CA) following manufacturer protocols. Complementary strand sequence data were generated by using ABI PRISM' M DyeDeoxy™ termina- tor cycle sequence chemistry on an automated sequencer (Applied Biosystems, Model 377, Foster City, CA). Cytochrome b sequence data (750 base pairs) from the four specimens were aligned with (previously gen- erated) orthologous sequences from 119 individuals representing 61 species of Sebastes (Rocha-Olivares et al., 1999b). Species identifications, based on cyt-fo data, were made by using distance-based cluster analyses in PAUP v4.0b2 (Phylogenetic Analysis Using Parsimony, 454 Fishery Bulletin 102(3) version 4, Sunderland, MA) and pairwise comparisons of sequence divergence (i.e., the number of nucleotide differences between two individuals expressed as a per- centage). A secondary data set, which included an ad- Table 1 Frequency of occurrence ( number of fish i and pectoral-fin ray counts, and gill ra 122 pygmy rockfish tSebastes wilsoni). of dorsal-, anal-, ker counts from Character Count Frequency of occurrence Percent occurrence Dorsal-fin rays 12 8 7 13 110 91 14 3 2 Anal-fin rays 5 2 2 6 116 95 7 4 3 Pectoral-fin rays 16 5 5 17 92 90 18 5 5 Gill rakers 36 11 14 37 15 18 38 22 28 39 20 25 40 10 13 41 1 1 42 1 1 ditional 450 base pairs of control region sequence, was generated for the four undetermined specimens and for a subgroup of known reference species with low levels of sequence divergence from the four putative pygmy rockfish specimens ( Puget Sound rockfish [S. empha- eus], redstripe rockfish [S. proriger], harlequin rockfish IS. variegatus]. sharpchin rockfish [S. zaeentrus], and pygmy rockfish). Species identifications, based on this extended (cyt-fe + CR) data subset, followed analyses described above. Results General development All 122 fish had completed notochord flexion and pos- sessed a full complement of segmented fin rays by 8.1 mm. The mode for dorsal-fin ray counts was 13, for anal-fin rays 6, and for pectoral-fin rays 17 (Table 1). The mode for gill raker counts was 38, and the range was 36-42. Anal- and dorsal-fin spines began to develop between 9.1 and 14.0 mm. Lateral line pores began to develop at 29 mm, although a full complement (37 to 46 pores) was not reached in our specimens. Morphometric measurements were taken from 16 individual pygmy rockfish of 8.1-29.6 mm (Table 2). Head spination At 8.1 mm, the postocular, parietal, nuchal, inferior post- temporal, supracleithral, superior opercular, preopercu- lars (with the exception of the 2nd anterior), and 1st and Table 2 Morphometric measurements (in mm) fr om 16 individual ; of pygmy rockfish tSebastes wilsoni). Snout-anus Head Snout Eye Body depth Body depth Pectoral-fin SL length length length diameter at pectoral base at anus length 8.1 5.2 3.3 0.8 1.3 2.8 2.3 1.5 9.0 5.3 3.0 1.0 1.5 3.0 2.3 1.7 10.8 6.5 4.2 1.2 1.7 3.5 2.8 2.3 12.1 7.0 4.3 1.3 2.0 3.8 2.8 2.5 12.8 7.7 4.8 1.3 2.2 3.7 3.0 2.5 14.2 8.3 4.7 1.3 2.2 4.3 3.5 3.3 15.2 9.2 5.7 1.7 2.0 4.2 3.5 3.5 16.2 9.7 5.2 1.5 2.0 4.5 3.8 3.8 17.5 10.8 6.2 2.0 2.3 5.0 3.8 4.2 18.6 11.5 6.0 2.0 2.7 5.5 4.3 5.0 20.7 12.7 6.2 2.0 2.7 6.2 5.2 5.0 22.3 14.2 6.7 2.2 2.8 6.8 5.7 6.2 23.8 14.5 6.7 2.0 3.2 7.3 6.3 5.9 24.3 14.2 7.0 2.2 2.7 7.0 5.8 6.3 28.9 17.0 8.0 2.6 3.3 8.5 7.3 7.0 29.6 16.9 7.8 2.6 3.5 8.7 7.5 6.9 Laidig et al.: Descriptions and growth of larval and juvenile Sebastes wilsoni 455 2nd inferior and 1st superior infraorbital spines were present (Table 3). The nasal, pterotic, and 4th superior infraorbital first appeared at 10.8 mm. At 12.1 mm, the inferior opercular spine became evident. Between 14.2 and 17.5 mm, the preocular, tympanic, superior postem- poral, 2nd anterior preopercular. and 3rd superior infraor- bital spines formed. After 17.5 mm, no further changes in head spination were noted. The supraocular, coronal, 3rd inferior infraorbital, and 2nd superior infraorbital spines did not occur on any of the fish examined. Body pigmentation At 9.1 mm, pygmy rockfish had no pigment along the lateral and ventral body surfaces (Fig. 1A, Table 4). Pigment was heavy on the top of the head and present on the operculum. The dorsal midline surface had a few melanophores under the soft dorsal fin. The ante- rior lower jaw was pigmented on the tip and one to two melanophores were present on each side of the snout near the tip. Pigment also was present at the base and on the distal half of the pectoral fin. By 14.0 mm, dense pigment along the dorsal midline stretched from the caudal peduncle to the first dorsal-fin spine (Fig. IB, Table 4). The only lateral pigment on the body consisted of a few melanophores along the midline near the caudal peduncle. The ventral surface, includ- ing the anal and pelvic fins, remained unpigmented. Pigment on the pectoral fins had mostly disappeared by 11.0 mm and was rarely observed at 14.0 mm. Opercu- lar and head pigment increased in density by 14.0 mm. Pigment along the lower edge of the orbit began to Table 3 Development of head spi aes in individua pygmy rockfish iSebastc • wilsoni ). "1' means spines present and "0" means spines absent. Spines Standard length (mm) 8.1 9.0 10.8 12.1 12.8 14.2 15.2 16.2 17.5 18.6 20.7 22.3 23.8 24.3 28.9 29.6 Nasal 0 0 1 1 1 1 1 1 111111 Preocular 0 0 0 0 0 0 1 1 111111 Supraocular 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Postocular 1 1 1 1 1 1 1 1 111111 Coronal 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tympanic 0 0 0 0 0 0 0 1 1 111111 Parietal 1 1 1 1 1 1 1 1 111111 Nuchal 1 1 1 1 1 1 1 1 111111 Pterotic 0 0 1 1 1 1 1 1 111111 Posttemporal Superior 0 0 0 0 0 0 1 1 111111 Inferior 1 1 1 1 1 1 1 1 111111 Supracleithral 1 1 1 1 1 1 1 1 111111 Opercular Superior 1 1 1 1 1 1 1 1 111111 Inferior 0 0 0 1 1 1 1 1 Preopercular 1st anterior 1 1 1 1 1 1 1 1 111111 2nd anterior 0 0 0 0 0 1 1 1 111111 3rd anterior 1 1 1 1 1 1 1 1 111111 1st posterior 1 1 1 1 1 1 1 1 111111 2nd posterior 1 1 1 1 1 1 1 1 111111 3rd posterior 1 1 1 1 1 1 1 1 111111 4th posterior 1 1 1 1 1 1 1 1 111111 5th posterior 1 1 1 1 1 1 1 1 111111 Infraorbital 1st inferior 1 1 1 1 1 1 1 1 111111 2nd inferior 1 1 1 1 1 1 1 1 111111 3rd inferior 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1st superior 1 1 1 1 1 1 1 1 111111 2nd superior 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3rd superior 0 0 0 0 0 0 1 1 111111 4th superior 0 0 1 1 1 1 1 1 111111 456 Fishery Bulletin 102(3) Figure 1 Developmental series of pygmy rockfish [Sebastes wilsoni) (drawn by authors). (A) 9.1-mm- SL larva; (B) 14.0-mm-SL larva; iC) 17.5-mm-SL larva; (D) 23.0-mm-SL pelagic juvenile; I E ) 28.6-mm-SL pelagic juvenile. Arrow marks ventral end of anterior body bar; I F ) 34.4- mm-SL pelagic juvenile. Note that not all head spines are included in the illustrations. form in some specimens by 12.0 mm and was visible in most specimens by 14.0 mm (Fig. IB). Snout pigment was represented by one or four melanophores. Anterior lower jaw pigment was heavy and confined to the tip of the jaw. By 17.5 mm, the dorsal midline pigment had become much darker and denser (Fig. IC, Table 4) and extended from the caudal fin to the head region, except for a gap where the nape pigment was beginning to form. All fins were unpigmented. Pigment along the ventral body midline began to form at 17.5 mm, with a few postanal melanophores. Lateral midline pigment formed in two locations. Melanophores near the peduncle increased anteriorly, and pigment began forming dorsal to the gut cavity and increased posteriorly toward the peduncle. A body bar began to form on the lateral surface above the pectoral fin between the spinous dorsal fin and the anterior lateral midline pigment. Opercular, eye, and head pigment all increased in density. Melanophores on the snout also became more prevalent between the tip of Laidig et al.: Descriptions and growth of larval and |uvenile Sebastes wilsoni 457 Figure 1 (continued) the upper jaw and top of the head. Pigment on the tip of the lower jaw spread posteriorly and became denser than in smaller specimens. When the pelagic juveniles reached 23.0 mm, the dorsal midline pigment was a dark strip extending from the caudal fin to the head (Fig. ID, Table 4). Nape pig- ment almost merged with the head pigment, except for a small unpigmented area below the insertion of the parietal and nuchal spines. Hypural pigment was pres- ent distally in all individuals at this size and at larger sizes (n = 30). Anterior and posterior lateral midline pig- ment merged to form a continuous line along the body. The number of melanophores increased on the ventral body surface, and a few melanophores were present at the anal-fin ray bases and along the ventral midbody posterior to the anal fin. The anterior body bar broad- ened and became more defined. A few melanophores on the flanks under the soft dorsal fin began to form a midbody bar. A third body bar began to form on the caudal peduncle. Pigment on the operculum, top of the 458 Fishery Bulletin 102(3) 2 5 f J3 .i +* TO g.| ^ II l g I 5 n -S « -5 J u 3 J _• a. 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Me- lanophores posteriorly around the eye socket increased in number. The fins remained unpigmented. Pygmy rockfish 28.6 mm long had dorsal pigment that stretched continuously from the jaws to the caudal fin (Fig. IE. Table 4). Pigmentation was heavy along the dorsal midline, head, and nape. Snout pigmenta- tion also intensified. More melanophores were present on the hypural margin. Pigment along the ventral body surface darkened, especially in the area posterior to the anal fin. More melanophores were observed at the anal-fin ray articulations than on smaller specimens. The three body bars increased in width and length and were better defined than on smaller specimens. The bar on the caudal peduncle began to exhibit a rectangular shape that is characteristic of the juvenile stage. The midbody bar also took on a rectangular shape, although the dorsal half was indented. The midbody bar and the caudal peduncle bars did not reach the ventral midline. The anterior body bar extended from the spinous dorsal fin to the vent (see arrow Fig. IE). Anteriorly, the bar formed a more or less rectangular pattern on the dorsal half of the body above the pectoral fin. In general, the lateral body surface became more heavily pigmented, especially on the dorsal half. The lateral midbody pig- ment line began to be incorporated into the body bars. Opercular pigment became denser and merged with the nape pigment. The area anterior to the nape and operculum was less pigmented than the surrounding areas. Pigment along the posteroventral portion of the orbit became denser than in smaller specimens. A cheek bar began to form ventral to the eye (as evidenced by the two melanophores in Fig. IE ). Melanophores formed along the ventral surface of the lower jaw and covered the lateral surface of the upper jaw. Pigment began to develop on the membranes of the spinous dorsal fin, typically with some unpigmented areas between the dorsal fin pigment and the dorsal body pigment. The largest specimen. 34.4 mm, had the densest and most distinctive pigmentation (Fig. IF, Table 4). Pig- ment was present on most of the body. Along the dorsal surface, the pigment formed a complete line from the tip of the upper jaw to the caudal fin. The number of melanophores increased along the hypural region, the postanal ventral midline, and at the anal-fin articula- tions. The mid- and caudal body bars were rectangular and still did not reach the ventral midline, leaving an unpigmented ventrolateral area. The anterior body bar comprised heavy pigment extending posteriorly between dorsal-fin spines VIII-XI and the vent, a lighter area just anterior to this, and another heavily pigmented area stretching from about dorsal-fin spines III— VI almost to the middle of the gut cavity. Anterior to this bar was an area of mottled pigmentation. Pigment was visible just anterior to the base of the pectoral fin. Pigment covered both the spinous and soft dorsal fins, except along the distal edge. All other fins remained unpigmented. Opercular pigment was dense and merged with the nape pigment, but these were separated from 40 35 E £ 30 _SL = 0.28(AGE)-5A5 _c ■ ms °> 25 S 20 ~o a 15 55 10 ■ / 20 40 60 80 100 120 140 160 Age (days) Figure 2 Standard length and age of pygmy rockfish i Sebastes wilsoni) in = 60 1. Solid line indicates predicted values from linear model. the head and eye pigment by an area of low pigment density. Two cheek bars radiated from the lower margin of the orbit. Pigment occurred along both jaws and cov- ered the snout and ventral portion of the lower jaw. Otolith examination A linear relationship between standard length and age (as estimated from otolith increment counts) resulted in a good estimate of growth of pygmy rockfish (slope = 0.28 mm/d; intercept=-5.15 mm; r2=0.91; ?i = 60; Fig. 2). The radius of the extrusion check ranged from 9.5 to 11.0 ^m, averaging 10.5 /jm (SD = 0.29; « = 60). Accessory pri- mordia first appeared in a 19.8-mm specimen and were observed in otoliths from all larger specimens. Based on this character, transition from larval to pelagic juvenile stage occurs at around 20 mm SL. Molecular confirmation Interspecific levels of divergence, calculated among adult reference species, ranged from 0.13% (rougheye rockfish [S. aleutianus] vs. shortraker rockfish [S. borealis]) to 9.7% (black rockfish [S. inermis] vs. bocaccio) with an average of 4.1%. Two of the specimens (FT2 and FT3; Fig. 3A) were identical to one of the adult pygmy rockfish references (i.e. 0% sequence divergence i and differed from the other verified adult pygmy rockfish by a single nucleotide substitution (0.13% seq. div. ). The remain- ing two specimens (FT1 and FT4; Fig. 3A) also were most similar to both adult pygmy rockfish references (0.13-0.40% seq. div.). Although all four specimens were most similar to pygmy rockfish based on cytochrome b data, only a small number of nucleotide differences separated them from Puget Sound, redstripe, harlequin, and sharpchin rockfish (0.27-1.87%; Fig. 3A). A secondary data subset that included control region sequence (cyt-6+CR) yield- ed concordant results; all four larval specimens were 460 Fishery Bulletin 102(3) 3.5- A Cyt-£> B Cyt-to + CR 3- • • • • X emphaeus (BU5) 2.5- X emphaeus (BU6) • pronger (BS4) >g □ vanegatus (BS5) 0) □ vanegatus (BS7) = 2- O zacentrus (BQ7) • A iv/fcoro(BP11) 0 o A wilsoni (BT1 1 ) > s -o • • 8 1.5- X H w 1- □ D X X D x o A o X x □ A 0.5- D O O H A A A H D □ * A a H H * A AAA 0- 1 1 t 1 12 3 4 12 3 4 Larval and juvenile specimens Figure 3 Percent sequence divergence based on the number of nucleotide differences in iAi cytochome 6 and (Bi cytochrome b + control region between the four putative pygmy rockfish I Sebastes wilsoni) specimens iFT1-FT4i and five closely related reference species of Sebastes, including two a dult S. wilsoni. Per- cent sequence divergence was calculated as the number of nucleotide differences over 750 base pairs (cyt-6) and 1200 base pairs (cyt-6+CRi. most similar to pygmy rockfish (0.25-0.83%; Fig. 3B). Increased levels of interspecific nucleotide variation, attributable to the faster evolving control region, re- sulted in more pronounced differences between the four specimens and the other species of Sebastes within the subset (range: 0.83-3.00%; Fig. 3B). Additionally, a distance-based analysis (UPGMA) of haplotypes (cyt- fr+CR) clustered all four specimens with pygmy rockfish reference material. Discussion Postflexion larval pygmy rockfish can be identified through a combination of pigment and meristic char- acters. At approximately 8-10 mm, the larval pigment pattern is similar to only four of the 30 Sebastes spe- cies illustrated in the literature that occur within our geographic area (Matarese et al., 1989; Moser, 1996; Laroche1): yellowtail (S. flavidus), blue (S. mystinus), canary (S. pinniger), and sharpchin rockfish. Yellowtail and blue rockfish can be separated from pygmy rockfish because they exhibit ventral body and hypural pigment at this size — pigment that does not show up in pygmy rockfish until approximately 14 and 15 mm, respectively. In canary rockfish, the presence of ventral body pigment and dorsal midline pigment posterior to the soft dorsal fin (instead of at the base of the soft dorsal-fin rays as in pygmy rockfish) can help differentiate this species from pygmy rockfish. Pigmentation patterns of sharpchin rockfish are very similar to pygmy rockfish at 10 mm; however, sharpchin rockfish retain pigmented pelvic fins until 12.7 mm (Laroche and Richardson. 1981). Counts of anal-fin rays often can be used to differentiate these two species because pygmy rockfish have six rays and sharp- chin have rockfish seven rays (Chen, 1986; Matarese et al., 1989; Moreland and Reilly, 1991; Laroche1!. There is a small overlap in anal-fin ray counts (approximately 1' i I, and, because of this, 100% certainty of identification cannot be reached by anal-fin ray counts alone. There- fore, in order to increase confidence in identifications, a combination of pigmentation and fin-ray counts should be employed. After approximately 15 mm. a full comple- ment of fin rays and gill rakers typically is present and can be used in combination with pigmentation patterns to differentiate pygmy rockfish from most other rockfish species. In these late-stage larvae, only three species (yellowtail. black (S. melanops), and blue rockfish) have a pigment pattern that could be confused with pygmy rockfish (Matarese et al., 1989; Moser, 1996; Laroche1). Laidig et al.: Descriptions and growth of larval and juvenile Sebostes wilsoni 461 but these patterns can be easily separated by using meristic characters. Pelagic juvenile pygmy rockfish have a distinctive pigment pattern consisting of three body bars that can be used to discriminate this species from other Sebastes species. Yellowtail, halfbanded, and redstripe rockfish are the only species that have a similar three-barred pigment pattern (Matarese et al., 1989; Moser, 1996; Laroche1). Yellowtail rockfish can be distinguished by the lack of cheek bars and the presence of body bars that extend all the way to the ventral surface. Also, in yellowtail rockfish, the body bars form at a larger size than in pygmy rockfish. In halfbanded rockfish, the most anterior body bar is more densely pigmented than the other bars and typically forms a diamond shape. The caudal body bar is much wider and covers the en- tire peduncle. Redstripe rockfish are the most similar and are difficult to separate from pygmy rockfish by using pigmentation alone. However, these two species can be separated with greater than 90% certainty by using meristic counts. Pygmy rockfish have a mean anal-fin ray count of 6 (95% from the present study, and 93% from Laroche1), whereas redstripe rockfish have an average of 7 anal-fin rays (100% from Chen, 1986; 97% from Laroche1). It should be noted that the only illustration of pygmy rockfish prior to our study was a 35.0-mm pelagic juve- nile by Laroche,1 which showed several pigment differ- ences from our specimens of equivalent size. Laroche's illustrated specimen had only faint body barring, no cheek bars, and no ventral pigment, whereas all our specimens had prominent body barring, at least one cheek bar, and ventral pigment along the anal-fin ar- ticulations. At this time we cannot determine whether these differences were due to geographic variability in pigment patterns (Laroche's specimen probably was collected farther north than all of our specimens), or a misidentification of the original specimen illustrated by Laroche.1 The identification of larval and pelagic juvenile pygmy rockfish used in our study was confirmed by using DNA sequence analyses. Previous molecular identifications and subsequent descriptions of juvenile starry rockfish (S. constellatus) and swordspine rockfish (S. ensifer) also were based on mitochondrial cytochrome b data (Rocha-Olivares et al., 2000). In our study, orthologous cytochrome b sequence was sufficient for identifica- tion purposes, particularly for those specimens exhibit- ing exact haplotype matches to reference adult pygmy rockfish (e.g., FT2/FT3: 0.0% sequence divergence). Relatively low levels of interspecific genetic variation occurred between larval specimens and several refer- ence species (pygmy, sharpchin. harlequin, and Puget Sound rockfish, and, to a lesser extent, redstripe rock- fish). Rocha-Olivares et al. (1999a) used control region sequence, in addition to cytochrome b, to resolve phy- logenetic relationships among recently diverged species of the Sebastes subgenus Sebastomus. In the present study, the control region sequence was used to increase divergence levels between species and to aid in insur- ing correct molecular identifications of specimens FT1 and FT4. Species assignment to pygmy rockfish was supported by the smallest divergence (based on cyt-6 and cyt-6+CR) from reference pygmy rockfish compared with the other Sebastes species. Larval and juvenile pygmy rockfish can also be sepa- rated from other Sebastes species by comparing the radius of the extrusion check on their otoliths. Of the fourteen other Sebastes species or species complexes with measured otolith extrusion check radii ( Laidig and Ralston, 1995; Laidig et al., 1996; Laidig and Sakuma, 1998), only four species and two complexes have radii close to the average extrusion check radii for pygmy rockfish (10.5 nm, SD = 0.3). Stripetail rockfish (S. saxi- cola) had an average extrusion check radius (11.6 (im, SD = 0.5) that was larger than the largest radius for pygmy rockfish (11.0 (jm). Quillback rockfish (S. ma- liger) had an average extrusion check radius of 9.1 jim (SD = 0.1). which was smaller than the smallest radius for pygmy rockfish (9.5 p.m). Species with extrusion check radii similar to pygmy rockfish were kelp rockfish (S. atrovirens) at 10.6 ^m (SD = 0.2), blue rockfish at 10.9 /./m (SD = 1.1), and the copper rockfish (S. caurinus, extrusion check radius = 10.5 /jm; SD = 0.4) and gopher rockfish (S. carnatus, extrusion check radius = 10.6 ,um; SD = 0.3) complexes (see Laidig et al., 1996. for complex definitions). Of these species, the only one that would be confused with pygmy rockfish, by pigmentation alone, would be blue rockfish at small sizes. However, pygmy rockfish and blue rockfish are easily separated by using meristic characters. Growth rates of larval rockfish generally are slow during the first month of life and increase thereafter (Laidig et al., 1991; Sakuma and Laidig, 1995; Laidig et al., 1996). Because the youngest fish in our study was estimated to be 40 days old, our linear model can not be used to estimate early larval growth rates. For pygmy rockfish older than 40 days, the growth rate of 0.28 mm/day was somewhat slower than that observed for other Sebastes. Woodbury and Ralston (1991) found that, for fish older than 40 days, growth rates varied from 0.30 for widow rockfish (S. entomelas) to 0.97 mm/ day for bocaccio. Other species exhibiting slightly faster growth rates after 40 days of age include stripetail rockfish (0.37 mm/day; Laidig et al., 1996), grass rock- fish (S. rastrelliger; 0.36 mm/day; Laidig and Sakuma, 1998), and shortbelly rockfish (S. jordani; 0.53 mm/day; Laidig et al., 1991). Yellowtail rockfish had a more similar growth rate, ranging from 0.19 to 0.46 mm/day (Woodbury and Ralston, 1991). These differences in growth may reflect genetic variability or responses to environmental variables. Woodbury and Ralston (1991) suggested that annual variability in growth rates of juvenile rockfish was related to year-to-year changes in environmental conditions, especially temperature. Boehlert (1981) determined that temperature greatly affected growth rate of young splitnose rockfish (S. dip- loproa) in the laboratory. Boehlert and Yoklavich (1983) observed slower growth rates for black rockfish in colder temperatures. Lenarz et al. (1991) analyzed the vertical 462 Fishery Bulletin 102(3) distribution of late larval and pelagic juvenile rockfish and determined that pygmy rockfish were present on average in deeper, colder water than that favored by other rockfish species. This spatial separation of pelagic juvenile pygmy rockfish and other Sebastes spp. may explain the slower growth observed in pygmy rockfish. Acknowledgments We would like to thank the scientists and crew from the Southwest Fisheries Science Center (SWFSC) who collected the samples aboard the NOAA RV David Starr Jordan. We thank Geoff Moser and Bill Watson (NOAA, SWFSC) for examining some of our pygmy rockfish specimens. Reference sequences of Sebastes were gener- ated and kindly provided by personnel at the Fisheries Resources Division of the SWFSC, La Jolla, CA (R. D. Vetter, A. Rocha-Olivares, B. J. Eitner, C. A. Kimbrell, and C. Taylor). In addition, we thank Mary Yoklavich for all her valuable comments and all the reviewers who contributed to this manuscript. Literature cited Boehlert. G. W. 1981. The effects of photoperiod and temperature on the laboratory growth of juvenile Sebastes diploproa and a comparison with growth in the field. Fish. Bull. 79:789-794. Boehlert, G. W„ and M. M. Yoklavich. 1983. Effects of temperature, ration, and fish size on growth of juvenile black rockfish, Sebastes melanops. Environ. Biol. Fish. 8:17-28. Chen, L. 1986. Meristic variation in Sebastes iScorpaenidaei. with an analysis of character association and bilateral pattern and their significance in species association. NOAA Tech. Rep. NMFS-45, 17 p. Hillis, D. M., B. K. Mable, A. Larson, S. K. Davis, and E. A. Zimmer. 1996. Nucleic acids IV: sequencing and cloning. Chapter 9 in Molecular systematics, 2nd ed. ID. M. Hillis. C. Moritz, and B. K. Mable, eds.l, p. 321-381. Sinauer Associates, Sunderland, MA. Hunter, J. R.. and N. C. -H. Lo. 1993. Ichthyoplankton methods for estimating fish bio- mass introduction and terminology. Bull. Mar. Sci. 53:723-727. Laidig. T E., and S. Ralston. 1995. The potential use of otolith characters in iden- tifying larval rockfish (Sebastes spp.). Fish. Bull. 93:166-171. Laidig, T. E., S. Ralston, and J. R. Bence. 1991. Dynamics of growth in tin- early life history of short- belly rockfish, Sebastes jordani. Fish. Bull. 89:611- 621. Laidig, T. E.. and K. M. Sakuma. 1998. Description of pelagic larval and juvenile grass rockfish, Sebastes rastrelliger (Family Scorpaenidaei. with an examination of age and growth Fish Bull. 96:788-796. Laidig, T. E., K. M. Sakuma, and M. M. Nishimoto. 1996. Description of pelagic larval and juvenile strip- etail rockfish, Sebastes saxicola (family Scorpaenidaei. with an examination of larval growth. Fish Bull. 94:289-299. Laroche, W. A., and S. L. Richardson. 1981. Development of larvae and juveniles of the rock- fishes Sebastes entomelas and S. zacentrus (Family Scorpaenidaei and occurrence off Oregon, with notes on head spines of S. mystinus, S. flavidus, and S. melanops. Fish. Bull. 79:231-257. Lee, T.-W.. and G.-C. Kim 2000. Microstructural growth in otoliths of black rock- fish, Sebastes schlegeli, from prenatal larval to early juvenile stages. Ichthyol. Res. 47:335-341. Lenarz, W. H., R. J. Larson. S. Ralston. 1991. Depth distributions of late larvae and pelagic juve- niles of some fishes of the California Current. CalCOFI (California Cooperative Oceanic Fisheries Investiga- tions! Rep. 32:41-46. Love. M. 1996. Probably more than you wanted to know about the fishes of the Pacific coast, 381 p. Really Big Press. Santa Barbara, CA. Love. M., M. Yoklavich. and L. Thorsteinson. 2002. The rockfishes of the northeast Pacific, 405 p. Univ. California Press. Berkeley, CA. Matarese. A. C, A. W. Kendall Jr., D. M. Blood, and B. M. Vinter. 1989. Laboratory guide to early life history stages of northeast Pacific fishes. NOAA Tech. Rep. NMFS- 80, 652 p. Moreland, S. L„ and C. A. Reilly. 1991. Key to the juvenile rockfishes of central California. In Methods used to identify pelagic juvenile rockfish (genus Sebastes) occurring along the coast of central California (T. E. Laidig and P. B. Adams, eds.l. p. 59-180. NOAA Tech. Memo.. NOAA-TM-NMFS- SWFSC-166. Moser, H. G. 1996. The early stages of fishes in the California Cur- rent region. Calif. Coop. Oceanic Fish. Invest. Atlas no. 33, 1505 p. Moser, H. G.. and J. L. Butler. 1987. Descriptions of reared larvae of six species of Sebastes. In Widow rockfish: proceedings of a work- shop, Tiburon, California, December 11-12, 1980 i W H Lenarz and D. R. Gunderson, eds.l, p. 19-29. NOAA Tech. Rep. NMFS-48. Pacific Fishery Management Council. 2000. Status of the Pacific coast groundfish fishery through 1999 and recommended acceptable biological catches for 2000, 165 p. Pacific Fishery Management Council. Portland. OR. Ralston, S., J. R. Bence, M. B. Eldridge, and W. H Lenarz. 2003. An approach to estimating rockfish biomass based on larval production, with application to Sebastes jordani. Fish. Bull. 101:129-146. Richardson. S. L.. and W. A. Laroche. 1979. Development and occurrence of larvae and juveniles of the rockfishes Sebastes erameri. Sebastes pinniger, and Sebastes helvomaculatus ' Family Scorpaenidae i 3 km I from the sanctuary into the fishing zone Number of lobsters tagged and released into the surrounding fishing zone Number of lobsters recovered that had moved (>3 km) within the fishing zone Number of lobsters ecovered that had not moved (>3 km) within the fishing zone Nf NF NF NF 413 29 3235 89 277 of tagging, roughly 100-140 mm CL for females, and 100-150 mm CL for males, with a noticeable shift to smaller sizes for both sexes on the southeast coast of South Australia where growth and thus size of maturity are known to be lower; 4) overall, most lobsters in the fished areas did not move large distances, about 15% moving more than 5 km; 5) two areas stood out as being habitats from where significant movement occurred, the coastal zone off the Coorong and Yorke Peninsula; and 6 ) for Yorke Peninsula, higher than proportional num- bers of tagged lobsters that moved significant distances were tagged and released inside Gleesons Sanctuary. In the present study study, a lobster was classified as having undergone movement if its measured distance from point of tagging to point of recapture was greater than 3 km. This definition of lobster "movement" was chosen for two reasons. 1) The mean width of MPA coastal zone to be protected in the currently proposed state representative system is assumed to be 5 km wide; that is, it is assumed that sanctuary areas will extend from the shore outward to sea across the full 3 nmi (which is about 5 km) of state territorial waters. Thus, a 3-km movement would represent slightly more than the mean distance needed for lobsters to leave the state- protected territorial waters of the reserve and enter wa- ters open for fishing. This assumption is strengthened by the knowledge that most longer-range movements of South Australian rock lobster are directed from inshore to offshore. 2) According to the geographical features of the present study, a 3-km movement seaward from any location in Gleesons Landing Sanctuary would place the tagged lobster well into the fished zone, i.e., it would constitute a movement out of the sanctuary. Of sanctuary-tagged lobsters, 4 of 33 recaptured lobsters in the first season after tagging exited the reserve but moved less than 3 km. These 4 recaptured lobsters were excluded from the data set. The mean distance moved by lobsters from the sanctuary was 37.4 km. Because movement of South Australian lobsters is directed strongly away from the inshore zone, the im- migration rate of lobsters back into the Gleesons Land- ing Sanctuary is likely to be quite low. Moreover, Jasus edwardsii seek shelter daily and remain on specific reefs through most of their life (MacDiarmid et al. 1991; Kelly 2001). Long-distance movements occur rarely more than once in a lifetime. Thus, in the fishing zone, where there is a continual removal of adult lobsters from reef habitat, the on-going creation of new shelter space is higher than in the sanctuary and thus lobsters that did stray inshore into the sanctuary would be less likely to find shelter, further reducing the probability of migration into the sanctuary. In the estimator pre- sented below, only the emigration rate (the movement rate out of the sanctuary) is calculated. The recapture data included lobsters at large for a wide range of times, many having been recaptured lon- ger than one year after tag release. However, to estimate emigration rate, we sought the proportion of lobsters emigrating out per year. Therefore, subsets of recapture data were selected that had a mean time at large of one year. The temporal distributions of recaptured lobsters showed distinct modes around 1 year at large (recap- tures between 0.5 and 1.5 years at large. Fig. 2), and the number of recaptures in these 1-year modes were used for estimating yearly movement rate (Table 1). Some tagged and released lobsters were recaptured more than once. For these lobsters, the single recapture was selected and used for which the time at large was closest to one full year. Notation The information on movement in each set of tag releases is taken to be binary: each recaptured animal is clas- sified as having moved or as having not moved during its approximately 1-year time at large (from time of tag release to time of recapture). To carry out the movement-rate estimation, it is use- ful to consider the complete set of four possible outcomes for each tagged and released animal: 1) it moved and was recovered after one year (denoted M,R); 2) it did not move and was recovered after one year (NM.R); 3) it moved and was not recovered after one year (M.NR); 4) it did not move and was not recovered after one year (NM.NR). These four possible recapture outcomes ap- plied to animals tagged and released in both strata, McGarvey: Estimating emigration rates from marine sanctuaries using tag-recovery data 467 80 60 50 40 20 12 10 8 6 4 2 0J Fished zone M }MM) 0 0 1 — JT-Th-n-i ~ Sanctuary firm \h m 0 12 3 4 5 6 Time at large (years) Figure 2 Histograms over time at large (in monthly bins! of recapture numbers from the fishing zone iMFA blocks 33 and 40 i and from the Gleesons Landing Lobster Sanctuary. The diamond mark- ers indicate divisions between modes at 0.5, 1.5., 2.5, etc. years at large; recaptures from the sanctuary and fishing zone that occurred between 0.5 and 1.5 years after release (between the black diamond markers! identify the subsets of data used to estimate yearly emigration rate from the sanctuary. inside and outside the sanctuary. The tag-recovery data provided direct measures for only three of these eight possible numbers of recaptures. We define "not recovered" to include both tagged ani- mals that were not recaptured, as well as those that were recaptured by a fisherman but whose tag informa- tion (notably the location of recapture) was not reported back to researchers and therefore was not included in the tag-recovery database. The movement-rate estimate is given in terms of the following data inputs: the number of lobsters tagged and released in li fished and 2) protected zones, and the numbers recovered that 3) moved (>3 km) or 4) did not move from the fished zone over one year after tag- ging, and the 5) number that moved (>3 km) from the sanctuary in one year. Superscripts 'F' and 'S' denote fished zone and sanc- tuary, respectively, for the location of tag release. Let N^MR and Nfj R denote the numbers of animals that were recovered after a year and that moved or that did not move in the fished zone. From animals tagged and released inside the sanctuary, only the number that moved and were recovered (A^;/?) is available as an un- biased measure. In addition, we know the total number of animals originally tagged and released in the fished zone and sanctuary, Nj. and N^. Input quantities from the tag-recovery data set will henceforth be indicated by a tilde ("): (iV® R iVf. N$MiRj N? R_ N% } (Table 1). Assumptions Three assumptions were used to derive an emigration- rate estimate: 1) The two ways to define an estimate for the proportion that moved within the fished zone, namely as a proportion by using only recapture numbers, and as a proportion over the number originally tagged, can be set equal. 2) Recapture probabilities of animals that were tagged and released inside the sanctuary and that moved are assumed to equal those that were tagged and released into the fished zone and that also moved. (The first two assumptions were employed explicitly in steps 2 and 3 below.) 3) A third assumption is implicit in step 2, specifically in the recapture-conditioned movement proportion in the fished zone (.PjtfR, Eq. 2): recapture probabilities of animals tagged and released in the fished zone that moved and of those that did not move are assumed to be equal. Assumptions 2 and 3 would both follow from assuming equal recapture probabilities for all lobsters in the fished zone. Emigration rate: derivation of the estimate formula In this section, an emigration-rate formula is derived. It provides a closed-form estimate of the yearly proportion of lobsters emigrating out of the sanctuary. The proportion of animals moving can be estimated from tag-recovery data in two ways, namely as "tag- 468 Fishery Bulletin 102(3) conditioned" and "recapture-conditioned" proportions. A tag-conditioned movement proportion (Eq. li is the total number of lobsters that moved (>3 km) divided by the number originally tagged and released. It includes, in the numerator, all tagged animals that moved, both those that were recovered, as well as those that were not recovered. With a recapture-conditioned movement- rate estimate (e.g., Eq. 2), only counts of recaptured lobsters are used. The estimate expresses the movement proportion as the number of tagged animals that were recaptured and that also moved l>3 km) divided by the total number recaptured. These two definitions for the movement proportion will be used to derive an estima- tion formula in terms of the five data inputs. Step 1 The derivation begins by writing the estimate for proportion of lobsters that moved (P|f) in tag-condi- tioned form: Ns + Ns pS _ JV MR T .U..YW N* (1) This estimate of movement rate from the sanctuary is based on a tag-conditioned proportion because we have no observations of recaptured lobsters from the sanctu- ary that did not move (no unbiased measure of NfjMM) which a recapture-conditioned movement proportion would have required. However we did have information about N§fNR, the nonrecovery of tagged animals that emigrate from the sanctuary into the fished zone. It can be estimated (steps 2 and 3) with the second assumption that recovery rate for lobsters moving from the sanctu- ary equals that of lobsters moving (>3 km) within the fished zone. Substituting Equations 2 and 3 into Equation 4 and solv- ing for NF, XR, the number of lobsters that moved >3 km within the fished zone but were not recovered, yields NF = NF NF NF +N 1 (5) Step 3 Assumption 2 permits the derivation of a for- mula forWS yR. We first define the recovery proportions of animals that moved within the fished zone (F) as fF = I m NF '^ MM Mr +MF 1 v U .XR A * M M (6) and from the sanctuary (S) as fs I M Nt iV.U XR T .V.fl (7i Assumption 2, that the recovery rate (necessarily in the fished zone) for animals that were tagged and released in the sanctuary and that moved into the fished zone is the same as for animals that were both released and recap- tured after moving within the fished zone becomes fF = fs I M I M ■ (8) Substituting Equations 6 and 7 into Equation 8 and rearranging terms, we have iy M.XR Ns (NF + Nf ) N n; (9) Step 2 Under assumption 1, the two ways in which movement proportion in the fished zone can be defined (as tag- and recapture-conditioned proportions) are equated. For fished zone releases, the recapture-condi- tioned Crc') movement proportion is written N* NF +NF (2) For the recapture-conditioned estimate formula (Eq. 2), all three quantities on the right-hand side are given as data inputs. With only numbers of lobsters recovered, the formula is, in this sense, conditional on recapture. The tag-conditioned I7c'» proportion of lobsters moving >3 km of those released in the fished zone is written Af^ +NF ,/• ,, _ lyM.R TJVM,.Vfl NF The first assumption is Din _ pf.l rM ~ rM (3) i4i Step 4 Substituting Equation 5 into Equation 9 and substituting the result into Equation 1 yields a closed- form estimation formula for the quantity we seek, the proportion moving from the sanctuary in one year: Ps=- Nf ■ArS /Vs' ■iNF +Nh ) (10) Numerical estimator: double-hypergeometric likelihood method A likelihood formulation of this estimator was also constructed. The likelihood function describing a single tag-recapture experiment is hypergeometric (Seber, 1982; Rice, 1995) because sampling is without replace- ment. The set of possible outcomes from each of the two tagging experiments can be formulated as a 2x2 contingency table for the experimental populations of all lobsters originally tagged and released. The two pairs of outcomes represented in each contingency table are "moved" or "not moved" and "recovered" or "not recov- ered," yielding the four possible outcomes from both sets of tag releases (see "Notation" section). McGarvey: Estimating emigration rates from marine sanctuaries using tag-recovery data 469 In this study the data from two interacting tag-recov- ery experiments were used to generate an estimate of reserve emigration rate, namely of lobsters tagged and released into the sanctuary and into the fished zone. Thus, the product of a pair of linked hypergeometric probability mass functions, each corresponding to a 2- way contingency table, is the natural form of the likeli- hood function for Pfj. The derivation of Equation 10 was made with two as- sumptions, namely Equations 4 and 8. Incorporated in the likelihood, the two assumptions constrain the eight recapture numbers in the contingency tables. In the likelihood formulation, a third constraint was needed which is analogous to assumption 1 but which applies to sanctuary releases. The derivation for constructing this likelihood from a pair of linked hypergeometric probability functions will proceed by 1) writing out the "raw" contingency tables in terms of the eight recapture numbers (TV), as denoted in the "Tag-recovery data" and "Notation" sections, 2) algebraically re-expressing the elements of the tables so that the parameter to be estimated is explicit. 3) imposing the three constraints, and 4) writ- ing out the likelihood, using the hypergeometric form for contingency tables. For the lobsters tagged and released in the sanctuary, the raw contingency table is Recovered Not recovered Totals Moved MS " M.R MS "M.NR fjS + MS "M.R * " M.NR Not moved MS "nm.r MS "nm.nr NST- Totals MS . MS " M.R + "NM.R NST- ftf. For the lobsters tagged in the fished zone Recovered Not recovered Totals Moved NF " Mil NF "m.nr "M.R + "M.NR Not moved MF "NM.R NF "nm.nr Totals ^M.R + ^NM.R NF- NF The two hypergeometric probability mass functions (pmfsi giving the model-predicted proportion of lobsters that moved and were recovered, based on the two con- tingency tables, are written as * Km)- N* +N ly M.R T ly M.NR NF N* -(N +N ) ylyM.R TJV M.NR' NF \\ NF (12) N +N ly MR * NM.R ) Because the goal is to estimate the movement propor- tion, Pft (rather than any specific value of N), this pro- portion will need to be made explicit in the likelihood function as the sole freely varying parameter. Substitut- ing from the definition of Py (Eq. 1), we have m" = ps . AT* _ MS lyM.NR 1M iyT lyM.R- (13) Substituting for all occurrences of Nfj NR, Equation 11 becomes p«o= PmK)(N^(1-P^^ Ni AT? N* Ns + Ns ly M.R T ly NM.R ) (14) Writing the full joint-likelihood expression formed by the product of the two hypergeometric pmfs gives L = NF +NF V NF -(NF +Nr ) ly M.R T ly M.NR ly T yly M.ff T ly M.NR ' NF NF ' NF 1 NF +NF {■ly M R ly NM.R ) (Pm-Nt) (N*-Q.-F*j\ { K.R J Ns \ lyNM.R ) ( Nt } k K, + NS ? T JY NM.R ) As formulated, the value of Nf!M R is still undeter- mined by data or constraint. A third constraint is therefore required. As with assumption 1 for the fished zone (Eq. 4), we apply the assumed equivalence of tag- and recapture-conditioned proportions to the sanctuary releases: ps.rc »>-& /(/Vs +/Vi ) = P ' rM ly M.R ' yly NM.R T ly M.R' *M -(MS +MS )/ fjs ~Xly MR + IV M.NR" lyT- P + ms , " T W,M.R ^lyM.NR' Ns Ms +MS ly M.R Tiv NM.R I In this application, Nf,M R is understood as the number of lobsters that would have been taken if fishing had 1 1 1 1 not been excluded from the sanctuary. Solving for N^M R yields the third constraint. MS -IMS .MS)/iMS ,JU* )_MS ly NM.R ~yly M.R lyT " WV M.R^ly M.NR1 lyM,R' 470 Fishery Bulletin 102(3) Table 2 Intermediate calculated quantities from the numerical estimation. The equalities of P[jr and 2. =P£" and /'(,=/'« state assumptions 1 Intermediate quantity Variable name Estimate The proportions of lobsters tagged in the fishing zone that moved (>3 km I; recapture-conditioned 3 km) within the fished zone. When independent estimates of exploitation rate are available, typically from stock assessment, the rate of tag reporting can be calculated from the tag-estimated recovery rate. The exploitation rate (yearly proportion of legal-size lobsters harvested) for the recapture year and McGarvey: Estimating emigration rates from marine sanctuaries using tag-recovery data 471 location of the present study (the 1995 northern zone rock lobster season) was estimated to be 26% (Ward et al.3) by using total yearly effort and catches by weight and number and a vector of weights at age. The tag-re- covery rate of 11.3% (Table 2) is the estimated propor- tion of tagged lobsters that were captured and for which tags were reported. Thus the estimated tag-reporting rate (of those recaptured) is 0.113/0.26 = 43%. If tag shedding and natural mortality were also incorporated as additional causes for nonrecovery, the estimate would fall in the neighborhood of a 50% tag-reporting rate. This estimated level of tag-reporting falls within the range considered probable by fishermen. Thus, the re- covery-rate estimate falls within a plausible range of values, adding confidence that the tag-recovery data are consistent with external estimates of exploitation rate. Substantial movement of Jasus edwardsii out of a ma- rine sanctuary was previously observed in New Zealand (Kelly and MacDiarmid, 2003) but not in Tasmania (Gardner and Ziegler4). Long-distance movement of this genus was also observed in New Zealand (Booth, 1997) but was much less common in Tasmanian Jasus edwardsii populations (Gardner et al., in press). Discussion The emigration-rate derivation above combined recap- ture-and tag-conditioned movement proportions. Both ways to define a movement rate were used to constrain the range of solutions for both analytic and numerical estimators. Equating these two definitions for movement proportion reduced the degrees of freedom by 1, thereby circumventing the absence of a count of recaptured lob- sters from within the fished zone. Previous estimators of movement rates among spatial cells from tag-recovery data have used either tag- or recapture-conditioned approaches. Hilborn (1990: see also Quinn and Deriso, 1999) developed a tag-condi- tioned movement-rate estimator. This estimator gen- erally requires prior knowledge of the tag reporting rate. Schwarz et al. (1993) employed data consisting of simultaneous tag releases and recaptures repeated over a number of years at the same time each year to estimate movement, survival, and recovery rates in each spatial stratum. Schwarz et al. (1993) presented a general formulation for modeling this multiple yearly 3 Ward, T. M., R. McGarvey. Y. Xiao, and D. J. Brock. 2002. Northern zone rock lobster [Jasus edwardsii) fish- ery. South Australian Fisheries Assessment Series Report 2002/04b. 109 p. Aquatic Sciences, South Australian Research and Development Institute (SARDI): RO. Box 120, Henley Beach, South Australia 5022. Australia. 4 Gardner, C, and P. Ziegler. 2001. Are catches of the south- ern rock lobster Jasus edwardsii a true reflection of their abundance underwater? Tasmanian Aquaculture and Fish- eries Institute Final Report. TAFI (Tasmanian Aquaculture and Fisheries Institute), University of Tasmania, Private Bag 49, Hobart TAS 7001, Australia. tag-recovery data set, extending a series of estimators for movement and survival (Arnason, 1972, 1973), and estimated the rate of tag recovery. Brownie et al. (1993) generalized the estimator of Schwarz et al. to non-Mar- kovian movement rates. McGarvey and Feenstra (2002), following Hilborn, used the less costly and more com- monly available single tag-recovery data employed in the present study but adopted a recapture-conditioned approach for estimating yearly movement rates. With "numbers recaptured" appearing in both the numerator and denominator, all nonspatially dependent sources of variation (such as tag reporting and shedding, short- and long-term tag-induced mortality, and natural mor- tality) cancel from the predicted recapture-conditioned likelihood proportions. This procedure permits a cor- responding reduction in the prior information required to obtain unbiased movement estimates. When recapture times vary, movement estimation is sensitive to spatial differences in mortality rate, no- tably between tag and recapture cells. Assuming that the nonreporting rate is unknown, mortality can be inferred from single tag-release information only impre- cisely, for example by using mean tagged time at large. For this reason externally obtained mortality estimates, typically from stock-assessment models using fishery data, can be usefully combined with single tag recover- ies in movement estimation. Hestbeck (1995) showed, when survival differs by cell, that ignoring the time of movement between yearly samples could bias movement estimates. McGarvey and Feenstra (2002) made explicit the variation in residence time and thus survival in source (tag-release) and destination (recapture) cells for each recaptured animal. By using prior knowledge of a migration season, migration source cell and destination cell residence times can be approximated as the time from the date of tag release to an assumed fixed (yearly) date of movement, and from that date to the date of re- capture. These residence times are used in exponential survival factors that differ spatially given externally- estimated fishing mortality rates in each cell. For the data set available from Gleesons Landing, all tagged animals were released during the peak fishing season (mid-summer). Thus recoveries from the fol- lowing fishing season had a mean and mode near the desired one-year-at-large. In future tag-recovery stud- ies, where a yearly movement rate is sought, a similar choice for timing of tag releases, namely during the season of highest fishery catches, should yield a peak in recaptures a year later. Schwarz et al. (1993) employed this strategy with their multiple yearly tag-recovery data sets. In the estimator presented above, variations in ex- pected recovery numbers versus time, notably due to sur- vival, were neglected. The small sample (33 recoveries between 0.5 and 1.5 years from the sanctuary) and lack of recaptures from within the sanctuary necessitated more modest estimation goals. Among data classes avail- able for movement analysis, notably 1) multiple yearly tag recaptures by researchers in all cells, 2) multiple yearly tag recoveries where recapture is by fishermen (or 472 Fishery Bulletin 102(3) hunters) in all cells, 3) single tag recoveries by fishermen in all cells, and 4) the data set employed in the present study of single-tag recoveries by fishermen in one of two cells, the latter represents the low end in quality and quantity of information about movement and survival. A time-dependent approach could theoretically extend the approach of McGarvey and Feenstra (2002) to make explicit the residence times of each recaptured indi- vidual in the fishing zone and sanctuary, respectively, and thus make explicit differences in the predicted survival rate before and after movement. However with- out prior knowledge of when movement took place for each recaptured lobster, a modified likelihood method is called for, requiring integration over the probable movement times between tag release and recapture. This extension of residence-time-dependent movement estimators to variable times of movement remains a topic for future research. Acknowledgments I thank Hugh Possingham, Andre Punt, and two review- ers for comments on the draft manuscript. Lobsters were tagged and released into Gleesons Landing sanctu- ary by Greg Ferguson, together with fishermen Lenny and Murray Williams, under Fisheries Research and Development Corporation Project 93/086. This work was supported by the Australian Fisheries Research and Development Corporation Project No. 2000/195, and by the South Australian rock lobster industry. Literature cited Abramowitz, M.. and I. R. Stegun. 1965. Handbook of mathematical functions, p. 257. Dover Publications, New York, NY. Arnason, A. N. 1972. Parameter estimates from mark-recapture exper- iments on two populations subject to migration and death. Res. Popul. Ecol. (Kyoto) 13:97-113. 1973. The estimation of population size, migration rates, and survival in a stratified population. Res. Popul. Ecol. (Kyoto) 15:1-8. Booth, J. D. 1997. Long-distance movements in Jasus spp. and their role in larval recruitment. B. Mar. Sci. 61ill: 1 11— 128. Brownie, C, J. E. Hines. J. D. Nichols, K. H. Pollock, and J. B. Hestbeck. 1993. Capture-recapture studies for multiple strata in- cluding non-Markovian transitions. Biometrics 49:1173- 1187. Gardner, C, S. Frusher, M. Haddon, and C. Buxton. 2003. Movements of the southern rock lobster Jasus edwardsii in Tasmania, Australia. Bull. Mar. Sci. 73(3): 653-671. Hestbeck, J. B. 1995. Bias in transition-specific survival and movement probabilities estimated using capture-recapture data. J. Appl. Stat. 22:737-750. Hilborn, R. 1990. Determination offish movement patterns from tag recoveries using maximum likelihood estimators. Can. J. Fish. Aquat. Sci. 47:635-643. Kelly, S. 2001. Temporal variation in the movement of the spiny lobster Jasus edwardsii. Mar. Freshw. Res. 53(31:323-331. Kelly, S., and A. B. MacDiarmid. 2003. Movement patterns of mature spiny lobsters, Jasus edwardsii, from a marine reserve. N.Z. J. Mar. Freshw. Res. 37:149-158. MacDiarmid, A. B.. B. Hickey, and R. A. Mailer. 1991. Daily movement patterns of the spiny lobster Jasus edwardsii (Hutton) on a shallow reef in northern New Zealand. J. Exp. Mar. Biol. Ecol. 147:185-205. McGarvey, R., and J. E. Feenstra. 2002. Estimating rates of fish movement from tag recov- eries: conditioning by recapture. Can. J. Fish. Aquat. Sci. 59:1054-1064. Quinn, T J. II, and R. B. Deriso. 1999. Quantitative fish dynamics, p. 414-419. Oxford Univ. Press, New York. NY. Rice, J. A. 1995. Mathematical statistics and data analysis, p. 13-14, 39-40. Duxbury Press, Belmont CA. Schwarz, C. J., J. F. Schweigert, and A. N. Arnason. 1993. Estimating migration rates using tag recovery data. Biometrics 49:177-193. Seber, G. A. F. 1982. The estimation of animal abundance and related parameters, 2nd ed., p. 59-60. Griffin, London. 473 Abstract— We describe reproduc- tive dynamics of female spotted sea- trout (Cynoscion nebulosus) in South Carolina (SC). Batch fecundity iBFl. spawning frequency (SF), relative fecundity (RF), and annual fecundity (AF> for age classes 1-3 were esti- mated during the spawning seasons of 1998, 1999, and 2000. Based on histo- logical evidence, spawning of spotted seatrout in SC was determined to take place from late April through early September. Size at first maturity was 248 mm total length (TL); 50% and 100% maturity occurred at 268 mm and 301 mm TL, respectively. Batch fecundity estimates from counts of oocytes in final maturation varied significantly among year classes. One- year-old spotted seatrout spawned an average of 145.452 oocytes per batch, whereas fish aged 2 and 3 had a mean BF of 291,123 and 529,976 oocytes, respectively. We determined monthly SF from the inverse of the proportion of ovaries with postovulatory follicles (POF) less than 24 hours old among mature and developing females. Over- all, spotted seatrout spawned every 4.4 days, an average of 28 times during the season. A chronology of POF atresia for water temperature >25°C is presented. Length, weight (ovary-free), and age explained 67%, 65%, and 58% of the variability in BF, respectively. Neither RF (number of oocytes/g ovary-free weight) nor oocyte diameter varied significantly with age. However. RF was signifi- cantly greater and oocyte diameter was smaller at the end of the spawn- ing season. Annual fecundity esti- mates were approximately 3.2, 9.5, and 17.6 million oocytes for each age class, respectively. Spotted seatrout ages 1-3 contributed an average of 29%, 39%, and 21% to the overall reproductive effort according to the relative abundance of each age class. Ages 4 and 5 contributed 7% and 4%, respectively, according to predicted AF values. Reproductive dynamics of female spotted seatrout (Cynoscion nebulosus) in South Carolina* William A. Roumillat Marine Resources Research Institute South Carolina Department of Natural Resources 217 Ft. Johnson Rd Charleston, South Carolina 29412 E-mail address roumillatbiSrnrd.dnr.state.sc.us Myra C. Brouwer South Atlantic Fishery Management Council One Southpark Center, suite 306 Charleston, South Carolina 29407 Manuscript submitted 13 May 2002 to Scientific Editor's Office. Manuscript approved for publication 19 March 2004 by the Scientific Editor. Fish. Bull. 102:473-487 (2004). The spotted seatrout iCynoscion neb- ulosus) is an estuarine-dependent member of the family Sciaenidae. Spot- ted seatrout are year-round residents of estuaries along the South Atlantic coast and spawning takes place inshore and in coastal areas (McMichael and Peters, 1989; Mercer1; Luczkovich et. al.2). As in many other sciaenids, spawning in this species occurs in the evening (Holt et al, 1985). Male spotted seatrout have the capacity to produce "drumming" sounds that are caused by the contraction of the swim- bladder by specialized muscles that are seasonally hypertrophied from the abdominal hypaxialis muscle mass (Fish and Mowbray, 1970; Mok and Gilmore. 1983). Direct involvement of sound production with spawning has been shown for this and other sciae- nids (Mok and Gilmore, 1983; Saucier et al., 1992; Saucier and Baltz, 1993; Luczkovich et al.2). We have collected information on the spawning behavior of spotted sea- trout in coastal South Carolina since 1990 (Saucier et al., 1992; Riekerk et al.3). Spawning aggregations were located by listening for drumming sounds from late afternoon until -2300 h with passive hydrophone equipment. Spawning activity was subsequently verified through collec- tions of newly spawned eggs and by the rearing of the larvae in the labo- ratory (Saucier et al., 1992). Spotted seatrout are group-synchro- nous spawners with indeterminate fe- cundity and the protracted spawning season extends from April through September along the South Atlantic and Gulf of Mexico coasts (Overstreet, 1983; Brown-Peterson et al.. 1988; McMichael and Peters, 1989; Saucier and Baltz, 1993; Brown-Peterson and Warren, 2001; Brown- Peterson et al., 2002; Nieland et al.. 2002, Brown- * Contribution 539 from the Marine Resources Research Institute of the South Carolina Department of Natu- ral Resources, Charleston. SC 29422- 2559. 1 Mercer. L. P. 1984. A biological and fisheries profile of spotted seatrout. Cynoscion nebulosus. Special Scien- tific Report 40, 87 p. North Carolina Department of Natural Resources and Community Development, Division of Marine Fisheries. Morehead City, NC 28577. 2 Luczkovich, J. J.. H. J. Daniel III and M. W. Sprague. 1999. Characterization of critical spawning habitats of weakfish. spotted seatrout and red drum in Pamlico Sound using hydrophone surveys. Final report and annual performance report F- 62-2 and F-62-2, p 65-68. North Caro- lina Department of Environment and Natural Resources, Division of Marine Fisheries, Morehead City, NC 28557. 3 Riekerk. G. H. M„ S. J. Tyree, and W. A. Roumillat. 1997. Spawning times and locations of spotted seatrout in the Charleston Harbor Estuarine System from acoustic surveys. 21 p. Final Report to Charleston Harbor Project, Bureau of Ocean and Coastal Resources Management, South Carolina Depart- ment of Health and Environmental Con- trol, 1362 McMillan Ave., Charleston, SC 29405. 474 Fishery Bulletin 102(3) Peterson, 2003; Wenner et al.4). As in other indetermi- nate spawning fish, annual fecundity in this species is determined by the number of oocytes released during each spawning event (batch fecundity) and the number of spawning events occurring during the course of the spawning season (spawning frequency!. Early efforts to estimate fecundity for spotted seatrout did not take into account the repetitive nature of spawning activities in this species (Pearson, 1929; Sundararaj and Suttkus, 1962; Overstreet, 1983) and only recently has an effort been made to coordinate batch fecundities with spawn- ing frequencies (Brown-Peterson et al., 1988; Brown- Peterson and Warren, 2001; Nieland et al., 2002). This procedure is intuitively necessary to estimate the re- productive output for an entire spawning season and is made even more useful for fisheries management if separated by size class or age cohort within a popula- tion (Prager et al.. 1987; Goodyear, 1993; Zhao and Wenner5). An important component of assessment for manage- ment involves determining the spawning potential ratio (SPR), a measure of the effect of fishing on the repro- ductive potential of a stock (Goodyear, 1993). This value is usually calculated as the ratio of spawning stock biomass per recruit (SSBR) in the presence of fishing mortality (F) to the SSBR when F is equal to zero (Ga- briel et al., 1989; Goodyear, 1993). Spawning potential ratio is currently used as a biological reference point for definition of recruitment overfishing (i.e., Vaughan et al., 1992). The calculation of SPR can be improved, however, by introducing egg production into the model. Fecundity is a much better predictor of reproductive po- tential than female biomass. Moreover, SPR calculations based on egg production may be more sensitive to the size-age composition of the spawning stock. However, accurate annual fecundity estimates for use in stock assessment do not exist for this or many other species in need of fisheries management. Therefore, our goal was to obtain batch fecundity (BF), spawning frequency (SF), and annual fecundity (AF) estimates for spotted seatrout by age class. Materials and methods Data to address the main objectives of this study were collected from late April through early September 1998- 1 Wenner, C. A., W. A. Roumillat. J. E. Moran Jr., M. B. Maddox, L. B. Daniel III, and J. W. Smith. 1990. Investigations on the life history and population dynamics of marine rec- reational lishes in South Carolina: part 1. Final Report F-37, 177 p. Marine Resources Research Institute, Marine Resources Division, South Carolina Department of Natural Resources, 217 Ft. Johnson Rd., Charleston, SC 29412 s Zhao, B., and C. A. Wenner. 1995. Stock assessment and fishery management of the spotted seatrout, Cynoscion nebu- losus, on the South Carolina coast, 90 p. Marine Resources K' i arch Institute, Marine Resources Division, South Caro- lina Department of Natural Resources, 217 Ft. Johnson Rd.. Charleston, SC 29412. 2000 as part of a long term monitoring effort 11991-pres- ent) to assess the relative abundance of age classes of recreationally important finfish in South Carolina estu- aries. The study followed a monthly stratified random sampling design in three estuarine systems. The Cape Romain system comprised two strata; Romain Harbor and northern Bulls Bay. The Charleston Harbor system contained four strata: the Wando, Cooper, and Ashley Rivers, and Charleston Harbor. The Ashepoo-Combahee- Edisto (ACE) Basin system comprised a single stratum (Fig. 1). The number of sampling sites within each stra- tum ranged from 23 to 30. A subset of 12-14 sites was randomly selected each month. Sampling was conducted only during the daytime ebbing tide (0700-1800 h), primarily over mud and oyster shell substrates adjacent to the Spartina alterniflora marsh. At each site, we deployed a trammel net (182.8 m long by 2.4 m deep; outer walls: 17.8 cm square (35.6 cm stretch]; inner wall: 3.2 cm square [6.4 cm stretch]) from a rapidly moving shallow water boat in an arc against the shoreline at depths ranging from 0.5 to 2.0 m. We disturbed the water within the site in an effort to frighten fishes into the entrapment gear. We then hauled the trammel net back into the boat and removed the catch, which was kept alive in a 70-liter oxygenated holding tank. Spot- ted seatrout were measured for total length (TL) and standard length (SL) and a subsample offish from each effort (5-10 individuals for each 20-mm size interval per month) were sacrificed, placed on ice, and transported to the laboratory for aging and reproductive data. Specimens were processed in the laboratory 2-12 hours after capture. We recorded standard life-history parameters (TL, SL, fish weight, gonad weight, sex, and maturity) for each specimen. The following equation was used to convert lengths when necessary: TL = 5.689 + 1.167ISL) (r- = 0.998) n = 1191. We removed sagittal otoliths for aging and preserved sections (<2% by weight) of each ovary in neutral buff- ered formalin for histological processing. The latter involved standard procedures for paraffin embedding and sectioning, and standard hematoxylin and eosin-y staining (Humason, 1972). Histological sections were viewed under a Nikon Labophot compound microscope equipped with a teaching head so that two readers could interpret sections simultaneously. Maturity estimation was modified from that of Wenner et al.4 (Table 1). Size at first maturity was histologically derived by first evidence of cortical alveoli stage oocytes. To ar- rive at estimates of 50% and 100% maturity, data were subjected to PROBIT analysis. Age determination The left sagittae were marked with a soft lead pencil through the core and embedded in epoxide resin. A transverse section (~0.5-mm thick) was taken through the core by using a low-speed saw equipped with a pair Roumillat and Brouwer: Reproductive dynamics of Cynoscion nebulosus 475 80 30W 80 O'W 79 30'W Figure 1 The three South Carolina estuarine systems (indicated by arrows) where C. nebulosus were collected. Table 1 Criteria used for microscopic staging of C. nebulosus ovaries. FOM = final oocyte maturation; POF = postovulatory follicle. Stage Description Immature Ovary small in cross section. Early stage with only oogonia evident; later stage with small (<0.08 mm) primary oocytes, tightly packed. No evidence of early vitellogenesis. Developing First appearance of cortical alveoli stage oocytes through late vitellogenesis but no evidence of early FOM (lipid and yolk globule coalescence). Ripe Ovary containing oocytes demonstrating FOM (lipid and yolk coalescence through hydration). Mature with day-0 POFs Ovary exhibits POFs <24 h (see Table 2 ). Found at all water temperatures throughout the spawning season. Mature with day-1 POFs Ovary exhibits POFs >24 h consisting of closely packed granulosa cells (0.08-0.1 mml. Only identified in water < 25°C. Spent Ovary containing alpha- and beta-stage oocytic atresia. Resting Ovary containing small primary oocytes and oocytes with perinuclear nucleoli (<0.12 mm); usually some remnants of oocytic atresia. of diamond wafering blades. The resulting section was was assumed for the process of aging. Spotted seatrout mounted on a labeled microscope slide and examined deposit an annulus in April or May (Murphy and Taylor, with a Nikon SMZ-U microscope. A 1 January birth date 1994; Wenner et al.4). Fish in the first three months of 476 Fishery Bulletin 102(3) Table 2 Criteria used for microscopic staging of C. nebulosus day-0 postovulatory follicles iPOFsl in water temperatures above 25°C. Measurements represent longest axis of POFs. POF chronology fin hours) Description 0-4 5-8 9-12 13-24 Regular arrangement of granulosa-cell nuclei proximal to the basement membrane and obvious multiple layering as described by Hunter and Macewicz 1 1985 ). 200-300 ^m i Fig. 4Ai. Early signs of atresia, loss of the obvious layering, hypertrophy of granulosa cells, and a general compaction with an investment of blood vessels. 180-250 jum (Fig. 4B). Well-defined lumen separating the internal granulosa cells from the outer wall of granulosa cells encompassed by theca. 150-200 jum (Fig. 4C). Lumen reduced primarily by loss of granulosa tissue and proximity of peripheral layers. 130- 175 ^m (Fig. 4, DandE). the year were aged by the addition of 1 year to the count of the number of annuli on the thin sections. In April or May, if the section had a large marginal increment, one was also added to the annular count. If the marginal increment was small or if the ring was detectable on the edge of the otolith section, age was equal to the number of annuli. Seasonality Spawning season for spotted seatrout in South Carolina was determined by using two techniques. The gonadoso- matic index (GSI) was calculated as (GW/OFWT) x 100, where GW = gonad weight (g); and OFWT = ovary-free weight (g). For years prior to this study (1991-97), mean monthly GSI was obtained for all females by using data from the South Caroline Department of Natural Resources inshore fisheries archives (Wenner6). Reproductive sea- sonality among female spotted seatrout throughout the year was also examined by using histology (Table 1). The first evidence of oocytes in final oocyte matura- tion (FOM) as evidenced by lipid and yolk coalescence; Brown-Peterson et al., 1988) or the occurrence of post- ovulatory follicles (POFs) defined the beginning of the spawning season. To determine the cessation of spawn- ing, the percent occurrence of females in spawning condition (ripe and repeat spawners) and those in post- spawning condition (spent and resting) were obtained for the months of August and September. To investigate the condition of females, we examined Fulton's condition factor (Ricker, 1975) over the spawning season using linear regression. 6 Wenner, C. 2002. Unpubl. data. Marine Resources Re- search Institute, Marine Resources Division, South Carolina Department of Natural Resources, 217 Ft. Johnson Rd., i harleston, SC 29412. Spawning frequency We obtained samples for spawning frequency ( SFi deter- mination from 1 May through 31 August 1998, 1999 and 2000. Although samples were routinely collected throughout the year, only from early May through late August did we capture enough animals in the appro- priate reproductive state for SF estimation. Spawning frequency was calculated as either the inverse of the proportion of ovaries with day-0 POFs (Hunter and Macewicz, 1985; Brown-Peterson et al., 1988) or with oocytes in FOM (Brown-Peterson et al., 1988; Liso- venko and Adrianov, 1991) among mature and develop- ing females. We designated two distinct morphological features of POFs based on time of specimen capture and water temperature. We interpreted the largest, least atro- phied POFs to be <24 h old and termed them "day-0" POFs (Hunter and Macewicz, 1985). The presence of day-0 POFs in the ovary indicated that spawning had occurred the previous night. The second category comprised smaller POFs, which primarily consisted of closely packed granulosa cells determined to be >24 h old. To complete the chronology of POF atresia we under- took round-the-clock sampling on 27-28 June and 26 July 2000. During these efforts, sampling continued beyond routine hours to encompass the period between dusk and dawn. The histological samples obtained al- lowed for the calibration of criteria used to age POFs (Table 2). To determine whether SF varied among months and age classes, Kruskal-Wallis tests were used. Because both factors (month and age) were fixed (model 1). it was not possible to test for their interac- tion by using a two-way parametric ANOVA without replication. As a result of targeting fish for batch fecundity esti- mates (see below), we had available numerous specimens with oocytes in FOM with which to establish monthly SF. However, we knew that these specimens were dis- appearing from our shallow sampling sites into deeper spawning areas as the day progressed (Riekerk et al.3), Roumillat and Brouwer: Reproductive dynamics of Cynoscion nebulosus All thus potentially adding bias to our SF estimates. Even though estimates of SF based on FOM were performed a posteriori, we chose to report them strictly for com- parison to other studies with this method. Because sampling for females exhibiting FOM was accomplished in a directed fashion, statistical comparisons were not attempted. Batch fecundity and relative fecundity Observations taken over a decade of sampling the Charleston Harbor estuarine system showed that in females captured in shallow water (<1.5 m) during the spawning season, FOM began at about 1200 h (Wenner6). Similarly, Crabtree and Adams' reported FOM begin- ning in Florida spotted seatrout at about mid-day. Low- erre-Barbieri et al.8 found hydrated females in shallow water in the vicinity of aggregations of drumming males in deeper water. We also speculated that from mid- to late afternoon hydrated females moved along the marsh edge toward deeper water spawning aggrega- tions (8-25 m). Hydrophone surveys conducted in the Charleston Harbor area over several years (Riekerk et al.3; Wenner6) indicated that noise production typically began around 1800 h and ceased around 2200 h. Because this behavior has been associated with spawning in this and other sciaenids (Mok and Gilmore, 1983; Holt et al., 1985; Saucier et al., 1992; Saucier and Baltz, 1993), we assumed that spawning began at 1800 h and stopped at 2200 h. Thus we were able to target spotted seatrout in the mid- to late afternoon specifically to capture females with oocytes in the late stages of FOM for batch fecun- dity (BF) estimation. Because we have consistently iden- tified recently spawned females in shallow areas, they apparently return to the marsh edge where they once again become available for capture with our sampling gear. Our stratified random sampling of estuarine areas along the coast (described previously) was designed to representatively sample these recently spawned females for SF estimation. We conducted BF sampling during two consecutive afternoons fortnightly from the middle of April through the first week of September 1998, 1999, and 2000. We deployed a trammel net from a shallow water boat as described above at preselected sites in Charleston Har- bor in depths ranging from 1.0 to 1.5 meters during the afternoon (1400-1800 h EDT) high tide. 7 Crabtree R. E., and D. H. Adams. 1998. Spawning and fecundity of spotted seatrout, Cynoscion nebulosus, in the Indian River Lagoon. Florida. In Investigations into near- shore and estuarine gamefish abundance, ecology, and life history in Florida, p. 526-566. Tech. Rep. for Fed. Aid in Sport Fish Rest. Act Project F-59. Florida Marine Research Institute, Department of Environmental Protection, 100 Eighth Ave. SE, St. Petersburg, FL 33701. 8 Lowerre-Barbieri, S. K., L. R. Barbieri, and J. J. Albers. 1999. Reproductive parameters needed to evaluate recruit- ment overfishing of spotted seatrout in the southeastern U.S. Final report to the Saltonstall-Kennedy (S-K) Grant Program (grant no. NA77FD0074), 23 p. Restricting our sampling to the hours immediately preceding the evening spawning event ensured that those females preparing to spawn were available for capture. Male spotted seatrout, identified by their drumming sounds, caught during this targeted effort were measured and released at the site of capture. We supplemented samples for BF estimation with specimens from local sportfishing tournaments held during sum- mer months in the Charleston Harbor area. We processed samples in the laboratory as previously described. If ovaries appeared by macroscopic examina- tion to contain hydrated oocytes, they were fixed in 10% buffered seawater formalin for potential counts (Hunter et al., 1985). The appropriateness of these ovaries for BF counts was subsequently determined by examining the corresponding histological preparation. To ensure that only those oocytes destined to be ovu- lated during the upcoming spawning event were counted, we chose to use only those oocytes undergoing FOM that could be easily separated by size from late vitellogenic oocytes (Nieland et al.. 2002; Lowerre-Barbieri et al.8). If we observed numerous recent POFs in the histologi- cal sample, the corresponding whole ovary was not used for oocyte counts (because their presence indicated that ovulation had occurred). We reweighed ovaries (approxi- mately 2 weeks after fixation) to the nearest 0.01 g and randomly extracted three 130-150 mg aliquots from eight potential locations in the ovary (each lobe was par- titioned into quarters lengthwise). We stored subsamples in 50% isopropyl and counted oocytes under a Nikon SMZ-U dissecting microscope at 12 x magnification. We counted each subsample twice by using a Bogorov tray and a hand-held counter and conducted a third count if the two initial counts were dissimilar by more than 10%. We used the mean number of oocytes in each subsample to calculate mean oocyte density (number of oocytes per gram preserved ovary weight) and total numbers of oo- cytes in the ovary. We compared mean oocyte densities among the four regions of each ovarian lobe and between the two lobes by using a two-way analysis of variance (ANOVA). Because our variances were heteroscedas- tic, we used nonparametric ANOVA (Kruskal-Wallis or ANOVA on ranks) for comparisons of mean BF among ages, months, and years. To investigate the relationships between BF and length, somatic weight (ovary-free body weight), and age, we used linear regression. Relative fecundity (RF) was calculated as the num- ber of oocytes per gram somatic weight (ovary-free). To select samples for inclusion in RF calculations, we looked for the presence of nuclear migration in histo- logical preparations. We used this criterion to ensure that oocytes of similar morphological dynamics would be used, minimizing the potential for error. We used the Kruskal-Wallis test to investigate the effect of age on RF. Because sample sizes were quite uneven among months, we chose to compare RF between the beginning and end of the spawning season (May and August). This comparison was done by using a Mann-Whitney test. To corroborate any trends in RF, we also conducted diameter measurements on the preserved (10% buffered 478 Fishery Bulletin 102(3) seawater formalin) oocytes. We used a video camera mounted on a Nikon SMZ-U dissecting microscope and coupled to a PC equipped with a frame-grabber and with OPTIMAS- Image Analysis software (ver- sion 6, Media Cybernetics, Bothell, WA). Two readers independently measured the diameter of approximately 30 preserved oocytes in each of three subsamples from 27 ovaries. To test for uniformity of size throughout the ovary, mean oocyte diame- ters were compared between ovarian lobes and among subsample locations within each lobe by using two-way ANOVA. We also compared mean oocyte diameters among months and ages by using two- way ANOVA. Annual fecundity Month Wiley (1996) demonstrated that spot- ted seatrout in South Carolina estuaries constitute a single population. Therefore, we felt justified in calculating monthly egg production (MEP) by multiplying the monthly SF (of specimens taken along the entire coast) by the mean monthly BF (of specimens from Charleston Harbor). Because not all age-1 female trout were mature at the beginning of the spawning season, the fraction of mature age-1 females obtained from previ- ous work in South Carolina (Wenner6) was used to refine the MEP estimate. Because the latter was calculated by using SF obtained from data pooled across years, any comparison of MEP among years was deemed invalid. Kruskal-Wallis tests were used to determine whether MEP varied among months for each age class. Monthly MEP estimates were summed to arrive at an annual fecundity (AF) estimate for each age class. Because the majority of individuals used in this study were aged 1-3, AF was estimated only for these age classes. We used linear regression to investigate the relationship between AF and age and thus predict AF for spotted seatrout aged 4 and 5. Using these predic- tions and the relative abundance of each age class in our samples, we estimated the contribution of each age class to the annual egg production. All statistical analyses were conducted with the Sta- tistical Package for the Social Sciences (version 9.0, SPSS Inc., Chicago, ID. The level of significance for all tests was 0.05. Results A total of 1038 spotted seatrout ranging in age from 1 to 5 was collected for this study. Because 97% of these belonged to age classes 1-3 we report reproductive parameters only for these ages. We examined a total of 941 mature and developing females, ranging in Length from 248 mm to 542 mm TL, to determine spawning Figure 2 Mean monthly gonadosomatic index iGSIl for spotted seatrout in South Carolina for years 1991-2000 (circles). Mean water temperature for 1991-2000 i triangles). Error bars are standard errors, n = 1185. frequency (569, 285, and 87 for ages 1-3, respectively). Of these, 135 specimens (12 from sportfishing tourna- ments) were used to conduct oocyte counts (62, 52, and 21 for ages 1-3. respectively). These fish ranged in length from 268 to 530 mm TL. Minimum size at first maturity, as indicated by the presence of cortical alveoli stage oocytes in histological sections, was 248 mm TL. Size at 50'/c maturity was 268 mm, whereas 100'; matu- rity was reached at 301 mm TL. Condition of females, as indicated by Fulton's condition factor, diminished over the course of the season (P<0.01, r2=0.24i. Seasonality Spawning in the Charleston Harbor area during the study period began in mid to late April as indicated by the presence of oocytes in late FOM or POFs in histo- logical samples. During the study period, mean water temperatures ranged from 16° to 34°C. Highest tem- peratures were recorded during July and August for all three years of the study. The lowest documented water temperature when spawning began was 20°C. Cessation of spawning occurred when water temperature was 28°C. Mean monthly GSI for spotted seatrout captured along the South Carolina coast since 1991 (Fig. 2) showed a marked increase from 4.6 in April to 9.4 in May. Mean GSI in June declined to 6.3 and remained around 5.0 in July and August. A sharp decline was noted in Septem- ber to 2.7, the lowest level for the season. Overall, mean gonadosomatic index (GSI) values followed the seasonal trend in water temperature (Fig. 2). Percent occurrence of females in spawning condition as evidenced from histological examination declined from approximately 87', in August to 12', in September. The percentage of Roumillat and Brouwer: Reproductive dynamics of Cynoscion nebulosus 479 Spawning Sampling Day 0 POFs • • • m A • •- • 1800 t 2200 0200 0600 t 1000 * 1400 t 1800 Fig. 4 A Fig. 4B Fig. 4C Fig. 4D Fig. 4E Sampling Day 1 POFs Fig. 4F A m A 1800 • 2200 0200 0600 Figure 3 1000 1400 1800 Forty-eight-hour chronological time line indicating C. nebulosus spawning time in Charleston Harbor, SC. Postovulatory follicles (POFs) observed at water temperatures >25 C were termed day-0 (<24 hours). Day-1 POFs (>24 hours) were observed only at water temperatures <25°C. Time of capture for specimens in Figure 4 are indicated. post-spawning females increased from 99r in August to 91% in September. Thus, the spawning season for spot- ted seatrout in South Carolina extends from late April through early September. Spawning frequency Day-0 POFs were found through 1800 h of the day fol- lowing a spawning event. Day-1 POFs were first observed in our routine samples when they were 36-37 hours old (the second day following a spawning event) only when water temperatures were below 25°C. Day-1 POFs were excluded from our analysis of SF because they did not provide evidence of a previous night's spawning event. Figure 3 illustrates the time line for POF atrophy in spotted seatrout from 1-42 h after the onset of spawn- ing at 1800 h. Because evidence of spawning for the first 12 h was documented only during a period when water temperatures were greater than 25°C, all of the exam- ples shown are indicative of atrophy in warmer tempera- tures (Fig. 4). As indicated in Table 2, there was a time- dependent deterioration of POFs such that only those <24 h were detectable at water temperatures >25°C. Small sample sizes prevented calculation of monthly SF for each age class by year. Therefore, we pooled data for all three years of this study to obtain a single monthly SF estimate by age class (Tables 3 and 4). The interaction between month and age on SF could not be statistically tested; however, age-3 fish spawned more frequently than younger fish (Kruskal-Wallis, P<0.05) and all seatrout spawned more frequently in June (Kruskal-Wallis, P<0.05). Peaks in SF observed for fish ages 2 and 3 in July and August, respectively (Tables 3 and 4), were not statistically significant. Monthly SF values based on the occurrence of ova- ries containing oocytes in FOM are also presented in Table 3 Spawning frequency (SF) expressed as the number of spawnings per month for C. nebulosus a ges 1-3 for the spawning seasons of 1998 -2000. Numbers in parenthe- ses repr esent days betweer spawnings. n -- = number offish in a sample. FOM = final oocyte maturation; POF = post- ovulatory follicle. Age SF SF (yr) Month n FOM method POF method 1 May 89 4.53(6.85) 4.18(7.42) June 166 4.53(6.62) 9.40(3.131 July 185 4.68(6.62) 6.54(4.74) August 129 6.26(4.95) 4.57(6.79) Total 569 19.9(6.18) 26.34(4.67) 2 May 114 11.5(2.78) 6.80(4.56) June 79 5.70(5.26) 7.60(3.95) July 48 0.65(47.62) 9.04(3.43) August 44 9.87(3.14) 6.34(4.89) Total 285 30.67(4.011 29.36(4.19) 3 May 46 10.10(3.07) 7.42(4.18) June 23 5.22(5.75) 9.12(3.29) July 10 3.10(10.00) 3.10(10.00) August 8 11.61 (2.67) 11.61(2.67) Total 87 32.54(2.67) 31.14(3.95) Overall 941 24.3(5.06) 27.7(4.44) Table 3. However, statistical comparisons were not fea- sible because of the nonrandom collection of specimens. Overall SF was estimated to be once every 4.4 days and once every 5.1 days with the POF and FOM methods, respectively. 480 Fishery Bulletin 102(3) l\»«. •*•?•*►. . Ml W • V 100 um Figure 4 Photomicrographs of C. nebulosus postovulatory follicles (POFs) showing chronology of atresia at water temperatures >25°C. (A) 0-4 hours after spawning. (B) 5-8 hours after spawning. 13-24 hours after spawning. Batch fecundity As expected, we found a significant difference in mean BF among age classes (ANOVA on ranks, P<0.05). Age-1 spotted seatrout produced an average of 145,452 oocytes per hatch spawned. Fish aged 2 and 3 spawned an aver- age of 291,123 and 529,976 oocytes per batch, respec- tively. Therefore, mean BF was compared among months Roumillat and Brouwer: Reproductive dynamics of Cynosaon nebulosus 481 Table 4 Fecundity parameters for C. nebulosus ages 1-3 from South Carolina estuaries BF = batch fecundity in numbe •s of oocytes; SF = spawning frequency based on the postovulatory follicle (POF) method and expressed as the nun tber of spawnin is per month : MEP = monthly egg production = (BF • SFi'i mature. Annual fecundity is the sum of mean month y MEP values ibr each vear class and represents the total number of oocytes produced by any given female from 1 May to 31 Aug jst. Numbers ir parentheses indicate sample sizes. Age (yrl Month Mean BF SF 'i mature Mean MEP 1 May 117.760(12) 4.18(89) 78.6 386,897 June 135,403(161 9.40(166) 94.0 1,196,418 July 141,237(16) 6.54(185) 97.0 895,978 August 176.594(18) 4.57(129) 100 807,035 Annual fecundity=3,286,328 oocytes 2 May 280,724(34) 6.80(114) 100 1,908,926 June 307.322(101 7.60(79) 100 2,335,650 July 370.170(1) 9.04(48) 100 3,346,337 August 307,195(7) 6.34(44) 100 1,947,620 Annual fecundity=9,538,533 oocytes 3 May 487,475(131 7.42(461 100 3.617,061 June 519,630(4) 9.12(23) 100 4,739,027 July 765.911(2) 3.1(10) 100 2,374,325 August 590,994(2) 11.61(8) 100 6,861,439 Annual fecundity= 17.591,852 oocytes Table 5 Monthly relative fecundity (number of oocytes 1998-2000. SD = standard deviation. /grams ovary-free weight) for C. nebu losus ages 1- -3 for the spawning seasons Month Mean Minimum Maximum SD n May 518.6 223.9 976.1 146.2 46 June 603.2 205.7 1306.1 241.8 20 July 820.9 662.2 1314.4 279.0 5 August 693.6 397.3 1021.8 207.9 12 and years for each age class separately (Table 4). There were no significant interannual or monthly variations in mean BF for any of the age classes (age-1: P=0.59, ;; = 62; age-2: P=0.17, n=52; age-3: P=0.07, n=21). However, BF analysis for age-2 fish excluded the month of July because only one two-year-old specimen was captured that month during the study period. We investigated the relationship between BF and total length by using linear regression analysis. After pooling data across years, we found that total length explained 67% of the variability in spotted seatrout BF (Fig. 5A). Batch fecundity showed a similarly strong relationship to female somatic (ovary-free) weight (Fig. 5B) but did not relate to age as strongly (Fig. 5C). The equations below describe these relationships: BF= 2179.65ITL) - 520597 BF = 530.60IOFWT) + 18537.77 BF = 169398. 21( Age) - 30956.33 (r2=0.67) P<0.001 (r2=0.65) P<0.001 (r2=0.58) P<0.001. Mean MEP was significantly different among months for age-1 spotted seatrout (Kruskal-Wallis, P<0.05). Age-1 fish spawned the least number of oocytes in May and most in June (Table 4). Statistical comparisons among months for ages 2 and 3 were inconclusive. Relative fecundity Relative fecundity among 83 spotted seatrout ages 1-3 ranged from 224 oocytes to 1314 oocytes/g OFWT (Table 5). Age did not have an effect on relative fecun- dity (Kruskal-Wallis, P=0.75). We found that spotted seatrout in South Carolina produced significantly more oocytes per gram ovary-free weight at the end than at the beginning of the spawning season (Mann Whit- ney, P<0.05). Mean oocyte diameters did not vary sig- nificantly between ovarian lobes or among locations within each lobe (ANOVA, P=0.28). A comparison among 482 Fishery Bulletin 102(3) CJ O 1200 - BF= 2179.65(71)- 520597 A X f = 0.67. P<0.001 • CO 0) n = 134 1000 - o o o 800 - • / 6 % S^ c % s^ >. • * /^ c 600 - •• •• */ 3 * ' s/9 400 ■ • > x^rT o CO •• u*wK 200 ■ CO 2 - • T"* 2! 1 — n i i 1 1 i 0 300 350 400 450 500 550 Total length (mm) O 1400 ■ B *"" SF= 530 60(O/=WT) + 1853777 Ul 1200 • f = 0 65. P<0.001 . • ■" ■6 600 ■ ' •'• ' %l^ ^ o • ^^^"^ J3) 00 n c ro CD 400 - 200 - ' — i r 1 1 1 i 200 400 600 800 1000 1200 Ovary-free body weight (g) o 1200 - c BF= 169398.21(age) - 3095633 ^ X ? = 0.58. P<0 001 • n = 134 >. 1000 - o o "o 800 - • 6 • ^ • >> i t5 600 ■ 1 o a> 400 ■ o 1 ro 200 • • • c ro 5 • • • 1 2 3 Age Figure 5 Re at Kinship between batch fecundity iBF) and total length (TL) lA), between BF and ovary-free weight (OFWTi (Bi, and bel ween BF and age (Cl for C. nebulosus ages 1-3. Linear re- gression on data pooled for spawning seasons 1998-2000. months and ages revealed that age had no effect on oocyte diameter (ANOVA, P=0.82). However, the effect of month corroborated the pattern of increasing RF as the spawning season progressed: oocytes were significantly smaller at the end of the season (ANOVA, P<0.05). Annual fecundity Annual fecundity estimates (summation of MEP) were approximately 3.2 million. 9.5 million, and 17.6 million oocytes for each age class, respec- tively (Table 4). The equation below describes the relationship between AF and age: AF = 7152762(Age) - 4166620 (r2=0.99> P<0.05. From this relationship, the predicted AF for ages 4 and 5 were 24,444,430 and 31,597,190 oo- cytes, respectively. We expanded AF in relation to the abundance of each age class in our standard random samples for the three years of the study. We estimated that the overall average contribu- tion from age-1 fish to the reproductive output for the season was approximately 29% . whereas fish aged 2 and 3 contributed 39% and 21', of oocytes, respectively. Ages 4-5 comprised less than 3% of specimens sampled and contributed 7% and 4% based on predicted AF values. Discussion Studies on the reproductive biology of Cynoscion nebulosus have established group-synchrony and indeterminate fecundity for this species through- out its range (i.e. Brown-Peterson et al., 1988; Brown-Peterson and Warren, 2001; Nieland et al., 2002; Mercer1 and references therein). Fish with these features release gametes in several batches over a protracted spawning season and annual fecundity is not fixed prior to the onset of spawning (Wallace and Selman. 1981). Based on mtDNA variation among spotted sea- trout, the existence of two populations, one in the Gulf of Mexico and one in the South Atlantic, was established by Gold et al. (1999). However, variations in reproductive parameters have been suggested among geographic locations within the Gulf of Mexico (Brown-Peterson et al., 2002). Wiley (1996) suggested that spotted seatrout com- prise a single stock in South Carolina: therefore reproductive parameters presented in the present study should be applicable only to the spotted seatrout population inhabiting coastal waters of this state. Further studies should be conducted to evaluate the applicability of these parameters to the entire southeast coast. Other investigators (Brown-Peterson et al., 1988; Wieting, 1989; Brown-Peterson and War- Roumillat and Brouwer: Reproductive dynamics of Cynosc/on nebu/osus 483 ren, 2001; Nieland et al., 2002; Lowerre-Barbieri et al.8) have used the gonadosomatic index (GSI) to delineate the spawning season in spotted seatrout. Even though the GSI provided a good approximation of the spawning season, histological data alone provided more precise evidence. Spotted seatrout in South Carolina began spawning near the end of April of each year and ceased by early September. Similarly, Lowerre-Barbieri et al.8 reported that the spawning season for spotted seatrout in Georgia extended from late April to mid-September. We found histological evidence of initial spawning in specimens captured in 20°C water, although approxi- mately 75% of spawning occurred when ambient water temperatures were greater than 25°C. In laboratory experiments, Brown-Peterson et al. (1988) found no suc- cessful spawning in water below 23°C but pointed out that others (McMichael and Peters, 1989) found eggs and larvae in 20.4°C water. We found that females became mature approximately one full year after their birth. A female born in May of one year would be reproductively active in May of the following year. Females born later in the season would not be mature as the same successive season began; therefore, not all one-year-old females were mature when the spawning season began in May, but became mature before that season ended. This maturity sched- ule has also been reported for spotted seatrout in Loui- siana (Nieland et al., 2002). However, Lowerre-Barbieri et al.8 found that all one-year-old females were mature in coastal Georgia. A limited sample size or habitat segregation of mature and immature trout (Lowerre- Barbieri et al.8) may have contributed to their result. The size at first maturity for spotted seatrout in this study was 248 mm TL. This size is comparable to what others have reported in other areas of the species' range (Brown-Peterson et al., 1988; Brown Peterson and War- ren, 2001; Nieland et al., 2002; Mercer1 and references therein; Lowerre-Barbieri et al.8). Our estimate of size at 50% maturity (268 mm TL) was larger than what Nieland et al. (2002) reported for 100% mature trout in Louisiana (250 mm TL). However, Nieland et al.'s (2002) statement that animals are 100% mature at 250 mm TL, does not agree with the growth equation they report for female trout when age = 1. Because we found size at 100% maturity among female spotted seatrout in South Carolina to be about 300 mm TL, we wonder whether Nieland et al.'s (2002) growth equation for female TL was meant to represent SL. Were this the case, they might have offered a different rationale for size at maturity among trout in Louisiana. Brown-Peterson et al. (1988) and Brown-Peterson and Warren (2001) reported size at 100% maturity of 356 mm and 309 mm TL (using the SL-TL conversion found in our "Methods: section) for spotted seatrout in Texas and Mississippi, respectively. Brown-Peterson et al. (1988), however, chose a combination of gears that may not have sampled the trout population in Texas representatively for size-at-maturity estimation. In Mis- sissippi, Brown-Peterson and Warren (2001) used a more appropriate gear for capture of late juvenile and early adult fish. Our estimate of size at lOO1* maturity was quite similar to theirs. Spawning frequency Determining the number of multiple spawning events during a single season for individual fish has been prob- lematic. Initially, there was little understanding of the reproductive dynamics of spotted seatrout, and BF esti- mates were reported to represent the output for a whole season (Pearson, 1929; Sundararaj and Suttkus, 1962; Overstreet, 1983). Hunter et al. (1985) and Hunter and Macewicz (1985) developed techniques to overcome these limitations by providing protocols for the use of hydrated oocytes in determining BF and SF among group-syn- chronous species. To use the techniques of Hunter (1985) and Hunter and Macewicz (1985) appropriately, it is critical to obtain a representative sample of the spawning population. DeMartini and Fountain (1981) and Lisovenko and Adri- anov (1991) maintained that the relative occurrence of hydrated oocytes (as determined macroscopically) was an effective measurement of SF when the spawning popula- tion was sampled representatively. However, when sam- pling a species that spawns in aggregations at specific geographic locations, as do many of the sciaenids, it is inherently impossible to obtain a statistically representa- tive sample of the spawning population for SF estimation based on FOM. Because the window of opportunity is temporally and spatially constrained, obtaining a sample that includes all sizes and ages involved is not feasible; the only choice in this situation is to sample in a directed fashion. This was the sampling strategy used to target females for BF counts; the majority of the animals cap- tured whose oocytes evidenced FOM were obtained in a nonrandom fashion. Additionally, we assumed that fishes demonstrating FOM were moving toward deeper water spawning aggregations and away from our capture gear. For these reasons, we felt that our SF estimates based on the proportion of females with oocytes in FOM were biased and we excluded them from AF estimation. This is an important matter to keep in mind when comparing frequencies of spawning based on different methods. Because obtaining representative numbers of ani- mals with late-maturing oocytes is not often feasible, researchers have relied on the relative abundance of postovulatory follicles (POFs) to calculate SF (Brown- Peterson et al., 1988; Brown-Peterson and Warren, 2001; Nieland et al., 2002; Lowerre-Barbieri et al.8). The POF method lacks the limitations (described above) of the FOM method. Because the method we chose allowed us to sample all sizes and ages of fish in the estuary, obtaining representative numbers of animals with POFs was accomplished effectively. Therefore, we felt that our estimates of SF based on the POF method were more precise and we chose to use them in deriving AF. The POF method depends on the ability to assess the disappearance of these structures. Hunter and Macewicz (1985) systematically sampled captive spawning ancho- vies to develop histological criteria for POF atrophy in 484 Fishery Bulletin 102(3) 19°C water. Their criteria have been used by others to estimate rates of POF atrophy in other species and thereby determine the percentage of a population un- dergoing spawning over a discrete time period (Brown- Peterson et al, 1988; Fitzhugh et al., 1993; Taylor et al., 1998; Macchi and Acha, 2000; Brown-Peterson and War- ren, 2001; Nieland et al., 2002). However, even though it has been demonstrated that the rate of POF atresia depends largely on ambient water temperature (Fitzhugh and Hettler, 1995), few (Brown-Peterson et al., 1988; Macchi and Acha, 2000; Nieland et al, 2002) have taken this into account when establishing the age of POFs for SF estimations. Our diurnal sampling of reproductively active spotted seatrout during warm water conditions en- abled us to establish criteria to accurately estimate the age of POFs throughout the spawning season. Further- more, we verified our assessments by sampling around the clock on two occasions to collect fish over the time period immediately following a spawning event. Spotted seatrout ages 1-3 in SC spawned less fre- quently than those from the Indian River Lagoon, Flor- ida (Crabtree and Adams7) but both studies showed that older fish spawned more frequently than younger animals. Our estimates for spotted seatrout aged 1-3 were 4.7, 4.2, and 4 days, respectively. Trout in these age classes in Florida were reported to spawn once ev- ery 4, 2.8, and 2.5 days, respectively. These differences probably not only reflect the distinct biological environ- ments of each region but also indicate potential discrep- ancies in aging methods. No age-specific estimates of SF are available for other areas in the species' range. Brown-Peterson and Warren (2001) found SF among spotted seatrout in Biloxi Bay, MS, to be significantly lower than that of fish inhabiting the other two areas included in their study. They suggested that Biloxi Bay was a less conducive spawning habitat because of sev- eral factors, including shoreline development and a reduced amount of aquatic vegetation. However, because we found that SF varied significantly among age classes (age-3 fish spawned more frequently), the relative age composition of fish sampled by Brown-Peterson and Warren (2001) in the three estuaries might also have played a critical role in the determination of SF. Batch fecundity The best approach for estimating BF is to use only oocytes in FOM (Hunter et al., 1985; Brown-Peterson et al., 1988; Brown-Peterson and Warren, 2001; Brown-Peterson et al., 2002; Nieland et al., 2002; Lowerre-Barbieri et al.8). When it is not possible to obtain these, BF estimations can and have been carried out in some species by using the largest vitellogenic oocytes (Overstreet, 1983; Hunter et al., 1985; Wieting 1989). These efforts have the poten- i ill of being less accurate because isolating those oocytes destined to be spawned is difficult if the latter have not yet reached final maturation (Nieland et al., 2002). Inevitably this scenario would result in a nonmeasurable overestimation of female reproductive output. Brown- Peterson et al. (1988) and Brown-Peterson and Warren (2001) used a modification of this approach to estimate BF of spotted seatrout in Texas and Mississippi, respec- tively. However, even though the potential existed for overestimating BF, their estimates fell well below those presented in the present study, as did those presented by Nieland et al. (2002) for spotted seatrout ages 2-4 in Barataria Bay, Louisiana. Mean BF for ages 1-3 (170 thousand, 226 thousand, and 274 thousand oocytes, respectively) spotted seatrout in Indian River Lagoon, Florida (Crabtree and Adams7), also differed from those reported here. Our estimate took into account that not all age-1 females were mature at the beginning of the season. Crabtree and Adams,7 however, did not adjust their estimate to reflect this discrepancy. Moreover, due to differences in aging methods, their age-1 and 2 cohorts possibly included ages 2 and 3, respectively. In addition, in the Florida study as well as in ours, relatively few numbers of older specimens were examined. The relationships between BF and length, weight, and age in the present study were significant and predictive. Of these, TL exhibited the most predictive relationship. This fact may explain why age-1 and age-2 spotted seatrout in Georgia had mean BFs considerably higher than ours (175 thousand and 407 thousand, respectively; Lowerre-Barbieri et al.8): the size ranges for age-1 and age-2 in the Georgia study were greater than ours. To- tal length seems to be the most reliable predictor of BF among spotted seatrout in Georgia and SC (Lowerre- Barbieri et al.,8 this study) and in Louisiana (Nieland et al., 2002). However, Crabtree and Adams7 found that BF related best to ovary-free weight among spotted seatrout in Florida. We found ovary-free weight to be the second best predictor of BF. Overall, it appeared that TL and ovary-free weight were better predictors of BF than age for this species (Brown-Peterson, 2003). As with SF, monthly egg production (MEP) estimates for SC spotted seatrout varied throughout the season. Because BF was not significantly different among months for any of our age classes, the variation in MEP resulted directly from the frequency of spawning. Monthly egg production estimates for age-1 fish were lowest in May and highest in June because SF was lowest in May and highest in June. Spawning frequency is a critical reproductive parameter because it seems to dictate annual reproductive output (DeMartini and Fountain, 1981; Brown-Peterson and Warren. 2001; Crabtree and Adams7); therefore, SF should be carefully considered, particularly for managed species. Relative fecundity We found that relative fecundity (RF), the number of oocytes per gram of somatic weight, did not show a sig- nificant relationship with female size. This finding was expected because dividing fecundity by ovary-free weight standardizes the values independently of size. However, this finding was in contrast to that of Brown-Peterson and Warren (2001). They collected specimens during the morning only, whereas we sampled ours throughout the day. This procedure allowed us to examine ovaries Roumillat and Brouwer: Reproductive dynamics of Cynosc/on nebulosus 485 over the entire range of maturation and to select only those clearly showing nuclear migration (based on his- tological observations) ensuring that only oocytes in the same phase of FOM (Brown-Peterson et al., 1988) were included in RF calculations. If sampling is conducted during a time period that is not close to active spawning (i.e., when oocytes are in different phases of FOM), then the number of oocytes per gram may be miscalculated. As with BF, our RF estimates were higher than those reported for seatrout in the Gulf of Mexico (Brown- Peterson et al., 1988; Brown-Peterson and Warren, 2001), although spotted seatrout reproductive para- meters appeared to vary considerably even within the Gulf of Mexico (Brown-Peterson et al., 2002). This was attributed to differential environmental conditions or food availability (or to both) (Brown-Peterson and War- ren, 2001; Brown-Peterson et al., 2002). The significant seasonal increase in RF that we observed for spotted seatrout in South Carolina, however, has not been re- ported elsewhere. Brown-Peterson et al. (1988) found no differences in mean monthly RF among spotted seatrout in Texas. Brown-Peterson and Warren (2001) found sig- nificantly higher RF values in June than in August. In both instances, however, a small sample size may have biased their results. Comparisons of mean oocyte diameters among months related the increase in RF to a general decrease in oocyte size over the course of the season. This phenom- enon is widespread among marine pelagic spawners, and scientists have put forth several explanations to account for it (see Chambers, 1997). Bagenal (1971) sug- gested that egg size decreased over the spawning sea- son owing to concurrent increased food availability for larvae. Others have suggested an inverse relationship between temperature and egg size (Ware, 1975; Woot- ton, 1994; Miller et al., 1995) or a seasonal decrease in egg size that is correlated to the condition of spawning females (DeMartini and Fountain, 1981; Chambers and Waiwood, 1996). The latter seems to apply to spotted seatrout in this study because a diminishing trend through the spawning season was observed in the con- dition factor of females. Annual fecundity Brown-Peterson (2003) presented AF estimates for spot- ted seatrout throughout their range. Our estimates were substantially below those for spotted seatrout in Indian River Lagoon (Crabtree and Adams7) but approximated those of Lowerre-Barbieri et al.8 for trout in Georgia. A possible reason for the higher values in Florida was the more protacted spawning season in that area (50 days longer). No comparisons of AF estimates presented in this study and those of spotted seatrout in the Gulf of Mexico (Brown-Peterson, 2003) were made because they were not specific to age classes. The main impetus behind the present study was to establish annual fecundity (AF) estimates by age class. We found that age-1 through age-3 spotted seatrout occurred abundantly in SC estuaries and that each of these age cohorts showed unique fecundity dynamics. The AF for an average age-1 fish was one-third that of age-2 (-3.28 million vs. 9.5 million). One year-old fish, however, constituted the majority offish in our samples; their abundance was twice that of 2-year-olds and seven times that of 3-year-old fish. Even though the average age-3 trout produced almost twice as many oocytes dur- ing the season (17.5 million) as the average age-2 fish, their reduced abundance in our estuaries made their overall contribution only half that of 2 year-olds. Ages 4 and 5 were estimated to produce approximately 24.4 million and 31.6 million oocytes per female, respective- ly; however, the oocyte production by the predominant age groups overshadowed theirs. When analyzed in re- lation to the occurrence of the other age classes in our estuaries, age-2 fish contributed the greatest number of fertilizable oocytes to the environment (39%). Reliable fecundities based on age and on length are optimal for stock assessment models (Williams9). This study provided AF estimates for three age classes that can be used in age-based models for the spotted seat- rout population in South Carolina. Annual fecundity estimates based on length, however, have not been at- tempted even though length appears to be the best pre- dictor of fecundity in spotted seatrout (see references in Brown-Peterson, 2003). Further analyses to investigate the relationship between egg production and fish length for each month of the spawning season would allow for more precise management efforts based on individual length-based estimates of AF. Acknowledgments We thank members of the Inshore Fisheries Section of the South Carolina Department of Natural Resources for assisting in field data collection throughout this study (C. Wenner, J. Archambault, H. von Kolnitz, W. Hegler, E. Levesque, L. Goss, C. McDonough, C. Johnson, A. Palmer). C. Wenner, H. von Kolnitz, and E. Levesque conducted age assessments. Histological processing was provided by C. McDonough, R. Evitt, A. Palmer, and W. Hegler. Assistance with oocyte counts was provided by C. McDonough, T. Piper, K. Maynard, and R. Evitt. Data management was coordinated by J. Archambault, C. Wenner, E. Levesque, and three anonymous reviewers provided helpful suggestions on the manuscript. Funding for this study was provided by the National Marine Fish- eries Service under MARFIN grant no. NA77FF0550. Literature cited Bagenal. T. B. 1971. The interrelation of the size offish eggs, the date of spawning, and the production cycle. J. Fish. Biol. 3:207-219. ;l Williams, E. 2003. Personal commun. National Marine Fisheries Service, 101 Pivers Island, Rd., Beaufort, NC 28516. 486 Fishery Bulletin 102(3) Brown-Peterson, N. J. 2003. The reproductive biology of spotted seatrout. In Biology of the spotted seatrout (S. A. Bortone, ed.i p. 99-133. CRC Press, Boca Raton, FL. Brown-Peterson, N. J., P. Thomas, and C. Arnold. 1988. Reproductive biology of the spotted seatrout, Cynoscion nebulosus, in South Texas. Fish. Bull. 86:3 73-387. Brown-Peterson, N. J., M. S. Peterson, D. L. Nieland, M. D. Murphy, R. G. Taylor, and J. R. Warren. 2001. Reproductive biology of female spotted seatrout, Cynoscion nebulosus, in the Gulf of Mexico: differences among estuaries? Environ. Biol. 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Fish. Res. Board Can. 32: 2503-512. Wieting, D. S. 1989. Age, growth and fecundity of spotted seatrout (Cynoscion nebulosus) in Louisiana. M.S. thesis, 94 p. Louisiana State Univ.. Baton Rouge, LA. Wiley, B. A. 1996. Use of polymorphic microsatellites to detect popu- lation structure in spotted seatrout, Cynoscion nebu- losus (Cuvier). M.S. thesis, 42 p. Univ. Charleston, Charleston, SC. Wootton, R. J. 1994. Life histories as sampling devices: optimum egg size in pelagic fishes. J. Fish Biol. 45:1067-1077. 488 Abstract— Dungeness crabs {Cancer magister) were sampled with commer- cial pots and counted by scuba divers on benthic transects at eight sites near Glacier Bay, Alaska. Catch per unit of effort (CPUE) from pots was compared to the density estimates from dives to evaluate the bias and power of the two techniques. Yearly sampling was conducted in two sea- sons: April and September, from 1992 to 2000. Male CPUE estimates from pots were significantly lower in April than in the following September; a step-wise regression demonstrated that season accounted for more of the variation in male CPUE than did temperature. In both April and September, pot sampling was signifi- cantly biased against females. When females were categorized as oviger- ous and nonovigerous, it was clear that ovigerous females accounted for the majority of the bias because pots were not biased against nonovigerous females. We compared the power of pots and dive transects in detecting trends in populations and found that pots had much higher power than dive transects. Despite their low power, the dive transects were very useful for detecting bias in our pot sampling and in identifying the optimal times of year to sample so that pot bias could be avoided. Estimating Dungeness crab (Cancer magister) abundance: crab pots and dive transects compared S. James Taggart Glacier Bay Field Station Alaska Science Center U.S. Geological Survey 3100 National Park Rd. Juneau, Alaska 99801 E-mail address |im_taggart(S!usgsgov Charles E. O'Clair National Marine Fisheries Service Auke Bay Laboratory 11305 Glacier Highway Juneau, Alaska 99801 Thomas C. Shirley Juneau Center, School of Fisheries & Ocean Sciences University of Alaska Fairbanks 11120 Glacier Highway Juneau, Alaska 99801 Jennifer Mondragon Glacier Bay Field Station Alaska Science Center U.S. Geological Survey 3100 National Park Rd. Juneau, AK 99801 Manuscript submitted 13 March 2000 to Scient ific Editor's Office. Manuscript approved for publication 25 March 2004 by the Scientific Kditor. Fish Bull. 102:488-497 (2004) Reliable population assessments are fundamental to the management and conservation of commercially har- vested crabs. Many crab populations are sampled with commercial crab pots to estimate population trends, to set harvest quotas, or to differentiate natural population fluctuations caused by anthropogenic changes to the eco- system. Pots are used, for example, to assess the population status of blue crabs, Callinectes sapidus (Abbe and Stagg, 1996), red king crabs, Para- lithodes camtschaticus (Zheng et al., 1993), snow crabs, Chionoecetes opilio (Dawe et al., 1996), and southern king crabs, Lithodes santolla i Wyngaard and Iorio. 1996). The Dungeness crab (Cancer ma- gister) fishery began in southeastern Alaska in 1916 and has been charac- terized by large fluctuations on an- nual and decadal scales tOrensanz et al., 1998). Large variation in the Dungeness crab harvest is not unique to Alaska; similar fluctuations have been documented in California and their causes are the subject of an on- going debate (Higgins et al., 1997a. 1997b). It is not clear whether the processes that cause fluctuations in California are the same as those re- sponsible for oscillations in Dunge- ness crab abundance in Alaska. Most of the Dungeness crab fisher- ies in Alaska are managed by regu- lating the size and sex of the crabs caught, and, in some places, the sea- son of the harvest. In southeastern Alaska, legal harvest is restricted to males with a carapace width greater than or equal to 165 mm (excluding the 10th anteriolateral spines) and the season is timed to avoid sensitive life Taggart et al.: Estimating abundance of Cancer magister 489 58°30'N. ^s 20' t r 135°50' 135 4n\\ Figure 1 Map of study area showing eight study sites in or near Glacier Bay in southeastern Alaska. history periods such as mating and molting (Kruse, 1993; Orensanz et al., 1998). Pre- and postseason stock assessment surveys using crab pots were initiated in southeastern Alaska in 2000 (Rumble and Bishop, 2002). The purpose of the latter management strategy is to assess the abundance of legal-size males before the fishing season, to estimate harvest rates, to define the timing of male and female mating and molting and to determine growth rate by tagging crabs. The usefulness of surveys with pots for Dungeness crab population assessment, however, depends on the accuracy of these surveys in measuring population pa- rameters. Factors that can bias catch per unit of effort (CPUE) and size-frequency estimates for Dungeness crabs are pot soak-time (Miller, 1974; High, 1976; Got- shall, 1978; Smith and Jamieson, 1989); freshness of bait (High, 1976; Smith and Jamieson, 1989); pot design (Miller, 1974; High, 1976; Smith and Jamieson, 1989); and agonistic interactions among conspecifics inside and at the entrance of pots (Caddy, 1979; Smith and Jamieson, 1989). Smith and Jamieson (1989) developed a standardized model to compensate for the effect of agonistic interactions, age of bait, and escapement. They also concluded that researchers could minimize these biases by measuring CPUE with standardized surveys with short soak times. These studies measured sampling bias with pots by comparing catch in pots among various experimental treatments. Opportunities for comparing surveys with pots to direct measures of abundance are rare. In our study, we compared the bias and power of CPUE estimates from surveys with pots to independent measures of abundance conducted by scuba divers on benthic dive transects. Methods Study area The study area included eight sites in southeastern Alaska, near Glacier Bay: North Beardslee Islands (58°33'N 135°54'W), South Beardslee Islands (58°30'N 135°55'W), Berg Bay (58°31'N 136°13'W), Bartlett Cove (58°27'N 135°53'W), Gustavus Flats (58°23'N 135°43'W), Secret Bay (58°29'N 135°56'W), inner Dundas Bay (58°27'N 136°31'W), and outer Dundas Bay (58°21'N 136°18'W) (Fig. 1). All study sites were located within Glacier Bay National Park and Preserve, with the excep- tion of Gustavus Flats, which was located adjacent to the Park boundary in Icy Strait. Glacier Bay is a large (1312 km2) glacial fjord system with high sedimentation rates of clay-silt particles from streams and tidewater glaciers (Cowan et al., 1988). The primarily unconsolidated rocky coastline is highly convoluted with numerous small bays. Dungeness crabs can be found throughout Glacier Bay; however the ma- jority of the population are found in the lower 40 km of the estuary where our sites were located (Taggart et al., 2003 ). The shallow water in and around our study sites was primarily characterized by mud bottom, but sand, pebble, cobble, and shell substrates were also common (Scheding et al., 2001). 490 Fishery Bulletin 102(3) Table 1 Sampling dates for yearly Sample size (n) is listed for spring and fall pot and dive surveys of Dungeness crabs (Cancer magister) pots and dives for each sampling event. in Glacier Bay, Al iska. Year Spring sampling Fall sampl ing Pots it Dives n Pots n Dives n 1992 7-12 April 248 7-12 April 69 17-22 Sept 250 17-22 Sep. 75 1993 20-27 April 350 20-27 April 105 23-28 Sept 250 23-28 Sep. 75 1994 20-27 April 350 23 April-1 May 105 13-18 Sept 249 13-18 Sept. 75 1995 19-26 April 350 23 April-1 May 105 9-14 Sept 236 15-19 Sept. 75 1996 15-21 April 350 22-28 April 105 13-18 Sept 242 19-23 Sept. 73 1997 17-22 April 300 23-28 April 115 14-19 Sept 298 20-25 Sept. 120 1998 — — — — 9-14 Sept 296 16-21 Sept. 91 1999 — — — — 9-14 Sept 299 17-22 Sept. 107 2000 — — — — 9-14 Sept 297 18-23 Sept. 60 Sampling dates Sampling was conducted biannually, in April and Sep- tember, from 1992 to 1997 and annually, in September, from 1998 to 2000 (Table 1). The spring and fall sam- pling periods were selected to coincide with crab life history events and to avoid sampling during commercial fishery operations. April sampling was scheduled to occur before larval hatching in May- June (Shirley et al., 1987) and before the summer commercial fishing season from 15 June to 15 August. September sampling began after the end of the fishing season (15 August) and ended before the beginning of the winter harvest (1 October to 30 November). During 1992, the study sites were sampled with pots (referred to as "pot sampling") and by divers (referred to as "dive sampling") concurrently (Table 1). In 1993 and 1994, sampling was conducted on nearby study sites and the dive sampling usually one day ahead of the pot sampling. For logistical reasons, starting in 1995. we separated the pot sampling and the dive-transect sampling into two separate research cruises. The pot sampling was conducted on the first cruise and the dive sampling occurred on the second cruise; pot and dive sampling were separated at each location by 2 to 12 days. Sampling with pots Crabs were sampled with commercial crab pots (0.91 m in diameter, 0.36 m tall, with 5-cm wire mesh). Escape rings were sealed with webbing on each pot to retain smaller crabs. Pots were baited with hanging bait com- prising salmon, cod, or halibut (depending on availabil- il nd bait jars that were filled with chopped herring and squid. We found that cod was predictably available; therefore from 1996 on, we consistently used cod for hanging bait. Pots were soaked for 24 hours. Within each study site, we set 25 pots in shallow water (0-9 m) and 25 pots in deep water (10-25 m). Each day we set 50 pots in one of the study sites and retrieved the 50 pots that had been set the previous day at one of the other study sites. The pots were set along strings parallel to shore at intervals of approximately 100 m. Within each study area, the strings of pots were located in prime Dungeness crab habitat determined by a local fisherman. We placed the pots at the same locations during subsequent sampling events by using a GPS (Rockwell PLGR+) with an accuracy of ±3 m. We estimate that the pots were set within 20 meters from the original waypoints. Water depth (standardized to mean lower low water), set and retrieval time, and GPS location were recorded for each pot. Water temperature and salinity profiles were measured at each study site during each sampling period with a SEABIRD SBE-19 Profiler. As the pots were retrieved, we counted and identified all organisms. For all Dungeness crabs we recorded the sex, carapace width, shell condition, and damage to appendages. For female crabs we also recorded repro- ductive status. Carapace width was measured to the nearest millimeter immediately anterior to the 10th anterolateral spine with vernier calipers (Shirley and Shirley, 1988; Shirley et al.. 1996). All organisms were returned to the water at the location where they were caught. A potential problem with returning the crabs to the water near the site of capture is the possibility that crabs could be resampled in subsequent pots, which would bias the catch per unit of effort. Beginning in April, 1995, all crabs collected in the South Beardslee Islands and Berg Bay were tagged with a sequentially numbered, double-T Floy tag (Floy Tag and Manufactur- ing Company, Seattle, WA) inserted along the postero- lateral margin of the epimeral suture. Tags placed in this location are retained through ecdysis (Smith and Jamieson, 1989). Of the 5226 crabs tagged, only a single Taggart et al.: Estimating abundance of Cancer magister 491 crab was recovered during the same sampling event. Thus, the probability of resampling crabs by returning them to the water was very low. Sampling by divers Divers using scuba equipment censused crabs on 15 to 20, 2x100 m belt transects within each study site. Approximately one day of sampling was required at each study site. The dive transects were conducted per- pendicular to the shoreline and they extended from the shallow subtidal (0 m, mean lower low water) to 18 m depth or to the end of the 100 m transect, whichever came first. Divers did not go below 18 m depth in an effort to reduce nitrogen accumulation in divers' blood and to reduce the surface intervals required between transects. From 1992 to 1997, transect locations were randomly selected in the same areas as the crab-pot sampling. The random locations selected in 1997 were resampled during the following years of the study. Divers counted all Dungeness crabs located within 1 m of each side of the transect. An effort was made to locate buried crabs by swimming close to the bottom and looking for irregularities in the bottom or protrud- ing crab eyestalks. Each crab was examined and the following were recorded: legal males >165 mm carapace width), sublegal males (<165 mm carapace width), ovig- enous females, and nonovigerous females. Data analysis For each year, we calculated the average pot CPUE for each site by reproductive class (males, nonovigerous females, and ovigerous females). The number of pots sometimes deviated from 50 when a pot was lost or when the degradable cotton string securing the pot lid broke (range: 44-50 pots). The number of crabs counted on dive transects was averaged for each reproductive class by site for each year. All dive transects were conducted perpendicular to shore; thus the transects crossed the shallow habitat where the shallow string of pots was set and terminated at 18 m which was the center of the depth we targeted for the deep pot set. Because the deep pot set was at or slightly beyond the deep end of the transect, we may have sampled more crabs from deepwater habitats than from the shallower transects. However, we did not think this was a significant bias because we sampled crabs from a relatively large area. We, therefore, pooled the pots from both depth strata for analysis. We tested for differences between April and Septem- ber for the pot CPUE data and the dive density data with paired f-tests. CPUE and density data were not normally distributed; therefore we transformed the data with a square-root transformation [Y=JiY + 3/8)] for statistical analyses (Zar, 1996). These analyses were conducted for males, nonovigerous females, and oviger- ous females. Because seasonal increases in water tem- perature could drive differences in CPUE between April and September, we calculated mean water temperatures by averaging the water temperatures at the 5 m and 15 m depths at each site and year. This analysis was limited to years and sites where we collected samples in both April and September (1992-97, from five sites: North Beardslee Islands, South Beardslee Islands, Berg Bay, Bartlett Cove, and Gustavus Flats). We assessed how CPUE was influenced by two independent vari- ables, water temperature and season, with stepwise regression. Because CPUE declined from 1992 to 1997 (Taggart et al., in press), we controlled for year so that it would not confound our analysis. In order to assess sampling bias between pots and dive transects, the percentages of females (females/all crabs), nonovigerous females (nonovigerous females/all crabs), and ovigerous females (ovigerous females/all crabs) were calculated for each site and sampling time. We also com- pared the percentage of the male population that was legal size (legal-size male crabs/all male crabs) from the pots and from the dives. The percentage estimates from the pot data were compared to estimates from the dive transects with a paired sign test (Zar, 1996). If percent- age estimates for pot data were unbiased when compared to estimates from dive data, the pot percentage esti- mates would have an equal chance of being higher or lower than the percentage estimates for the dive data. Because small sample sizes exaggerate percentage com- parisons, we excluded samples where the total number of crabs collected was less than 25 crabs/site. The power of pots and dive transects to detect trends in populations was compared with Monitor, a power analysis program (Gibbs and Melvin, 1997; Gibbs, 1998). For our analyses, we varied the number of tran- sects and pots, compared males and nonovigerous fe- males, and varied the duration of the study. For all analyses the following input parameters of the model were held constant: "survey occasions" = annual, "type" = linear, "significance level" = 0.05, "number of tails" = 2, "constant added" = 1, "trend variation" = 0, "round- ing" = decimal, "trend coverage" = complete, and "rep- lications" = 10,000. To estimate power, the model requires "count" and "variance" for each plot across years for at least three years. Pot and transect data collected from 1992 to 1998 from five sites (North Beardslee Islands, South Beardslee Islands, Berg Bay, Bartlett Cove, and Gusta- vus Flats) were used for these analyses. The data were limited to September to avoid seasonal bias. The aver- age across years was calculated for each transect and each pot. These averages were input into the model's variable called "plot count." For each pot and transect a linear regression was calculated among years (CPUE vs. year for pots; density vs. year for dive transects) and the residual mean square was the "plot variance" variable (Thomas and Krebs, 1997). To estimate the effect of sample size on power we set the "number [surveys] conducted" to four and limited the analysis to males. We varied the number of "plots" (pots and transects). For pots, we randomly selected subsamples of the 250 pots and ran simulations from 25 pots to 250 pots in 25-pot increments. The number 492 Fishery Bulletin 102(3) of dive transects for which data were collected for mul- tiple years was 75. For simulations with a sample size less than 75, we randomly subsampled the data in the same manner as we did with pots. For simulations with sample sizes greater than 75, we amplified the samples with simple bootstrapping to obtain samples from 100 to 250 transects in 25-transect increments (Wonnacott and Wonnacott, 1990). For each sample size, we modeled three annual rates of change (0.02, 0.03, and 0.05). To evaluate how study duration affects power, we lim- ited the analysis to males, varied study duration ("num- ber [surveys] conducted") from two years to 12 years in two-year increments, and compared three annual rates of change (0.02, 0.03, and 0.05) for both pots and tran- sects. To hold effort constant between the two sampling techniques, we set the pot and transect sample size to the number we could accomplish in a five-day research cruise (250 pots and 75 transects). To explore the relationship between annual trend in population and power, we held effort constant (250 pots and 75 transects) and varied the annual trend (from -0.10 to +0.10 in 0.01 increments) for both males and nonovigerous females. It was not possible to conduct a power analysis for ovigerous females because a large proportion of the pots and transects had no ovigerous female crabs. Results The pot CPUE estimates for males, nonovigerous females, and ovigerous females was significantly different in April than in the following September (Fig. 2, A, C, and E). Male and nonovigerous female CPUE was higher in September (Fig. 2, A and C) and ovigerous female CPUE was lower in September (Fig. 2E). In contrast, April den- sity estimates from dive transects were not significantly different from the following September density estimates for males (Fig. 2B). Dive density estimates for nonovig- erous females were higher in September than in April (Fig. 2D); density estimates for ovigerous females were lower in September than in April (Fig. 2F). When we tested the influence of temperature and season on male CPUE with stepwise regression, season was selected first; temperature was not selected because it did not have a significant additional effect (Table 2). Because no significant difference was found between the April and September density estimates from dive transects (Fig. 2B), we did not conduct a stepwise re- gression for the dive data. Percentage estimates of females from sampling with pots were lower than percentage estimates from dive transects for a significant number of samples for both April and September (Fig. 3Ai; therefore pots were bi- ased against sampling females. When females were split by reproductive status, no bias was detected for sampling nonovigerous females with pots (Fig. 3B). In contrast, the percentage estimates for ovigerous fe- males remained biased and the magnitude of the bias increased (Fig. 3C). To test potential sampling bias co 2.5 • 3.25 2.75 ■ 2.25 O 1.5 • 1.75 2.75 2.25 P=0.40 P=0.04 0.75 1.75 3.75 2.75 \ 2.75 P=0.04 April September April September Figure 2 Within-year paired comparisons by site of catch in pots (left column) and density on dive transects (right column) for: (A and B) male Dungeness crabs {Cancer magister); (C and D) nonovigerous female crabs; and (E and F) ovigerous female crabs. Catch and density data were transformed with a square-root transforma- tion. P-values indicate results from paired /-tests and significant results show differences between April and September. Lines on the graphs are parallel if measure- ments at sites were consistently higher or lower in April and September. related to crab size, we compared the proportion of the male population that was legal size sampled with pots and dives (Fig. 4). There was no significant bias when pots and transects were compared with a sign test (April. P>0.999; September, P=0.06). CPUE estimates from pots had a higher power than density estimates from dive transects for the same sample size (Fig. 5). Because more time is required to conduct a dive transect than to set and pull a crab pot, the power of transects compared to pots was even lower when effort was incorporated into the analysis (Fig 6). The power can be increased for both pots and Taggart et al.: Estimating abundance of Cancer magister 493 Table 2 Stepwise regression results of CPUE (male crabs/pot) versus three independent variables (year, season, and temperature). Step Model parameters r2 P-value ( parameter 1 1 2 3 Year 0.1493 Year and month 0.5589 Year, month. 0.5589 and temperature 0.001 (year) 0.04 (month I 0.98 (temperature) transects by increasing the study duration or increasing the amount of change in the population that the study is attempting to detect (Fig. 6). Although pots had more power than dive transects, there was only slightly more power to detect change in abundance of male crabs versus nonovigerous females (Fig. 7). Discussion For male Dungeness crabs, the density estimates from the dive transects showed no difference between April and September (Fig. 2B). The male CPUE estimates from pots, however, were consistently lower in April than in the following September (Fig. 2A). Because feeding rates of Dungeness crabs are correlated with tempera- ture (Kondzela and Shirley, 1993), we thought that tem- perature was likely to explain the differences in CPUE between April and September. We found, however, that season had a larger effect than temperature (Table 2). This result suggests that seasonal factors other than temperature influence catchability. Stone and O'Clair (2001) followed the seasonal movements of Dungeness crabs in a glacial estuary in southeastern Alaska and reported that mean movement of male crabs was lower during the spring than in the late summer and fall. It is possible that our spring sampling schedule coincided with low male activity and male crabs were less likely to encounter a bait plume and be attracted to a pot. These results indicate that if pots are used for sampling, late summer and early fall is the time of year to conduct population assessment surveys of male crabs. Similar seasonal differences in CPUE have also been described for edible crabs (Cancer pagurus) and American lobsters (Homarus americanus) (Bennett, 1974). These data dem- onstrate the importance of controlling for season when comparing CPUE among years or sites. The proportion of large crabs caught in pots increased with longer soak time for Dungeness crabs in British Columbia (Smith and Jamieson, 1989) and red king crabs in Britstol Bay, Alaska (Pengilly and Tracy, 1998). We found no bias when we measured the legal- size proportion of the male population caught in pots and compared it to the proportion sampled on dives 1 0.8 -I 0.6 0.4 -I 0.2 0 1 0.8 i A April P<0.0001 / Sept. P<0.0001 / / @ O ' A / ° O »/i*AA /O n A ~ / a4o o A <3$»# / u ' 1 1 1 i 0.4- 0.2- B April P=0.17 Sept. P=0.49 ' A 0.6 9 o o i>4. O " Oft /A *«0«' *A 0°A^ a * A A AA o O April A September 0- i-i c 0.8 0.6 0.4 0.2 0* April P<0.0001 Sept. P<0.0001 O o cr o 0 0.2 0.4 0.6 0.8 1 Percent crabs on transects Figure 3 The percentage of (A) female Dungeness crabs (C. magister). (B) nonovigerous female crabs, and (Ci ovigerous female crabs estimated from pots and from dive transects. The dashed line in each graph has a slope of 1: thus half of the data points should be above and half should be below the dashed line if percentage estimates for dives and pots are unbiased. Pot and dive transect data for each sex class and season were compared with a paired sign test and P-values are reported. (Fig. 4). We expect, however, that the bias observed in British Columbia and Bristol Bay would occur for our study sites if the soak time of pots were increased. 494 Fishery Bulletin 102(3) O April A September (P>0.999) (P=0.06) 1.0 0.8 0.6 CD CD Q- E • 0A\f i, 02OA O A. °^° O o A O A ' C*°J 0D 0.2 0.4 0.6 0.1 1.0 Percent legal-size male crabs on transects Figure 4 The percentage of male crabs of the Dungeness crab (C. magister) popu- lation that were legal size (>165 mm) estimated from pots compared to the percentage of male crabs estimated from dive transects. Data from pots and dive transects were compared for each season with a paired sign test and P-values are reported. 0.8 06 g o 0.2 Pots Transects -o- •*■ 0 05 change/year 003 change/year 50 100 150 Sample size (n) 250 Figure 5 Relationship between power and sample size (/!) in comparing catch from pots and density on dive transects for male Dungeness crabs (C. magister) at three levels of population change. In both April and September, pot sampling was signif- icantly biased against females (Fig. 3A). When females were categorized as ovigerous and nonovigerous, it was clear that ovigerous females accounted for the major- ity of the bias because pots were not biased against nonovigerous females (Fig. 3B). Similar results have been found for a closely related species, Cancer pagurus; female C. pagurus readily enter pots when they are in a nonovigerous reproductive state but are rarely cap- tured when they are ovigerous (Bennett, 1995). Move- ment studies of Dungeness crabs tagged with sonic transmitters have demonstrated that ovigerous females move less frequently and move slower than males or nonovigerous females (O'Clair et al., 1990). Thus, one explanation for the bias against ovigerous female crabs is that their restricted movements make it less likely they will be able to locate and become entrapped in pots. In addition to being less mobile, ovigerous females may be less attracted to bait than nonovigerous crabs. In controlled feeding experiments, ovigerous females had lower feeding rates than nonovigerous females, and ovigerous females took longer to begin feeding (Schultz et al., 1996; Schultz and Shirley, 1997 i. Therefore, ovig- erous females may be less responsive to the bait plume from a pot. The estimate of nonovigerous females from both pot CPUE and dive transect density increased from April to September (Fig. 2, C and D). As with males, the in- crease in CPUE for nonovigerous females may be partly due to an increase in catchability in September. How- ever, the fact that the density estimates from dives also increased suggests that the number of nonovigerous females actually increased between April and Septem- ber. This explanation is supported by the decrease in ovigerous crabs from April to September for both CPUE (Fig. 2E) and density estimates (Fig. 2F i. The low catchability of ovigerous females makes it problematic to monitor relative abundance of females or changes in sex ratio through time. However, be- cause pots were not biased against nonovigerous fe- males (Fig. 3), the solution may be to estimate the rela- tive abundance of females by sampling after females hatch their eggs and before they extrude a new clutch of eggs in the fall. In southeastern Alaska, most females are nonovigerous in late July and early August (Stone and O'Clair, 2001; Swiney et al., 2003); therefore this would be the optimal time of year to sample females or to measure sex ratio of Dungeness crab populations. Unfortunately, this timing coincides with the summer commercial fishing season, which could bias sampling if there was "competition" between survey pots and commercial pots. For both males and females, the power analyses of the pot and dive data indicated that for most population assessment applications it would be extremely difficult to conduct enough dive transects to obtain sufficient statistical power. Even if it were possible to conduct as many dive transects as pot samples, the power of a dive transect was still lower than that of a pot; the higher power of the pots was due to lower variance among pots. Pots work by attracting crabs with a bait plume; thus the area and number of crabs sampled is Taggart et al.: Estimating abundance of Cancer magister 495 0 6- 04- 0.2 Pots Transects (n=250) (n=75) -O- -•- 0 05 change/year -O ♦ 0 03 change/year •V- -T- 0 02 change/year 0 2 4 6 8 10 Study duration (years) 12 Figure 6 Relationship between power and study duration in comparing catch from crab pots and density on dive transects for male Dungeness crabs (C. magister) at three levels of population change. To hold effort constant, we set the sample size (n ) to the number of pots and dives that could be accomplished in five days. 1 1 0.8- 0.6- 53 s o Q_ 04- \ \\ II ff ^ots Transects 0.2- \ \\ // J ("=250) (n=75) >. » 3 y ~D~ "*" Males w^\ A?f -^- •+. Nonovigerous females -.C 12 -0.1 -0 08-0.06-0 04-0 02 0 0 02 0.04 0.06 0.08 0 1 0.12 Trand (change/year) Figure 7 Relationship between power and trend in population in comparing catch in crab pots and density on dive transects for male and nonovigerous female Dung eness crabs iC. magister). larger with pots than with transects and the variance with pots is lower. Despite their low power, the independent measures of abundance provided by dives helped us identify bias in our Dungeness crab survey method. Our analysis of these two techniques demonstrates that it is possible to avoid most biases with pots if sampling is conducted at optimal times of year. Similar comparisons could be conducted in other areas to identify sampling biases so that they could be minimized and important param- eters, such as abundance, size, and sex ratio, could be monitored effectively. 496 Fishery Bulletin 102(3) Acknowledgments This long-term study was made possible by the support of a large number of people. J. de La Bruere made the field work efficient and enjoyable through his expert ability to operate the RV Alaskan Gyre. We thank A. Andrews for large efforts during the field work, data manage- ment, and analysis. G. Bishop, C. Dezan, E. Hooge, P. Hooge, E. Leder, J. Luthy, J. Nielsen, C. Schroth, D. Schultz, L. Solomon, and K. Swiney each participated in the project for several years. The manuscript was improved by comments from E. Mathews, E. Knudsen, and three anonymous reviewers. We thank M. Jensen, J. Brady, T. Lee, M. Moss, and S. Rice for their contin- ued support. We especially thank the large number of unnamed graduate students, faculty, state and federal agency researchers — over 70 people total — who gener- ously donated their time and efforts to this long-term project. This project was funded by the United States Geological Survey and the National Park Service. Literature cited Abbe, G. R„ and C. Stagg. 1996. Trends in blue crab {Callinectes sapidus Rathbun) catches near Calvert Cliffs. Maryland, from 1968 to 1995 and their relationship to the Maryland commercial fishery. J. Shellfish Res. 15:751-758. Bennett, D. B. 1974 The effects of pot immersion time on catches of crabs. Cancer pagurus L. and lobster, Homarus gammarus (L.). J. Cons. Int. Explor. Mer 35:332-336. 1995. Factors in the life history of the edible crab [Cancer pagurus L.) that influence modelling and manage- ment. ICES Mar. Sci. Symp. 199:89-98. Caddy. J. F. 1979. Some considerations underlying definitions of catch- ability and fishing effort in shellfish fisheries, and their relevance for stock assessment purposes. Manuscript Report, 1489, 1-18 p. Department of Fisheries and Oceans Canada, Halifax, Nova Scotia, Canada. Cowan. E. A., R. D. Powell, and N. D. Smith. 1988. Rainstorm-induced event sedimentation at the tidewater front of a temperate glacier. Geology 16: 409-412. Dawe, E. G., D. M. Taylor, and J. M. Hoenig. 1996. Evaluating an index of snow crab (Chionocetes opilio) biomass from trapping surveys. In Proceedings of the international symposium on biology, management, and economics of crabs from high latitude habitats. Anchorage, Alaska, October 11-13, 1995, vol. 96-02, p. 301-314. Alaska Sea Grant College Program, Univ. Alaska, Anchorage, AK. Gibbs, J.P. 1998. Monitoring populations of plants and animals. Bio- Science 48:935-940. Gibbs, J. P., and S. M. Melvin. 1997. Power to detect trends in water bird abundance with call-response surveys. J. Wildl. Manag. 61(4): 1262-1267. Gotshall, D. W. 1978. Catch-per-unit-of-effort studies of northern Cali- fornia Dungeness crabs. Cancer magister. Calif. Fish Game 64(31:189-199. Higgins, K., A. Hastings, and L. W. Botsford. 1997a. Density dependence and age structure: non- linear dynamics and population behavior. Am. Nat. 149(21:247-269. Higgins, K., A. Hastings, J. N. Sarvela, and L. W. Botsford. 1997b. Stochastic dynamics and deterministic skele- tons: population behavior of Dungeness crab. Science 276(53171:1431-1435. High, W. L. 1976. Escape of Dungeness crabs from pots. Mar. Fish. Rev. 38(41:19-23. Kondzela, C. M., and T C. Shirley. 1993. Survival, feeding, and growth of juvenile Dunge- ness crabs from southeastern Alaska reared at different temperatures. J. Crustacean Biol. 13(11:25-35. Kruse, G. H. 1993. Biological perspectives on crab management in Alaska. In Proceedings of the international symposium on management strategies for exploited fish populations (G. Kruse, D. M. Eggers. R. J. Marasco, C. Pautzke, and T J. Quinn II, eds. i, 355-384 p. Lowell Wakefield Fish- eries Symposium. Alaska Sea Grant College Program Report 93-02, Univ. Alaska, Fairbanks, AK. Miller, R. J. 1974. Saturation of crab traps: reduced entry and es- capement. J. Cons. Int. Explor. Mer 38(31:338-345. O'Clair. C. E., R. P. Stone, and J. L. Freese. 1990. Movements and habitat use of Dungeness crabs and the Glacier Bay fishery. In Second Glacier Bay science symposium. Glacier Bay National Park & Pre- serve, AK, Sept. 19-22, 1988 (A. M. Milner and J. D. Wood Jr., eds. I, 74-77 p. U.S. National Park Service, Anchorage, AK. Orensanz, J. M., J. Armstrong, D. Armstrong, and R. Hilborn. 1998. Crustacean resources are vulnerable to serial depletion — the multifaceted decline of crab and shrimp fisheries in the Greater Gulf of Alaska. Rev. Fish Biol. Fish. 8:117-176. Pengilly. D„ and D. Tracy. 1998. Experimental effects of soak time on catch of legal- sized and nonlegal red king crab by commercial king crab pots. Alaska Fish. Res. Bull. 5(2):81-87. Rumble. J., and G. Bishop. 2002. Report to the Board of Fisheries, Southeast Alaska Dungeness Crab Fishery. Alaska Department of Fish and Game, Regional Information Report 1J02-45. p. 2.2-2.16. Alaska Dep. Fish and Game. Juneau, AK. Scheding, K., T C. Shirley, C. E. O'Clair. and S. J. Taggart. 2001. Critical habitat for ovigerous Dungeness crabs. In Spatial processes and management offish populations October 27-30 (G. H. Kruse, N. Bez, A. Booth. M W. Dorn, S. Hills, R. N. Lipcius, D. Pelletier, C. Roy, S. J. Smith, and D. Withercll. eds.), 431-446 p. Alaska Sea Grant, report AK-SG-01-02. Univ. Alaska, Fair- banks, AK. Schultz. D. A., and T. C. Shirlej 1997. Feeding, foraging and starvation capability of ovig- erous Dungeness crabs in laboratory conditions. J. Crustacean Res. 26:26-37. Schultz, D. A., T. C. Shirley, C. E. O'Clair, and S. J. Taggart. 1996. Activity and feeding of ovigerous Dungeness crabs in Glacier Bay, Alaska. In High latitude crabs: biology. management, and economics. October 11-13. 1995. vol. Taggart et al.: Estimating abundance of Cancer magister 497 96-02, p. 411-424. Alaska Sea Grant College Program, AK-SG-96-02, Univ. Alaska, Anchorage, AK. Shirley, S. M., and T. C. Shirley. 1988. Appendage injury in Dungeness crabs, Cancer magister, in southeastern Alaska. Fish. Bull. 86:156- 160. Shirley, T. C, G. Bishop, C. E. O'Clair, S. J. Taggart, and J. L. Bodkin. 1996. Sea otter predation on Dungeness crabs in Glacier Bay, Alaska. //; High latitude crabs: biology, manage- ment, and economics, 563-576 p. Lowell Wakefield Fish- eries Symposium. Alaska Sea Grant College Program Report 96-02, Univ. Alaska, Fairbanks, AK. Shirley, S. M., T. C. Shirley, and S. D. Rice. 1987. Latitudinal variation in the Dungeness crab. Cancer magister: zoeal morphology explained by incubation temperature. Mar. Biol. 95(31:371-376. Smith, B. D., and G. S. Jamieson. 1989. A model for standardizing Dungeness crab (Cancer magister\ catch rates among traps which experienced different soak times. Can. J. Fish. Aquat. Sci. 46:1600-1608. Stone, R. P., and C. E. O'Clair. 2001. Seasonal movements and distribution of Dungeness crabs, Cancer magister, in a glacial southeastern Alaska estuary. Mar. Ecol. Prog. Ser. 214:167-176. Swiney, K. M., T. C. Shirley. S. J. Taggart. and C. E. O'Clair. 2003. Dungeness crab, Cancer magister, do not extrude eggs annually in southeastern Alaska: An in situ study. J. Crustacean Biol. 23(21:280-288. Taggart, S. J., P. N. Hooge, J. Mondragon, E. R. Hooge, and A. G. Andrews. 2003. Living on the edge: the distribution of Dunge- ness crab, Cancer magister, in a recently deglaciated fjord. Mar. Ecol. Prog. Ser. 246:241-252. Taggart, S. J., T. C. Shirley. C. E. O'Clair, and J. Mondragon. In press. Dramatic increase in the relative abundance of large male Dungeness crabs, Cancer magister, following closure of commercial fishing in Glacier Bay. Alaska. In Aquatic protected areas as fisheries management tools (J. B. Shipley, ed.). Am. Fish. Soc. Bethesda, MD. Thomas, L., and C. J. Krebs. 1997. A review of statistical power analysis software. Bull. Ecol. Soc. A. 78(21:128-139. Wonnacott, T. H., and R. J. Wonnacott. 1990. Introductory statistics for business and economics, 815 p. John Wiley & Sons, New York. NY. Wyngaard, J. G., and M. I. Iorio. 1996. Status of the southern king crab (Lithodes san- tolla) fishery of the Beagle Channel. Argentina. In Proceedings of the international symposium on biology, management, and economics of crabs from high latitude habitats. Anchorage, Alaska, October 11-13, 1995, vol. 96-02, 25-39 p. Alaska Sea Grant College Program, Univ. Alaska. Anchorage, AK. Zar, J. H. 1996. Biostatistical analysis, 663 p. Prentice-Hall, Inc.. Upper Saddle River, NJ. Zheng, J.. T. J. Quinn II. T. J., and G. H. Kruse. 1993. Comparison and evaluation of threshold estimation methods for exploited fish populations. In Proceedings of the international symposium on mnagement strate- gies for exploited fish populations, Anchorage, Alaska, October 21-24, 1992 iG. H. Kruse. D. M. Eggers, R. J. Marasco, C. Pautzke. and T. J. Quinn II. eds.i. vol. 93-02, 267-289 p. Alaska Sea Grant College Program, Anchorage, AK. 498 Abstract— The lengths of otoliths and other skeletal structures recovered from the scats of pinnipeds, such as Steller sea lions iEumetopias juba- tus), correlate with body size and can be used to estimate the length of prey consumed. Unfortunately, otoliths are often found in too few scats or are too digested to usefully estimate prey size. Alternative diag- nostic bones are frequently recovered, but few bone-size to prey-size cor- relations exist and bones are also reduced in size by various degrees owing to digestion. To prevent under- estimates in prey sizes consumed techniques are required to account for the degree of digestion of alternative bones prior to estimating prey size. We developed a method (using defined criteria and photo-reference material) to assign the degree of digestion for key cranial structures of two prey species: walleye pollock (Theragra chalcogramma) and Atka mackerel (Pleurogrammus monopterygius). The method grades each structure into one of three condition categories; good, fair or poor. We also conducted feeding trials with captive Steller sea lions, feeding both fish species to determine the extent of erosion of each structure and to derive condition-specific diges- tion correction factors to reconstruct the original sizes of the structures consumed. In general, larger struc- tures were relatively more digested than smaller ones. Mean size reduc- tion varied between different types of structures (3.3-26.3%), but was not influenced by the size of the prey consumed. Results from the observa- tions and experiments were combined to be able to reconstruct the size of prey consumed by sea lions and other pinnipeds. The proposed method has four steps: 1) measure the recovered structures and grade the extent of digestion by using defined criteria and photo-reference collection; 2) exclude structures graded in poor con- dition; 3) multiply measurements of structures in good and fair condition by their appropriate digestion correc- tion factors to derive their original size; and 4) calculate the size of prey from allometric regressions relating corrected structure measurements to body lengths. This technique can be readily applied to piscivore dietary studies that use hard remains of fish. Manuscript submitted 28 April 2003 I" Scientific Editor's Office. Manuscript approved for publication 25 March 2004 by the Scientific Editor. Fish. Bull. 102:498-508(2004). A method to improve size estimates of walleye pollock (Theragra chalcogramma) and Atka mackerel (Pleurogrammus monopterygius) consumed by pinnipeds: digestion correction factors applied to bones and otoliths recovered in scats Dominic J. Tollit ' Susan G. Heaslip1 Tonya K. Zeppelin2 Ruth Joy' Katherine A. Call2 Andrew W. Trites1 1 Marine Mammal Research Unit, Fisheries Centre University of British Columbia, Room 18, Hut B-3 6248 Biological Sciences Road Vancouver, British Columbia, Canada, V6T 1Z4 E-mail address (for D. J Tollit): tollit 5zoology.ubc.ca 2 National Marine Mammal Laboratory Alaska Fisheries Science Center National Marine Fisheries Service, NOAA 7600 Sand Point Way NE Seattle, Washington 98115 Prey skeletal remnants from stom- ach samples and more recently from fecal (scat) samples are widely used to determine what pinnipeds eat (Pitcher, 1981; Olesiuk et al., 1990; Tollit and Thompson, 1996; Browne et al., 2002). Prey can usually be identified from taxon-specific hard remains, the sizes of which often cor- relate with the length and mass of the prey (Harkonen, 1986; Desse and Desse-Berset, 1996). In the past, sag- ittal otoliths were commonly used to estimate prey size (Frost and Lowry, 1981) but were recognized to erode or become completely digested (Prime and Hammond, 1987; Harvey, 1989). Thus, otolith measurements likely underestimated sizes and numbers of fish ingested (Jobling and Breiby, 1986), thereby preventing a reliable assessment of overlap of prey con- sumed with catch taken by commercial fisheries (Beverton, 1985). Accurate estimates of size of prey consumed by pinnipeds are also important in order to understand foraging behavior and to explain spatial and temporal vari- ability in diet composition. There are at least three potential ways to deal with the effect of diges- tion on estimates of prey size. One is to measure only relatively uneroded otoliths and assume that eroded oto- liths are from the same size fish as uneroded otoliths (Frost and Lowry, 1986; Bowen and Harrison, 1994). Another is to apply a single species- specific digestion coefficient or correc- tion factor (DCF), derived from feed- ing experiments with captive seals fed fish of known sizes and using measurements of all the eroded oto- liths recovered in the scats produced (Prime and Hammond, 1987; Harvey, 1989). The third is to estimate and correct for the degree of digestion (based on defined losses of morpho- logical features) of each recovered otolith by using estimates from ref- erence material (Sinclair et al.. 1994; Antonelis et al., 1997) or by applying condition-specific DCFs derived from fish fed in captive seal feeding studies (Tollit et al„ 1997). Of the three approaches to correctly estimate prey size from skeletal re- mains, there is the assumption with To Hit et al.: A method to improve size estimates of Theragra chalcogramma and Pleurogrammus monopterygius 499 the use of only uneroded otoliths that recovery and the degree of digestion is independent of otolith size, result- ing in a potentially biased fraction. For certain species it can also result in a notable reduction in sample size because relatively few otoliths pass through the gut in good condition. The second approach of applying mean species-specific DCFs is an improvement to not accounting for size reduction (Laake et al., 2002); how- ever, there is the assumption with this approach that all structures are reduced in size by the same amount. Consequently, mean fish mass may be overestimated if such correction factors are applied to relatively undi- gested otoliths, or they may be underestimated if ap- plied to very digested otoliths (Hammond et al., 1994; Tollit et al., 1997). The third method accounts for the intraspecific variation in size reduction caused by di- gestion, reduces systematic error (see Hammond and Rothery, 19961. yields estimates of mass that compare favorably to those fed to captive animals (Tollit et al., 1997). and hence may well be the most promising ap- proach to reconstructing prey size. The dramatic decline of the western population of Steller sea lions (Eumetopias jubatus) in the 1980s (Loughlin et al., 1992; Trites and Larkin, 1996) has prompted a number of studies to determine what they eat and the extent of dietary overlap (prey consumed) with catch taken by commercial fisheries. Stomach con- tents analysis was used to determine diet until the late 1980s when scat analysis became the preferred method (e.g., Pitcher, 1981; Frost and Lowry, 1986; Sinclair and Zeppelin, 2002). However, unlike in stomachs, there is an overall sparsity of otoliths in Steller sea lion scats (Sinclair and Zeppelin, 2002) and, therefore there is a need to also use other skeletal structures to describe the size of prey consumed. The following outlines a method (using defined crite- ria and photo-reference material) to assign the degree of digestion for otoliths and alternative key skeletal structures of walleye pollock (Theragra chalcogramma) and Atka mackerel (Pleurogrammus monopterygius) recovered from scats. We also present the results of a feeding study with captive Steller sea lions used to determine the extent of erosion and to derive condition- specific digestion correction factors to reconstruct the original sizes of the pollock and Atka mackerel struc- tures consumed. Finally, we combine these DCFs with newly developed regression formulae that estimate fish length to derive a more accurate method of estimating size of pollock and Atka mackerel consumed by Steller sea lions and other pinnipeds (see Zeppelin et al., 2004, this issue; Tollit et al., 2004, this issue). Materials and methods Experimentally derived digestion correction factors Feeding experiments were conducted with two 3-year- old female Steller sea lions: Steller sea lion 1 (SSL11 [ID no. F97HA], mean mass 129 kg; steller sea lion 2 [SSL2] [ID no. F97SI], mean mass 150 kg) between October 2000 and April 2002 at the Vancouver Aquarium Marine Science Centre. Over the experimental period, the sea lions were fed pollock for 52 days in 16 separate feeding experiments, and Atka mackerel for 31 days in 5 separate feeding experiments, at between -4-8% of body mass per day. Fork length (FL) and weight of all fish were measured to ±0.1 cm and ±1 g. Sea lions were fed meals of pollock of three size categories (small, 28.5-32.5 cm FL; medium. 33.5-38.7 cm FL; large, 40-45 cm FLi and meals of Atka mackerel of one size category (30-36 cm FL). Fish of one particular size cat- egory were fed either as a single meal or as a seven-day block of meals. Full details of a typical experimental protocol can be found in Tollit et al. (2003). Size ranges for any category offish fed within separate experiments were usually <3 cm. Fecal material was collected until no other remains of experimental meals were found (7 days after feeding), and was washed through a 0.5-mm sieve to remove hard parts. Each animal was maintained on whole Pacific herring (Clupea pallasi) between experi- ments at ~6'7( body mass per day. The strong relationship between fish size and otolith size also exists for other skeletal structures (Desse and Desse-Berset, 1996). Thus, we quantified the types and numbers of the prey structures recovered in the scats of free-ranging Steller sea lions (from the collections of Trites et al.1 and Sinclair and Zeppelin, 2000) and selected seven of the most commonly occurring struc- tures for pollock and Atka mackerel. These were the sagittal otolith (OTO), as well as the interhyal (INTEl, hypobranchial 3 (HYPO), pharyngobranchial 2 (PHAR), angular (ANGU), quadrate (QUAD), and the dentary (DENT). The structures selected also had particular morphological features that seemed to be relatively resistant to digestion and could effectively be used to estimate fish size (Figs. 1 and 2, Table 1). Concurrent with our feeding study, we measured selected structures (Figs. 1 and 2) from randomly subsampled fresh fish and combined these data with unpublished NMFS data to generate allometric regres- sion formulae relating structural measurements to fish length (see Zeppelin et al., this issue). Fork lengths (±0.1 cm) and weights (±1 gl of an extended subsample of pollock (8.3-47.7 cm FL) were measured to generate an appropriate regression formula for estimating fish mass from fork length estimates. All selected structures are located in the cranium as illustrated in Zeppelin et al. (2004, this issue). Naming offish structures follows Rojo (1991). Initial inspection of selected structures found in scats from the wild revealed high intraspecific variation in the degree of digestion, ranging from no apparent size reduction to about a 60% size reduction (heavily digest- ed material). Consequently, we extended the condition- Trites, A. W., D. G. Calkins, and A. J. Winship. 2003. Un- publ. data. Marine Mammal Research Unit, Fisheries Centre, University of British Columbia, Hut B-3, 6248 Bio- logical Sciences Road, Vancouver, B.C., Canada, V6T 1Z4. 500 Fishery Bulletin 102(3) Figure 1 Photographs showing the changes in morphological features in seven cranial structures of walleye pol- lock iTheragra chalcogramma) resulting from digestion. Within each section of the figure three condi- tion categories (good, fair, and poor) are represented from left to right for (A) interhyal (INTEi. iBi hypobranchial 3 (HYPOl, (Ci pharyngobranchial 2 (PHARl, (D) angular (ANGU), lE i quadrate (QUAD), (F) dentary (DENTi and (G) sagittal otolith (OTO). Key features used in classification are labeled (see Table 1 for details), and the measurements taken to calculate fish length (solid line between dashed lines i. specific DCF technique described by Tollit et al. (1997). We began by examining the external morphological features and surface topography of selected structures from undigested fish (<12 cm to >53 cm) and compared these with the topography of the same structures recov- ered from scats collected from wild and captive animals "To Hit et al.: A method to improve size estimates of Theragra chakogramma and Pleurogrammus monopterygius 501 Figure 2 Photographs showing the changes in morphological features in seven cranial structures of Atka mack- erel (Pleurogrammus monopterygius) resulting from digestion. Within each section of the figure three condition categories (good, fair, and poor) are represented from left to right for (A) interhyal (INTE), (B) hypobranchial 3 (HYPO), (C) angular (ANGU), (D) quadrate (QUAD), (E) dentary (DENT) and (F) sagittal otolith (OTO). Key features used in classification are labeled (see Table 1 for details) and measurements taken to calculate fish length (solid line between dashed lines). (Figs. 1 and 2). The morphological features used to as- sess level of digestion showed no differences in relative shape, structure, or in proportion across the size range of fresh fish examined. We then devised a criteria-based method to assign a condition category to each structure depending on the degree of digestion. These criteria 502 Fishery Bulletin 102(3) take into account only the loss of size to the relevant feature being measured to estimate fish length (Figs. 1 and 2). The grading criteria for otoliths (OTO) were based on the condition categories developed by Sinclair et al. (1996) to investigate prey selection by northern fur seals (Callorhinus ursinus). As seen in Sinclair et al. (1996) and other studies (Frost and Lowry, 1986; Tollit et al., 1997), external features such as lobation and the general shape and definition of the sulcus were found in our study to be good indicators of the degree of otolith digestion. For the remaining cranial bones, digestion indicators included the loss of definition or breakage of defined structural features such as the horns and ridge (QUAD), hammerhead and stock (DENT), swan neck, notch and ridge (INTE), honeycomb and crown iPHAR), cap, neck, and head (ANGU) and tube and cone (HYPO). We used changes in the described condi- tion-category criteria (see Table 1 for full details) in tandem with photo-reference material (Figs. 1 and 2) to classify all structures into one of three digestion grades or condition categories: "good", "fair," or "poor." Hard parts recovered from feeding experiments were sorted, and all selected cranial structures were as- signed a condition category and measured with cali- pers to within ±0.01 mm. Because otoliths were often chipped or partly broken lengthwise, both length and width were measured. To test our grading technique, an independent observer (T.Z.) reassigned a random sub- sample of each condition category of pollock structures (n = 158) in a blind test. On initial investigation, high intraspecific variation was observed within the selected structures assigned in poor condition in our feeding study with captive Steller sea lions. Consequently, structures in poor condition were not used to calculate DCFs for this category. Our basis for exclusion was supported by the work of Sin- clair et al. (1994) and Tollit et al. (1997). Captive sea lions in our study occasionally regurgitated prey in the swim tank. Recovered structures that we considered to have been regurgitated were excluded from DCF calcu- lations (i.e., vertebrae still articulated, bones that had flesh attached or that were of a size to exclude passage through the pyloric sphincter). Mean reduction (MR) in the metric of each structure (s) recovered from our feeding experiment was estimated for each remaining condition category (c) according to MR T xlOO, where the mean size of egested structures (E) of each condition category was calculated from measurements of those recovered from the captive feeding experiments. and the mean size of each ingested structure (/.) was estimated from the fork length of fish fed by using inverse predictions of the regression formulae derived from fresh material (Zeppelin et al., 2004, this issue). Mean ingested size was estimated by using bootstrap simulations (1000 runs) that randomly sampled with replacement and selected the median (500th value I from the sorted bootstrapped values (Reynolds and Aebischer, 1991). For pollock, mean reduction for each condition cat- egory was compared across size ranges by using a Krus- kal-Wallis analysis of variance. A significance level of P<0.0056 was set based on the Bonferroni adjusted probability for nine multiple comparisons (Siegel and Castellan, 1988). Failing to find any significant differ- ences resulted in pooling the data from each size range to calculate specific condition category MR values. Con- dition category DCFs were calculated for each selected structure as / JESC except for PHAR structures of Atka mackerel because too few elements were recovered from the scats of captive animals. Estimating confidence limits around digestion correction factors We used a bootstrap simulation to estimate upper and lower bounds of the 95% confidence interval (CI) given that the DCF is a ratio of two means (Reynolds and Aebischer. 1991 1. This technique allows different sources of error to be combined or partitioned. There were two major sources of error associated with calculating DCFs (Tollit et al., 1997). The first were those associated with the regression formulae used to calculate the mean size of structure ingested from the original fish fed. and the second were those associated with the errors around the mean size of egested structure (i.e., resampling errors). We assessed errors associated with the regression formulae using a parametric bootstrapping procedure (Manly, 1997) that involved regressing structure size against fork length. This was repeated 1000 times and 95% confidence intervals were taken as the 25th and 975th values of the sorted bootstrapped regression coef- ficient values. Results were compared to those computed analytically by using the resultant standard error (Eq. 17.23 in Zar, 1984) and were found to be consistent (see Zeppelin et al., 2004, this issue). We estimated resampling errors related to the vari- ability in digestion of egested structures by repeatedly selecting n structures, at random, with replacement from the original sample set of n egested structures. Mean egested size was recalculated in this way 1000 times, as were a mean DCF and 95% CI as described above. Both regression and resampling errors were combined in sequence to derive overall 95% CIs around DCFs. Our recommended procedure for applying our DCFs to cranial structures recovered from scats collected in the wild has four steps: 1) measure the recovered structures and grade the extent of digestion using defined criteria and photo-reference collection; 2) exclude structures graded in poor condition; 3) multiply measurements of structures in good and fair condition by their appropriate digestion correction factors to derive their original size; and 4) calculate the size of prey from allometric regres- sions relating corrected structure measurements to fish fork lengths (see also Tollit et al.. 2004, this issue). "To Hit et al.: A method to improve size estimates of Theragra cha/cogramma and Pleurogrammus monopterygius 503 Results A relatively objective method to estimate the degree of digestion of dominant structures of pollock and Atka mackerel was derived by using defined criteria (Table 1) and photo-reference material (Figs. 1 and 2). Condition- specific digestion correction factors (and derived confi- dence intervals) calculated for each structure augmented our method of estimating size of prey from bones and otoliths recovered in scats, as well as potentially from bones and otoliths taken from stomach contents. Mean reduction (MR) in the size of pollock DENT and QUAD in good condition and ANGU, HYPO, IN- TE, OTO, and PHAR in fair condition were between 12.2-18.5%, and larger values were found for QUAD (22.8%) and DENT (24.7%) in fair condition (Table 2). Our overall 95% confidence intervals were generally symmetrical and converted to a mean range of ±2.2% (±0.5, SD) around MR values. Mean DCFs ranged be- tween 1.14 and 1.33, and lower bounds of 95% CIs ex- ceeded 1.11 in all instances, confirming that egested structures of these condition categories were significant- ly smaller than the size at which they were ingested (Table 2). Partitioning errors showed that resampling of egested structures was the major source of error (>73% across structures) within the overall total. Our overall 95% CIs resulted in a maximum total error of ±1.7 cm around an estimated mean of 40 cm for pollock. Mean reduction in the size of Atka mackerel struc- tures varied more widely (3.3-26.3%), leading to DCFs ranging between 1.03 and 1.36. QUAD in good condition provided the smallest DCF, and DENT in fair condition the largest. Overall, our 95% CIs converted to a mean range of ±2.4% (±0.6) around MR values, and all lower 95% CI bounds exceeded 1.0 (Table 2). As seen when errors were partitioned for pollock, errors owing to resa- mpling of egested structures were the major source of error (>83'7t across structures) within the overall total for Atka mackerel. Our overall 95% CIs resulted in a maximum total error of ±1.2 cm around an estimated mean of 40 cm for Atka mackerel. With the exception of the two largest skeletal struc- tures (DENT and QUAD, Table 2), some selected struc- tures (INTE, HYPO, PHAR, ANGU, and OTO) occurred in scats with no clear loss in size or loss of morphologi- cal features related to digestion. For these five struc- tures, we ascribed the condition category good and as- signed a DCF of 1.0 (i.e., no correction for partial size reduction due to digestion required). Of the 158 structures in our blind test, 141 (89.2%) were assigned identical condition categories. Of the remaining 17 structures, 11 (65%) were noted as be- ing borderline between categories. Angulars (ANGU) accounted for the majority (-60%) of all differences, with all but one re-assigned in good condition as op- posed to fair condition. On review, differences in as- signing angulars were mainly the result of differences in opinion on what constituted a well-defined and sharp point (Fig. 1, Table 1). Clarification through the addi- tional use of reference material (including both pristine structures and examples of each condition category) is advised, particularly for angulars. Comparison of the same 158 bones between two observers (D.T. and S.H.) using the same structure reference collection resulted in assigning more than 93% (147/158) of structures to an identical category. The regression formula for estimating pollock mass (M) from fork length (FL) estimates was best described by using an exponential equation (M=0.0051 xFL3n, n = 981. r2 = 0.987). Discussion The size of prey consumed by pinnipeds can usually be reliably estimated from otoliths recovered in scats if partial digestion is accounted for (Tollit et al., 1997). However, otoliths from Steller sea lion scats are often found in too few numbers, or are too digested or broken to be useful (Sinclair and Zeppelin, 2002; Tollit et al., 2004, this issue). It was, therefore, necessary to use alternative skeletal structures to estimate the size of prey selected by Steller sea lions. Zeppelin et al. (2004, this issue) documented good relationships (r2=0.78-0.99) between the size of selected alternative structures and fork length for pollock and Atka mackerel. However, all skeletal structures are susceptible to digestion in the stomach (our study, and Murie and Lavigne, 1986). Thus, techniques are required to account for the degree of digestion of alternative structures prior to estimating prey size. Reductions in the size of otoliths during passage through the digestive tract of pinnipeds have been widely reported (e.g., da Silva and Neilson, 1985; Prime and Hammond. 1987; Harvey, 1989; Tollit et al., 1997). Similarly, we found significant reduction in the sizes of all selected cranial structures from pollock and Atka mackerel. Size reduction also showed great variability. Relatively small structures were found with no obvious loss in size due to digestion, but were also frequently heavily eroded. The degree of digestion on different otoliths and bones may be related to species, size of fish (Bowen, 2000). or even its shape, but seems to be random in any one meal (Murie and Lavigne, 1986). Degree of diges- tion likely depends on a range of factors such as meal size, meal frequency, meal composition, and method of consumption. In the face of these multiple factors we feel our method for classifying the degree of digestion into one of three condition categories is practical and relatively objective. However, our technique does not consider potential biases of enumeration associated with smaller prey being more susceptible to complete digestion than relatively larger prey, or of individual fish being counted more than once if all multiple struc- tures are used. Nevertheless, resolution to these biases have been advocated (see Tollit et al., 1997; Laake et al., 2002; Tollit et al., 2003; Tollit et al., 2004, this is- sue). The category selections chosen with our criteria showed good agreement among independent observers. 504 Fishery Bulletin 102(3) Table 1 Distinctive external morphological features for defining the degree of digestion (condition category) as good(G), fair (F), and poor (Pi for selected cranial structures of walleye pollock and Atka mackerel. Features are given in order of importance. See Table 2 for definition of structure codes and Figure 1 and 2 for illustrations. WP = walleye pollock. Species and structure code Category Distinctive external morphological features Walleye pollock INTE HYPO PHAR ANGU QUAD DENT OTO G 1) Retains characteristic shape, notably the ridge and swan neck. 2) Both ends show no damage (except for the loss of the point and minor nicks) and do not affect length measurement. F li Ridge and swan neck clearly defined. 2) One end can show limited damage with <159c reduction. Minor nicks on opposite end acceptable, if there is no further loss in length measurement. P 1) Loss of characteristic shape, with ridge or swan neck (or both) ill defined. Body of structure contains holes. 2) Both ends show clear damage. G 1) Retains characteristic shape, with cone ~2x the length of the tube. 2) Tube end and area 1 show no damage (except for minor nicks) and do no affect the total length measurement. 3) Cone end angled when viewed from the front elevation (back elevation shown in Fig. 1). F 1) Tube end or area 1 shows limited damage (cone end no longer angled) clearly preventing an accurate length measurement. P 1) Both tube end and area 1 show damage, and a general loss of characteristic shape evident. G 1) Retains characteristic shape, notably a raised spout, honeycomb, and crown. 2) Crown clearly projects above honeycomb (front elevation) and is intact at area 2. 3) Clear projection of honeycomb (back elevation — see area 3). 4) No affect on measurement. F 1) No clear projection of honeycomb at area 3 or crown shows damage at area 2 (preventing an accurate width measurement). 2) Crown or spout (or both) can show minor damage. P li Characteristic shape lost, often only honeycomb present. 2) Honeycomb smooth, crown heavily eroded with areas 2 and 3 eroded or damaged. 3) Both ends show clear damage. G 1) Point sharp and well defined with no impact to measurement. 2) Area 4 in good condition and angled curve complete. 3) Neck present, but with minor damage. 4) Material of cap continues to point tip. F 1 ) Point no longer extensive or sharp or area 4 damaged and poorly defined. 2 ) Neck usually present, but with wear. P 1 ) Characteristic shape lost with neck often absent. 2 ) Point heavily eroded. 3 ) Area 4 shows damage or no definition. G 1) Groove defined from all angles and observable with the naked eye. 2) Horns rounded. 3) Angle of area 5 is clearly curvilinear. 4) Evidence of ridge and spike often observable. F 1) Groove unclear, forming only an indisctinct notch. 2) Horns have lost rounded definition and may- be pointed or worn on one side. 3) Ridge and spike often only residual. P 1) Horns pointed, notch absent. 2) Ridge and spike often absent. 3) Angle of area 5 flattened. 4 1 Unable to determine side with assurance. G 1) Hammerhead retains rounded end elevation features (note: both sides are not exactly symmetrical), allowing full width measurement. 2) Material in addition to the stock may be present. 3) Stock clearly curved from side elevation. 4) Width and breadth of "rounded" stock similar. F 1) Hammerhead shows erosion on one side, affecting full width measurement. 2) Breadth of stock reduced, but not flattened. P li Hammerhead eroded and flattened with both sides showing erosion. 2) Breadth of stock flattened, stock less rounded and less robust. 3) Unable to determine side with assurance. P 1) Sulcus and scalloping (on most margins) well defined, and no obvious reduction in size due to digestion. 2) Able to determine side. 3) Inside strongly convex, retains characteristic shape. F 1) Sulcus worn but shows definition. 2) Able to determine side. 3) Scalloping worn but shows no reverse scalloping. P 1) Unable to determine side. 2) Scalloping worn completely smooth and reverse scalloping present. 3) Clearly broken, worn, flattened, and unable to obtain an accurate measurement. continued "To Hit et al.: A method to improve size estimates of Theragra chalcogramma and P/eurogrammus monopterygius 505 Table 1 (continued) Species and structure code Category Distinctive external morphological features Atka mackerel INTE HYPO ANGU QUAD DENT OTO G Like that of walleye pollock (WP) (except no point), with ridge, neck and notch clearly defined. F li Ridge present, but shows signs of wear. 2) Swan neck shows wear resulting in a "horseshoe" shape. 3 1 Notch shows only minor wear or chipping and does not prevent accurate measurement. P 1) Loss of characteristic ridge and neck with body worn (may contain holes). 2) Both neck and notch show clear damage. G 1) Cone rounded and complete, tube complete, retains characteristic shape. 2) Minor nicks on cone and tube may be present but do not impact total length measurement. F Cone worn, loss of rounded shape, and area 1 shows minor chipping or damage or tip of tube is broken or clearly chipped. P 1) Cone body and area 1 show major wear, chips, and breaks. 2) Tube broken or absent entirely, unable to measure length. G Like WP. additionally cap rounded and head shows only minor wear. F Like WP, additionally 1) cap worn with loss of shape. 2) Head worn, chipped, and often has holes, 3) Ridge on dorsal side above neck worn smooth. P Like WP. additionally 1 ) head shows major damage, wear, breaks, and holes, 2 ) Difficult to determine side with confidence. G It Horns rounded and in good condition, with angle between horns clearly curvilinear. (Note: Horns are of unequal size and shape and one side is more robust, rounded, and sloped.) 2 1 Evidence of ridge and spike observable. 3) Definition of left and right sides is easily achievable. F 1) Horns have lost rounded definition and may be pointed or worn on one side, making distinction between sides difficult. 2) Ridge and spike often only residual. P Like WP, additionally no distinction between horns easily achievable. G Like WP (except no hammerhead), additionally 1) head retains characteristic features, tooth sockets present, 2) Ventral side of head a defined point. F Like WP, additionally 1) head eroded or chipped with tooth sockets noticeably worn, 2) Point on ventral side of head eroded or chipped. P Like WP, additionally head eroded or flattened with point often heavily eroded or badly chipped, accurate measurement unattainable. G 1 1 Rostrum not chipped or broken. 2 ) Sulcus clearly defined, as are anterior and posterior colliculums. 3 ) Scalloping on antirostrum and posterior end clearly distinguishable. 4 ) No obvious wear or chipping with no obvious reduction in length (width). 5) Cristae of antirostrum forms a well-defined ridge. F 1) Rostrum shows some wear but remains unbroken and retains characteristic shape. 2 1 Sulcus still has definition despite wear, shown as a uniform channel, anterior and posterior colliculums indistinct. 3) Cristae and scalloping on antirostrum and posterior end worn smooth. P 1 ) Rostrum or posterior end broken or worn to such a degree that accurate measurement cannot be obtained. 2) Sulcus difficult to distinguish or worn smooth. 3) Cristae and scalloping on antirostrum and posterior end worn completely smooth. 4) Side cannot be easily obtained. Nevertheless, we recommend that a hands-on reference collection be used. The procedure we recommend to estimate fish length after classification involves excluding structures considered heavily digested (condition category poor) and applying specific condition-category DCFs (Ta- ble 2) to the remaining structure prior to calculating fish length from allometric regressions (see Tollit et al., 2004, this issue). The exclusion of structures in poor condition was necessary because of the large and variable size reduction observed in this category. Our technique uses changes noted in the morphological features of the structures themselves and is therefore not specific to Steller sea lions. Because structures are likely to erode in a predictable manner whatever the species of the stomach they are held within, it seems probable that they can also be classified into a particular condition category for use with DCFs. Consequently, our technique may be appropriate to marine piscivore dietary studies where prey size needs 506 Fishery Bulletin 102(3) Table 2 Condition-specific digestion correction factors (DCFs) for selected cranial structures of walleye pollock and Atka mackerel with associated condition categories good (G) and fair (F ). Lower and upper bounds of the 95^ confidence intervals (CIs) were calcu- lated by using bootstrap ret ampling procedures. Structure CI Species and structure code Grade n DCF SD Lower Upper Walleye pollock Interhyal INTE F 54 1.1423 0.054 1.1168 1.1714 Hypobranchial 3 HYPO F 22 1.1658 0.063 1.1343 1.1970 Pharyngobranchial 2 PHAR F 39 1.2109 0.067 1.1717 1.2566 Angular ANGU F 85 1.2065 0.103 1.1670 1.2462 Quadrate QUAD G 20 1.2272 0.039 1.2025 1.2512 F 27 1.2958 0.074 1.2623 1.3280 Dentary DENT G 17 1.1950 0.074 1.1546 1.2337 F 31 1.3285 0.071 1.2941 1.3649 Otolith (length) OTOL F 37 1.1593 0.059 1.1400 1.1788 Otolith (width) OTOW F 49 1.2107 0.089 1.1901 1.2419 Atka mackerel Interhyal INTE F 37 1.0729 0.089 1.0374 1.1085 Hypobranchial 3 HYPO F 23 1.1361 0.040 1.1160 1.1568 Angular ANGU F 40 1.1361 0.097 1.1053 1.1700 Quadrate QUAD G 23 1.0343 0.053 1.0070 1.0597 F 23 1.0886 0.078 1.0551 1.1213 Dentary DENT G 34 1.2068 0.098 1.1666 1.2466 F 37 1.3563 0.143 1.3063 1.4119 Otolith (length) OTOL F 109 1.1691 0.109 1.1459 1.1921 Otolith (width) OTOW F 115 1.2062 0.104 1.1837 1.2277 to be determined from partially digested prey hard remains. Experimentally derived pollock DCFs were deter- mined from three distinct size ranges of fish (28.5-45 cm FL), but the degree of erosion for each structure within each condition category did not show any sig- nificant differences across this range. We also found the relative shape, structure, and proportion of the morpho- logical features used to estimate erosion were consistent for both smaller and larger fish. We therefore believe DCFs can be used for fish outside of the experimental size range of this study. Average size reduction varied between different pollock structures (12.2-24.7%) and also between condition categories, as they did for Atka mackerel (Table 2). We determined that pollock otoliths in fair condition were reduced by 149J in length, close to the 20% value estimated from reference material (Sin- clair et al., 1994). Our criteria for defining a condition category of fair for pollock otoliths equates to a grade between low amounts and medium amounts of diges- tion as defined by Tollit et al. (1997) for Atlantic cod (which has a similar looking otolith). Our value of 14% lies midway between those determined for cod otoliths graded low and medium. Jaw bones (DENT) were by far the largest structure used in our study but do not appear to pass through the pyloric sphincter without some level of digestion. Usually only the hammerhead and stock (representing less than a third of the whole structure) are recov- ered in scats. The large size accounts for the relatively greater percent mean reduction and hence higher DCF of DENT structures graded either in good or fair con- dition (Table 2). Although quadrates (QUAD) are also relatively large structures with a projecting ridge that is often much reduced when found in scats, we found QUAD structures of Atka mackerel recovered in scats from field studies and captive sea lion studies in rela- tively better condition than those of pollock, leading to differences in grading criteria and resulting DCFs (Tables 1 and 2). Part of the reason may be that the horns on a pollock QUAD project widthwise more than those of Atka mackerel, presenting a greater surface area for digestive erosion of the structural feature that is measured to estimate size (Fig. 1). Our overall 95^f confidence intervals around DCFs were generally narrow (Table 2), highlighting the tight fit of the regression formulae used and the benefits of partitioning the data into specific categories. Our boot- strap analysis suggests that resampling errors were the major source of error in calculating DCFs. Future research should concentrate on improving sample sizes for data on percentage size reduction of bones for each Tollit et al.: A method to improve size estimates of Theragra chalcogramma and Pleurogrammus monopterygius 507 category, rather than on improving regression formulae. For both prey species, QUAD in good condition and OTO in fair condition, in addition to pollock INTE in fair condition and Atka mackerel HYPO in fair condi- tion, provided the most reliable estimates of prey size (Table 2). DENT in fair condition, particularly for Atka mackerel, provided the least reliable estimate of prey size (Table 2). Measurement error was relatively insig- nificant, but attention should be taken when measuring ANGU and HYPO (Tollit et al., 2004, this issue). Companion studies by Tollit et al. (2004, this issue) and Zeppelin et al. (2004, this issue) demonstrate the feasibility of applying DCFs to structures other than otoliths and the need to consider the degree of diges- tion to correctly estimate the length of prey eaten by pinnipeds and other piscivores. Applying appropriate digestion correction factors will lead to more refined estimates of consumption (mass of prey) by marine mammals, as well as the extent of potential overlap (length of prey) with the length of fish caught by com- mercial fisheries. Acknowledgments Funding was provided to the North Pacific Universities Marine Mammal Research Consortium by the National Oceanographic Atmospheric Administration and the North Pacific Marine Science Foundation. We would like to thank the marine mammal trainers and staff of the Vancouver Aquarium Marine Science Centre, the contribution of personnel of the UBC Marine Mammal Research Unit of the UBC EM facility, J. L. Laake for statistical advice, T. J. Orchard, C. J. Gudmundson, S. J. Crockford, M. Wong, E. H. Sinclair, and two anonymous reviewers. We would also like to express gratitude to the organizations and companies that have donated fish to the project. Work was undertaken in accordance with UBC Animal Care Committee guidelines. Literature cited Antonelis, G. A., E. H. Sinclair. R. R. Ream, and B. W. Robson. 1997. Inter-island variation in the diet of female northern fur seals tCallorhinus ursinus) in the Bering Sea. J. Zool. lLond.l 242(31:435-51. Beverton, R. J. H. 1985. Analysis of marine mammal-fisheries interaction. In Marine mammals and fisheries (J. R. Beddington, R. J. H. Beverton, and D. M. Lavigne eds.l, p. 3-33. George Allen & Unwin, Boston, MA. Bowen, W. D. 2000. Reconstruction of pinniped diets: Accounting for complete digestion of otoliths and cephalopod beaks. Can. J. Fish. Aquat. Sci. 57:898-905. Bowen, W. D., and G. D. Harrison. 1994. Offshore diet of grey seals Halichoerus grypus near Sable Island, Canada. Mar. Ecol. Prog. Ser. 112 (1-2):1-11. Browne, R, J. L. Laake. and R. L. DeLong. 2002. Improving pinniped diet analyses through identification of multiple skeletal structures in fecal samples. Fish. Bull. 100:423-433. da Silva, J., and J. D. Neilson. 1985. Limitations of using otoliths recovered in scats to estimate prey consumption in seals. Can. J. Fish. Aquat. Sci. 42:1439-1442. Desse, J., and N. Desse-Berset. 1996. On the boundaries of osteometry applied to fish. Archaeofauna 5:171-179. Frost, K. J., and L. F. Lowry. 1981. Trophic importance of some marine gadids in northern Alaska and their body-otolith size relation- ships. Fish. Bull. 79:187-192. 1986. Sizes of walleye pollock, Theragra chalcogramma, consumed by marine mammals in the Bering Sea. Fish. Bull. 84:192-197. Hammond, P. S„ A. J. Hall, and J. H. Prime. 1994. The diet of grey seals around Orkney and other island and mainland sites in North-eastern Scotland. J. Appl. Ecol. 31:340-350. Hammond, P. S., and P. Rothery. 1996. Application of computer sampling in the estimation of seal diet. J. Appl. Stat. 23:525-533. Harkonen, T. 1986. Guide to the otoliths of the bony fishes of the North- east Atlantic, 256 p. Danbiu ApS. Biological Consul- tants, Hellerup, Denmark. Harvey, J. T. 1989. Assessment of errors associated with harbor seal (Phoca vitulina) fecal sampling. J. Zool. (Lond.) 219:101-111. Jobling, M., and A. Breiby. 1986. The use and abuse offish otoliths in studies of feed- ing habits of marine piscivores. Sarsia 71:265-274. Laake. J. L., P. Browne, R. L. DeLong. and H. R. Huber. 2002. Pinniped diet composition: a comparison of esti- mation models. Fish. Bull. 100:434-477. Loughlin, T. R., A. S. Perlov, and V. A. Vladimirov. 1992. Range-wide survey and estimation of total number of Steller sea lions in 1989. Mar. Mamm. Sci. 8:220- 239. Manly, B. F. J. 1997. Randomization, bootstrap and Monte Carlo meth- ods in biology, 2nd ed., 399 p. Chapman and Hall, New York. NY. Murie, D. J., and D. M. Lavigne. 1986. Interpretation of otoliths in stomach content analy- ses of phocid seals quantifying fish consumption. Can. J. Zool. 64:1152-1157. Olesiuk, P. F., M. A. Bigg, G. M. Ellis, S. J. Crockford, and R. J. Wigen. 1990. An assessment of the feeding habits of harbour seals [Phoca vitulina) in the Strait of Georgia, British Columbia, based on scat analysis. Can. Tech. Rep. Fish. Aquat. Sci. 1730. 135 p. Pitcher. K. W. 1981. Prey of the Steller sea lion. Eumetopias jubatus, in the Gulf of Alaska. Fish. Bull. 79:467-472. Prime, J. H., and P. S. Hammond. 1987. Quantitative assessment of gray seal diet from fecal analysis. In Approaches to marine mammal ener- getics (A. C. Huntley, D. P. Costa. G. A. J. Worthy, and M. A. Castellini. eds.), p. 165-181. Allen Press, Lawrence, KS. 508 Fishery Bulletin 102(3) Reynolds, J. O, and N. J. Aebischer. 1991. Comparison and quantification of carnivore diet by fecal analysis — a critique, with recommendations, based on a study of the fox Vulpes vulpes. Mamm. Rev. 21:97-122. Rojo, A. L. 1991. Dictionary of evolutionary fish osteology, 273 p. CRC Press, Boca Raton. Siegel, S., and N. J. Castellan. 1988. Nonparametric statistics for the behavioral sci- ences, 2nd ed., 399 p. McGraw-Hill, New York, NY. Sinclair, E. H., G. A. Antonelis. B. W. Robson, R. R. Ream, and T. R. Loughlin. 1996. Northern fur seal, Callorhinus ursinus, predation on juvenile walleye pollock, Theragra chalcogramma. In Ecology of juvenile walleye pollock, Theragra chalco- gramma. Papers from the workshop "The importance of prerecruit walleye pollock to the Bering Sea and North Pacific ecosystems" Seattle, WA, October 28-30, 1993 iR. D. Brodeur, P. A. Livingston, T. R. Loughlin, and A. B. Hollowed, eds.), p. 167-178. NOAA Tech. Rep. NMFS 126. [NTIS No. PB97-155188.] Sinclair, E., T. Loughlin, and W. Pearcy. 1994. Prey selection by northern fur seals {Callorhi- nus ursinus) in the eastern Bering Sea. Fish. Bull. 92:144-156. Sinclair, E. H.. and T. K. Zeppelin. 2002. Seasonal and spatial differences in diet in the west- ern stock of Steller sea lions (Eumetopias jubatus). J. Mammal. 83(4):973-990. Tollit, D. J., S. G. Heaslip, and A. W. Trites. 2004. Sizes of walleye pollock (Theragra chalcogramma) consumed by the eastern stock of Steller sea lions (Eumetopias jubatus) in Southeast Alaska from 1994 to 1999. Fish. Bull. 102:522-532. Tollit, D. J., M. J. Steward, P. M. Thompson, G. J. Pierce, M. B. Santos, and S. Hughes. 1997. Species and size differences in the digestion of otoliths and beaks: implications for estimates of pin- niped diet composition. Can. J. Fish. Aquat. Sci. 54:105-119. Tollit, D. J., and P. M. Thompson. 1996. Seasonal and between-year variations in the diet of harbour seals in the Moray Firth, Scotland. Can. J. Zool. 74:1110-1121. Tollit, D. J., M. Wong. A. J. Winship, D. A. S. Rosen, and A. W. Trites. 2003. Quantifying errors associated with using prey skeletal structures from fecal samples to determine the diet of the Steller's sea lion (Eumetopias jubatus). Mar. Mamm. Sci. 19l4):724-744. Trites, A. W., and P. A. Larkin. 1996. Changes in the abundance of Steller sea lions (Eumetopias jubatus) in Alaska from 1956 to 1992: how many were there? Aquat. Mamm. 22:153-166. Zar. J. H. 1984. Biostatistical analysis, 2nd ed., 718 p. Prentice- Hall, Englewood Cliffs, N.J. Zeppelin, T. K., D. J. Tollit. K. A. Call. T. J. Orchard, and C. J. Gudmundson. 2004. Sizes of walleye pollock (Theragra chalcogramma i and Atka mackerel (Pleurogrammus monopterygius) con- sumed by the western stock of Steller sea lions (Eume- topias jubatus) in Alaska from 1998 to 2000. Fish. Bull. 102:509-521. 509 Abstract— Prey-size selectivity by Steller sea lions lEumetopias juba- tus) is relevant for understanding the foraging behavior of this declin- ing predator, but studies have been problematic because of the absence and erosion of otoliths usually used to estimate fish length. Therefore, we developed regression formulae to estimate fish length from seven diag- nostic cranial structures of walleye pollock (Theragra chalcogramma) and Atka mackerel (Pleurogrammus monopterygius). For both species, all structure measurements were related with fork length of prey (r2 range: 0.78-0.99). Fork length (FL) of walleye pollock and Atka mackerel consumed by Steller sea lions was estimated by applying these regres- sion models to cranial structures recovered from scats (feces) collected between 1998 and 2000 across the range of the Alaskan western stock of Steller sea lions. Experimentally derived digestion correction factors were applied to take into account loss of size due to digestion. Fork lengths of walleye pollock consumed by Steller sea lions ranged from 3.7 to 70.8 cm (mean=39.3 cm, SD = 14.3 cm, n = 666l and Atka mackerel ranged from 15.3 to 49.6 cm (mean = 32.3 cm, SD = 5.9 cm, rc = 1685). Although sample sizes were limited, a greater propor- tion of juvenile (<20 cm) walleye pol- lock were found in samples collected during the summer (June-September) on haul-out sites (64^ juveniles, ;;=11 scats) than on summer rookeries (9% juveniles, n = 132 scats) or winter l February-March) haul-out sites (3% juveniles, n = 69 scats). Annual changes in the size of Atka mackerel consumed by Steller sea lions cor- responded to changes in the length distribution of Atka mackerel result- ing from exceptionally strong year classes. Considerable overlap (>51%) in the size of walleye pollock and Atka mackerel taken by Steller sea lions and the sizes of these species caught by the commercial trawl fishery were demonstrated. Sizes of walleye pollock (Theragra chalcogramma) and Atka mackerel (Pleurogrammus monopterygius) consumed by the western stock of Steller sea lions (Eumetopias jubatus) in Alaska from 1998 to 2000 Tonya K. Zeppelin' Dominic J. Tollit2 Katherine A. Call1 Trevor J. Orchard3 Carolyn J. Gudmundson' E-mail address: Tonya Zeppelin ifflnoaa gov 1 National Marine Mammal Laboratory Alaska Fisheries Science Center National Marine Fisheries Service, NOAA 7600 Sand Point Way NE Seattle, Washington 98115 2 Marine Mammal Research Unit Fisheries Center, Room 18, Hut B-3 University ot British Columbia 6248 Biological Sciences Road Vancouver, British Columbia, Canada V6T 1Z4 3 Department of Anthropology University of Toronto 100 St. George Street Toronto, Ontario, Canada M5S 3G3 Manuscript submitted 28 April 2003 to Scientific Editor's Office. Manuscript approved for publication 25 March 2004 by the Scientific Editor. Fish. Bull. 102:509-521 (2004). The western stock of Steller sea lions (Eumetopias jubatus) in the Gulf of Alaska and the Bering Sea has experi- enced dramatic and continued declines since the mid-1970s (Loughlin et al„ 1992; Loughlin and York, 2000). It is likely that changes in prey availabil- ity linked to commercial fisheries and large-scale oceanographic changes are among the reasons for the continued decline (Loughlin and Merrick, 1989; NRC, 1996). The diet of the western stock of Steller sea lions has been recently assessed (Sinclair and Zep- pelin, 2002), but discrete selection of prey by size has not been described. The size of prey is relevant for under- standing the foraging behavior of the predator as well as the ecological role of the prey (e.g., mortality at a given life history stage). In the case of the Steller sea lion, prey-size selectivity is particularly important for understand- ing spatial and temporal changes in diet and is needed for making fishery management decisions. Size of fish prey consumed by ma- rine mammals has been estimated by using sagittal otoliths recovered from stomach and more recently scat samples (Pitcher, 1981; Frost and Lowry, 1986; Browne et al., 2002). Significant relationships have been demonstrated between fish fork length (FL) and otolith length (Templeman and Squires, 1956; Frost and Low- ry, 1981; Harvey et al., 2000). The use of otoliths to describe the size of prey taken by Steller sea lions has proved useful in data collected from stomach samples (e.g., Pitcher, 1981; Calkins and Goodwin1). However, few 1 Calkins, D. G., and E. Goodwin. 1988. Unpubl. report. Investigation of the declining sea lion population in the Gulf of Alaska, 76 p. Alaska Department of Fish and Game, 333 Raspberry Road, Anchorage, Alaska, 99518-1599. 510 Fishery Bulletin 102(3) otoliths are recovered from Steller sea lion scat, and measurements of otoliths recovered from scats likely underestimate prey size because of partial erosion from digestion (Prime and Hammond, 1987; Del- linger and Trillmich, 1988; Harvey, 1989). Because of the impracticality of collecting stomachs and the low number and poor quality of otoliths found in scats, alternative methods are needed to accurately describe the size of prey consumed by Steller sea lions. Archaeological studies routinely use skeletal struc- tures other than otoliths to estimate either fish length or mass (Keys, 1928; Casteel, 1976; Owen and Merrick, 1994; Desse and Desse-Berset, 1996). Wise 11980) used a regression offish length on ver- tebrae length to estimate prey size from scat samples of otters (Lutra lutra) and mink tMustela vison). The regression approach relies on the assumption that the overall size of a given fish and the size of skeletal structures are highly correlated. This as- sumption has been substantiated for cranial and skeletal structures other than otoliths in various North Pacific fish species (Orchard, 2001). Thus, the use of cranial structures appear to be a viable alter- native to the use of otoliths for studying prey size of Steller sea lions. Walleye pollock (Theragra chalcogramma) and At- ka mackerel (Pleurogrammus monopterygius) rank among the top prey items of Steller sea lions (Sin- clair and Zeppelin, 2002) as well as being valuable in the U.S. commercial fishery (NMFS, 2003). We estimated fork length for these two primary prey species from scats collected between 1998 and 2000 across the range of the Alaskan western stock of sea lions. Fish length was estimated by using regres- sion formulae relating bone or otolith measurement to fork length for seven cranial structures found in sufficient quantities and in good and fair condition in scat samples. Experimentally derived digestion cor- rection factors (Tollit et al., 2004b, this issue) were applied to bone and otolith measurements to account for loss of size due to erosion. The methods developed here proved to be an effective tool to estimate size of prey selected by Steller sea lions and are applicable for other marine mammal diet studies particularly where otoliths are highly eroded. Materials and methods Development of regression formulae Fork-length to bone and otolith-length regression equations were developed for seven cranial struc- tures from walleye pollock and Atka mackerel. Bones and otoliths were selected according to species-specific features, predictability in condition, and prevalence in scats. Bones included the angular (ANG), quadrate (QUAD), interhyal (INTE), dentary (DENT), pharyn- gobranchial 2 (PHAR), and hypobranchial 3 (HYPO) A Walleye pollock Pharyngobranchial #2 Quadrate Interhyal B Atka mackerel Otolith I Hypobranchial #3 Pharyngobranchial #2 Interhyal Otolith Figure 1 Illustrations of the various planes for bone and otolith measurements used to solve the bone-length to fish-length regression equations for (A) walleye pollock and (Bl Atka mackerel. The structures from the right side of the body are shown for all structures except for quadrates. (Fig. 1). Fork length regressions were developed for sagit- tal otolith length (OTOL), as well as for width (OTOW) measurements. All selected cranial structures were paired (having a left and right side) which allowed for enumeration of prey species. Only right-sided bones and otoliths were used to develop the regression equations. Zeppelin et al.: Sizes of walleye pollock and Atka mackerel consumed by Eumetopias jubatus 511 In symmetrical fishes such as walleye pollock and Atka mackerel the left and right otoliths are mirror images of each other (Harkonen, 1986). We compared the left and right-sided measurements for all seven structures using a subsample of the structures used to develop the regression equations. There was no significant difference for either walleye pollock (paired Ntest, P<0.05, /? = 13 for HYPO, 15 for QUAD, and 14 for all other structures) or Atka mackerel (paired t-test, P<0.05, ra=14 for OTOS and 17 for all other structures). Fish specimens used for regressions were collected from the Gulf of Alaska and Bering Sea. Standard length (SLi was converted to fork length for walleye pollock (when fork length was not available for a small number of otoliths included in the regressions) by using the following equation: FL = 0.40+1. 07(SL) (Wilson2). We chose to use FL over SL for the regressions because all fish were in good condition, thus allowing for ac- curate measurements. Additionally, FL is the standard used for commercial fishery and survey data by the National Marine Fisheries Service for direct compari- sons. A partial analysis of these data was previously reported in Orchard (2001). We expanded the data set reported in Orchard (2001) to reflect the size range of bones found in Steller sea lion scats and included only fish specimens collected within our study area. Linear regression models were fitted for most cranial structures by using the following equation: Y= a+ PX, where Y = the fork length of the fish; X = the measurement of the cranial structure; and a and P are constants that define the regression formula. However, some cranial structures provided a better fit with the following quadratic regression equation: Y = a + PX + pX2. The strength of the relationship of the regression models was assessed by using a coefficient of determination (r2). Erosion is a potential source of bias when estimating prey body size from digested otoliths (Prime and Ham- mond, 1987; Dellinger and Trillmich, 1988; Harvey, 1989). We used condition-specific digestion correction factors (DCFs) developed by Tollit et al. (2004b, this issue) to correct for the high degree of variation in the erosion of cranial structures. DCFs were obtained from feeding experiments on captive juvenile Steller sea lions by using a subsample of fish collected for the regres- sion analysis (Tollit et al., 2004b, this issue). Selected cranial structures from three size groups of pollock 2 Wilson, M. 2003. Persona] commun. Alaska Fisheries Sci- ence Center, Natl. Mar. Fish. Serv., NOAA. Seattle, WA. (28.5-45.0 cm FL) and one size group of Atka mackerel (30-36 cm FL) were used to develop the DCFs. Estimation of size of walleye pollock and Atka mackerel consumed by Steller sea lions in the Bering Sea and Gulf of Alaska Steller sea lion scats were collected from 1998 to 2000 along most of the U.S. range of the Alaskan western stock. Scats were collected from rookery (breeding) and haul- out (nonbreeding) sites in summer (June-September) and haul-out sites in winter (February-March). We assumed that scats collected on summer rookery sites primarily represent the diet of adult females because adult males present on rookeries usually fast during this time. Juveniles of both sexes come ashore on rook- eries during summer and undoubtedly are represented in the data, but to a lesser degree than adult females. Scats from juvenile Steller sea lions are more likely to be sampled on haul-out sites during summer, where juveniles make up the greatest proportion of individuals. Scats collected on summer haul-out sites or any winter site presumably represent a greater cross-section of ages and sexes than collections from rookeries during summer. Scats were rinsed through nested sieves of 4.8-, 1.4-, 0.7-, and 0.5-mm mesh. Bones and otoliths were iden- tified to the lowest possible taxon by using reference collection specimens. All recovered otoliths and selected bones identified as either walleye pollock or Atka mack- erel were given a condition grade based on the degree of erosion (Tollit et al., 2004b, this issue). In general, cra- nial structures considered in "good" condition had little or no erosion, "fair" were moderately eroded (generally up to about 20%), and "poor" were heavily digested (Tollit et al., 2004b, this issue). All structures that were given a condition grade of "good" or "fair" were identi- fied as being from the left or right side and measured to the nearest 0.01 mm with digital calipers. Cranial structures graded as "poor" were not measured and ex- cluded from further analyses because of high observed intraspecific variation (Tollit et al., 1997; Tollit et al., 2004b, this issue). Fork-length estimates with and without DCFs applied were calculated for each cranial structure and for all structures combined. Otoliths were treated separate- ly because most diet studies currently rely on otolith length to estimate fish fork length. Ninety-five percent confidence intervals around all mean size estimates were calculated by using parametric bootstrapping pro- cedures (Manly, 1997) in which error associated with the regression equation and resampling error resulting from variability within correction factors, and variabil- ity in scats were taken into account. Full details of the bootstrapping procedure are presented in Tollit et al. (2004b, this issue). The same fish may be represented by multiple cranial structures within a scat; therefore, in order to avoid pseudoreplication. we selected a minimum number of individuals (MNI; Ringrose, 1993) for each scat sample. 512 Fishery Bulletin 102(3) Table 1 Relationship between bone measui •ement and fish fork length (FL) in millimeters. For each equation the number of bones mea- sured In), coefficient of determination (r2), standard error of the regression coefficient (SE and SE2for quadratic regression coef- ficients), range offish lengths and mean of fork lengths are g ven. All measurements are given in millimeters. Species Structure code Regression r2 ii SE, SE2 Range of FL Mean FL Walleye pollock INTE FL = 49.78* + 5.12 0.98 49 1.12 83-477 201.61 HYPO FL = 43.14* + 14.12 0.99 49 0.78 83-477 231.58 PHAR FL = 80.19a- + 19.43 0.95 51 2.58 83-477 204.37 ANGU FL = 59.25* +15.27 0.96 44 1.82 83-477 208.75 QUAD FL = 89.47* + 6.77 0.99 59 1.32 83-477 203.92 DENT FL = 108.46x- 1.52 0.99 60 1.75 83-477 206.61 OTOL FL = 0.50*2 + 15.74* + 13.3 0.99 504 0.68, 0.34 49-530 187.35 OTOW FL = 2.32*2 + 44.74* + 3.73 0.99 508 1.54,0.19 49-530 188.66 Atka mackerel INTE FL = 57.38* + 95.57 0.86 106 2.26 185-500 355.37 HYPO FL = 38.58* 80.64 0.95 105 0.85 185-500 355.62 PHAR FL = 81.32* + 70.40 0.91 107 2.48 185-500 354.90 ANGU FL = 58.38* + 73.86 0.91 105 1.85 185-500 355.34 QUAD FL = -8.90*2 + 129.38* + 9.16 0.96 108 7.07,0.96 185-500 354.69 DENT FL = -7.10*2 + 115.83* -21.68 0.94 108 7.08, 0.73 185-500 354.69 OTOL FL = 62.54* +24.24 0.83 165 2.19 185-500 349.82 OTOW FL= 188.19* -77.71 0.78 170 7.71 185-500 350.09 Minimum number of individuals for each species in each scat was estimated by counting species-specific sided elements and choosing the greatest number of left or right elements. If more than one structure had the same number, the structure with the highest r2 value in its regression on fork length (Table 1) was selected as a representative length estimate for that fish. If an equal number of left and right bones were present, right bones were selected. Temporal variation in size of walleye pollock and Atka mackerel consumed by Steller sea lions Temporal differences were assessed by grouping fish into stage-class categories. Stage-class categories were defined for pollock as follows: juvenile or 1-year-old fish (<20 cm FL), adolescent (20.1-34 cm FL), subadult (34.1-45 cm FL), and adult (>45.1 cm FL; Dorn et al., 2001; Smith, 1981; Walline, 1983). Walleye pollock sub- adults are likely 3-4 years old, of which -50% have matured and recruited into the fishery, whereas adults are sexually mature fish, likely 5 years or older. Stage- class categories for Atka mackeral were defined as fol- lows: juvenile up to 2-year-old fish (<30 cm), adolescent or 3-year-old fish (30.1-35.2 cm), subadult or 4-year-old fish (35.3-45 cm), and adults (>45.1 cm; Lowe et al., 2001; McDermott and Lowe, 1997). Atka mackerel ado- lescents are -50% sexually mature and adult-size fish are fully mature. A chi-squared contingency test was used to test for differences in the proportion offish stage-classes occur- ring in scats among rookeries and haul-out sites, years, and seasons by using corrected fork-length estimates from all cranial structures (S-PLUS 2000, Mathsoft, Inc., Cambridge, MA). To avoid pseudoreplication, we used presence or absence of cranial elements of a stage class in a scat particilarily because multiple elements from the same stage-class within a sample may not be independent (Hunt et al., 1996). By using presence- absence data we also avoided the problems associated with the variability in passage and recovery rates of different size structures (Tollit et al., 1997). Because sample sizes were small, juvenile and adolescent wall- eye pollock stage classes and recruiting adult and adult Atka mackerel stage classes were combined for seasonal comparisons among years. Fisher's exact test was used for comparisons when samples sizes for any stage class were less than 5 (S-PLUS 2000, Mathsoft, Inc., Cam- bridge, MA). We obtained size composition data from commercial bottom trawls of walleye pollock and Atka mackerel from the NMFS North Pacific Groundfish Observer Pro- gram. Data were divided into winter (January-April I and fall (August-November) seasons and compared with our seasonal scat data (February-March and June-September). The percentage of overlap in sizes of fish caught by the commercial groundfish fishery with sizes of fish consumed by Steller sea lions was calculated by comparing size-frequency distributions. Two-cm size bins were used for the overlap calculation and Steller sea lion prey-size data were rounded to the nearest integer to be consistent with the fishery data. Zeppelin et al.: Sizes of walleye pollock and Atka mackerel consumed by Eumetopias lubatus 513 Results Regression formulae A total of 517 pollock and 191 Atka mackerel samples were used to develop the regression equations of bone and otolith measurement to fork length. The sample size and range of fish lengths used for the regressions varied between species and cranial structures (Table 1). No clear indications of sample size required for regression analysis are currently provided in the literature; how- ever, Owen and Merrick (1994) recommend a minimum sample size of 30-40. Sample sizes used to develop equa- tions presented here ranged from 44 to 508. In general, linear models were used for regression equations; however, several cranial structures were best fitted with a quadratic model. For both species, all structures were strongly related to fork length (r2 range: 0.78-0.99; Table 1). The regressions encompassed the majority of sizes of bones and otoliths found in Stell- er sea lion scat samples for this study. However, a small proportion of walleye pollock bones from scats were larger than those used to develop the regressions. Frost and Lowry (1981) developed otolith linear re- gression equations for walleye pollock from the Bering Sea using a double-regression approach that produced an inflection point at 10 mm. We examined the double regression approach but found a higher degree of corre- lation using a quadratic regression model. We compared the results of our model with Frost and Lowry 's (1981) model and found that estimated fork lengths of walleye pollock differed less than 2 cm across the length range in our samples. Estimation of size of walleye pollock and Atka mackerel consumed by Steller sea lions in the Bering Sea and Gulf of Alaska A total of 714 scats from 39 sites contained 3646 selected cranial elements from either walleye pollock or Atka mackerel. Of those, 212 scats contained 666 walleye pollock cranial elements with a condition grade of either "good" (ft =236) or "fair" (n = 430). The minimum number of individual pollock per scat ranged from 1 to 18 with a mean of 1.6 (SD = 1.7). For Atka mackerel, 379 scats contained 1685 skeletal elements with condition grade of either "good" (;?=755) or "fair" (rc=930). The minimum number of individual Atka mackerel per scat ranged from 1 to 14 with a mean of 1.9 (SD = 1.6). The mean fork length of walleye pollock consumed by Steller sea lions in the Bering Sea and Gulf of Alaska es- timated from uncorrected otoliths found in scats was 23.7 cm (SD=12.0; « = 88). Application of the DCF increased the mean estimate to 28.4 cm (SD=14.75; rc=88). The size distribution estimated from corrected otoliths had three modes: a major mode around 32 cm and minor modes around 5 cm and 13 cm (Fig. 2A). Confidence intervals for all grade-corrected estimates can be found in Table 1. The mean fork length of walleye pollock estimated from all seven structures was 39.8% greater than the mean estimated from otoliths alone. The uncorrected mean was 33.1 cm. Applying the DCF increased the mean length of walleye pollock by 18.7% to 39.3 cm (paired t test, ?665=37.9, P<0.001). Mean grade-corrected size estimates for cranial structures other than otoliths ranged from 34.5 cm (PHAR) to 47.2 cm (HYPO) and 95% confidence intervals ranged from 25.2 to 50.6 cm (Table 2). The condition-specific DCFs increased length estimates between 6.8% (HYPO) and 28.3% (DENT). The size distribution estimated from all grade-corrected structures had three modes: a major mode around 44 cm and minor modes around 5 cm and 15 cm (Fig. 2A). The mean fork length of Atka mackerel consumed by Steller sea lions in the Bering Sea and Gulf of Alas- ka estimated from uncorrected otoliths was 30.3 cm (SD=4.0; n=117). Application of the DCF increased the mean estimate to 34.7 (SD = 4.8; n=U7). The mean fork length of Atka mackerel estimated from all structures (30.7 cm; SD = 5.9 cm, corrected 32.3 cm; SD = 5.9 cm, rc=1685, paired t test, f1684=39.1, P<0.001) was similar to the mean estimated from oto- liths (6.9% less without a DCF and 1.3% less with a DCF; Fig. 2B). Mean grade-corrected size estimates for structures other than otoliths ranged from 26.6 cm (QUAD) to 34.2 cm (INTE) and 95% confidence inter- vals ranged from 24.0 cm (DENT) to 35.0 cm (INTE; Table 2). Use of the condition-specific DCFs increased length estimates between 2.1% (INTE) and 24.0% (DENT). Fork length estimates for all structures did not include PHAR because too few were recovered in scats in the feeding studies of captive Steller sea lions to develop a correction factor. When mean prey size was calculated by using MNI, the mean corrected and uncorrected size estimate of both walleye pollock and Atka mackerel differed by less than 0.2 cm from estimates derived by using all struc- tures. There was little difference in the standard devia- tions or distributions when MNI estimates were used compared with all structures (Table 2). Unsurprisingly, the use of MNI estimates did substantially reduce the sample size (336/666 for walleye pollock and 722/1685 for Atka mackerel). Spatial and temporal variation in size of pollock and Atka mackerel consumed by Steller sea lions No statistical difference was found in the proportion of pollock stage classes among years on summer rookery sites (P=0.29, ^2 = 4.9, df=3) or winter haul-out sites (P=0.10; Fisher's exact test). Scats were collected only on summer haul-out sites during 2000. Although sample sizes were limited, we found significant differences in the proportion of pollock stage classes between summer rookery and haul-out scats (P=0.02; Fisher's exact test) and between summer and winter haul-out sites (P=0.018; Fisher's exact test) for year 2000. A greater proportion of juvenile pollock were found on summer haul-outs (64%' juveniles, n=ll scats) than on summer rookeries (9% juveniles, n = 132 scats) or winter haul-out sites (3% juveniles, n = 69 scats, Fig. 3). No statistical difference 514 Fishery Bulletin 102(3) Walleye pollock All structures MNI 8 6 1 n = 666 No DCFs DCFs applied — i — i — i — i — i — i — i — 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 Estimated fork length (cm) B Atka mackerel All structures 1NI No DCFs DCFs applied 0 10 20 30 40 50 60 70 80 0 i — i — r 10 20 30 40 50 60 70 80 Estimated fork length (cm) Figure 2 Relative frequency histograms of the estimated fork length of walleye pol- lock and iB) Atka mackerel consumed by Steller sea lions. Fork lengths were predicted from cranial structures in good and fair condition. Comparisons were made on the application of correction factors (DCFs) which account for digestion and for using minimum number I MNI I estimates as a selection tech- nique versus using all structures. Otoliths (black bars) are stacked beneath all other structures (gray bars). was found in the proportion of stage classes between summer rookery (9.09% juvenile; 20.45% adolescent: 53.03$ subadult; 65.15'^ adult) and winter haul-out (2.90% juvenile; 21.74r; adolescent; 57.97% subadult; 46.38% adult) sites for all years combined (P=0.32, *2=2.3, df=2). Zeppelin et al.: Sizes of walleye pollock and Atka mackerel consumed by Eumetopias jubatus 515 Table 2 Estimated mean fork length of walleye pollock and Atka mackerel consumed by Steller sea lions based on selected structures with or without application of condition-specific digestion correction factors (DCFsl. Data sets exclude all structures graded as heavily digested. Remaining total sample sizes of elements in1) are given along with proportion of grade "good" structures (n«). For data sets where DCFs were applied, 95'r confidence intervals (95% CI) were estimated by using bootstrap resampling pro- cedures (Tollit et al., 2004b, this issue). Species Structure code DCF /!' Mean FLlcml SDlcm) Range (cm) 95% CI Walleye pollock Atka mackerel INTE HYPO PHAR ANGU QUAD DENT OTOL All INTE HYPO ANGU QUAD DENT OTOL All No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes 60 60 38 38 23 23 136 136 134 134 187 187 88 88 666 666 601 601 238 238 488 488 161 161 80 80 117 117 1685 1685 0.45 0.45 0.55 0.55 0.61 0.61 0.40 0.40 0.34 0.34 0.37 0.37 0.03 0.03 0.35 0.35 0.58 0.58 0.42 0.42 0.45 0.45 0.37 0.37 0.28 0.28 0.06 0.06 0.45 0.45 43.7 47.0 44.2 47.2 32.2 34.5 36.1 40.2 35.1 44.5 28.6 36.7 23.7 28.4 33.1 39.3 33.5 34.2 31.1 32.9 30.2 31.8 25.3 26.6 22.5 27.9 30.3 34.7 30.7 32.3 8.0 8.5 7.2 7.8 14.3 14.8 8.4 9.0 12.0 15.3 11.8 15.1 12.0 14.8 12.4 14.3 5.0 5.1 4.8 5.5 4.7 5.1 5.4 5.6 7.7 8.0 4.0 4.8 5.9 5.9 16.7- 16.7- 30.5- 34.9- 9.7- 10.9- 10.6- 10.6- 9.4- 11.9- 3.1- 3.7- 4.6- 4.6- 3.1- 3.7- 19.5- 19.5- 18.8- 19.3- 17.3- 17.3- 14.8- 15.3- 13.0- 17.7- 21.2- 21.2 13.0- 15.3- 59.4 65.9 60.4 62.7 53.1 5.3.1 55.3 60.6 ■57.8 ■70.8 -57.2 ■70.2 46.8 -57.1 60.4 -70.8 46.8 49.6 46.2 48.3 43.0 ■46.1 40.6 -41.4 -38.7 ■44.1 -40.6 -47.0 ■46.9 -49.6 44.9-49.8 44.5-50.6 25.2-44.5 38.5-42.4 38.8-49.6 30.3-42.4 17.0-32.4 35.9-42.4 33.4-35.0 32.4-34.6 31.7-33.3 25.1-28.4 24.0-33.0 33.5-35.8 31.7-33.4 Significant differences were found in the proportion of Atka mackerel stage classes between 1998 and 1999 on summer rookery sites (P=0.05, x2 =6.0, df=2 ) and winter haul-out sites (P=0.01, ^ = 9.9, df=2) and be- tween 1998 and 2000 winter haul-out sites (P=<0.01, Fisher's exact test). Significant seasonal differences were found only in 1998 (P=0.03, r = 7.1, df=2) which may be the result of the small sample size in winter 2000. In summer and winter, annual differences in size of Atka mackerel consumed by Steller sea lions corresponded to changes in the length distribution of Atka mackerel resulting from exceptionally strong year classes in 1995 and 1998 (Lowe et al., 2001). The 1995 year class is represented as a mode around 30 cm in 1998 (3-year-old fish), 35 cm in 1999 and >40 cm in 2000 (Fig. 4). The 1998 year class is represented most clearly as 2 year olds (mode 20-25 cm) in summer 2000 (Fig. 4). Strong annual modes found in our data match those recorded in surveys of Atka mackerel in the Ber- ing Sea and Gulf of Alaska (Lowe et al., 2001). For walleye pollock and Atka mackerel there was no difference in the mean size of fish caught by the com- mercial fishery among years (P>0.4, one-way ANOVA). There was a significant difference (P<0.05, one-way ANOVA) in the size of fish caught between seasons. This difference is likely due to aggregations of spawning adult fish caught during the roe fishery. In the winter there is a 56% overlap between the size of fish caught in the commercial pollock fishery and those taken by sea lions and a 54% overlap in the size taken by the Atka 516 Fishery Bulletin 102(3) 25 - 20 15 10 -\ 5 0 Winter 1998 ns = 5 n = 8 25 20 15 10 5 0 0 102030405060 7080 25 - 20 - 15 - 10 5 0 Summer 1998 (rookey) n, = 81 n„ = 226 0 1020304050607080 Winter 1999 ns = 24 n =46 Winter 2000 ns = 40 n =96 0 1020304050607080 0 1020304050607080 Summer 2000 (rookery) ns = 24 a, = 81 Summer 2000 (haul-out) ns= 1 1 n„ = 77 l j I l! I. . 0 1020304050607080 0 1020304050607080 0 1020304050607080 Estimated fork length (cm) Figure 3 Relative frequency histograms of the estimated fork length of walleye pollock consumed by Steller sea lions across seasons and years for rookeries and haul-outs. Fork lengths are predicted from corrected cranial struc- tures in good and fair condition. Sample sizes for cranial elements inel and scats (ra ) are provided. All winter sites are considered haul-out sites. Winter 1999 ns = 48 n =218 Winter 2000 ns = 13 n„ = 32 - 1 " 1 0 10 20 30 40 50 Summer 1998 _ ns = 201 ne = 965 0 10 20 30 40 50 Summer 1999 ns = 44 n =193 0 10 20 30 40 50 Summer 2000 n, = 35 n . = 142 J 0 10 20 30 40 50 0 10 20 30 40 50 Estimated fork length (cm) 0 10 20 30 40 50 Figure 4 Relative frequency histograms of the estimated fork length of Atka mackerel of consumed by Steller sea lions by season and year. Fork lengths are predicted from corrected cranial structures in good and fair condition. Sample sizes for cranial elements (ne) and scats (ns) are provided. All summer sites are rookeries and winter sites are haul-out sites. Zeppelin et al.: Sizes of walleye pollock and Atka mackerel consumed by Eumetopias jubatus 517 12-1 A Walleye pollock Overlap 68% 10- SSL, n=666 ■ trawl. n=92133 8- 6 - 4 - ll 2- I III I ll.. n - 10 20 30 40 - 25 20 B Atka mackerel 50 60 70 Overlap 53°o 80 15 10 SSL. n=1685 ■ trawl. n=92877 ill .III 1. 10 20 30 Estimated fork length (cm) 40 50 Figure 5 Relative frequency histograms of the estimated fork length of wall- eye pollock and Atka mackerel consumed by Steller sea lions (SSL) compared with relative frequency histograms offish caught by the walleye pollock and Atka mackerel commercial trawl fishery. mackerel fishery. In the summer the overlap in size of fish consumed by sea lions and the size of fish caught in the pollock fishery is 67% and there is a 51% overlap in the size of fish caught in the Atka mackerel fishery. When seasonal data were pooled, overlap between the size of fish caught in the commercial fishery and the size of fish consumed by sea lions was 68% for walleye pollock (Fig. 5A) and 53% for Atka mackerel (Fig. 5B). Discussion Regression formulae Regressions of cranial structure measurement on fish fork length with the use of multiple structures was an effective tool for estimating size of fish consumed by Steller sea lions. Sample sizes of measurable prey remains from scats were enhanced by using a number of cranial structures in addition to otoliths. Body size estimates of only 13.2% of the pollock and 6.9% of the Atka mackerel prey in this study were based on otoliths alone. Fork-length estimates can be considered accurate regardless of which structure was used in the estimate because all r2 values were high (range: 0.78-0.99). Like- wise errors associated with the application of DCFs were consistent across structures (Tollit et al., 2004b, this issue). Confidence intervals around size estimates generally overlapped across structures; however, it was not surprising that different structures yielded slightly different mean sizes because different bones can origi- nate from different scats. The use of multiple cranial structures may also re- duce bias resulting from variability in recovery and passage rates of structures from different species or sizes of fish (Pierce and Boyle, 1991; Browne et al., 518 Fishery Bulletin 102(3) 2002; Tollit et al., 2003). Even after applying a DCF, the estimated mean size of walleye pollock based on otoliths was 10.9 cm smaller than the mean size esti- mated by using all cranial structures. Because walleye pollock otoliths are relatively large and have a different composition than other cranial structures, the larger otoliths may be regurgitated, fully digested, or crushed by rocks in the stomach and not pass through in scat as readily as smaller otoliths or other cranial structures, thereby reducing their occurrence in scat and use in generating prey-size estimates. Atka mackerel otoliths are much smaller at older ages in relation to walleye pollock, which may explain why the size of prey esti- mated from otoliths was similar to the size estimated from other cranial structures. The use of DCFs for all structures, including otoliths, to account for erosion increased mean size estimates for both walleye pollock (33.1 vs. 39.3 cm FL) and Atka mackerel (30.7 vs. 32.3 cm FLi. The relatively small increase in the corrected size of Atka mackerel re- flects that the structures from this species were found in better condition than those from pollock (Table 2), as well as that correction factors were found to be species-, structure-, and condition-specific (Tollit et al., 2004b, this issue). Overall, our results emphasize the importance of using appropriate condition-specific DCFs. Other studies with captive sea lions have also demonstrated that grade-specific DCFs can reduce sys- tematic error and increase precision of body mass es- timates (Tollit et al. 1997). For walleye pollock, there was no significant difference in the degree of erosion across the three size ranges for each structure within each condition category (Tollit et al., 2004b, this issue). We assume the DCFs can be used for fish outside of this size range because the relative shape, structure, and proportion of the morphological features are con- sistent for both smaller and larger fish (Tollit et al., 2004b, this issue). Further research is necessary to test whether there are differences across the size range for Atka mackerel. Size of walleye pollock and Atka mackerel consumed by Steller sea lions in the Bering Sea and Gulf of Alaska In general, Steller sea lions on summer rookery and winter haul-out sites consumed primarily subadult and adult-size walleye pollock and Atka mackerel year-round in 1998-2000. Steller sea lions typically forage near shore, in shallow water (<50 m) and at night (Raura- Suryan et al., 2002; Loughlin et al., 2003). Likewise, adult walleye pollock migrate vertically to shallower depths during the night (Smith, 1981). Adult-size Atka mackerel also are commonly found in nearshore coastal areas during their spawning season (Zolotov, 1993). Juvenile walleye pollock were found in relatively high numbers only in scats collected on summer haul-out sites. Scats collected from summer haul-out sites likely represent a larger proportion of juvenile Steller sea li- ons than those collected on summer rookery or winter haul-out sites. Previous studies indicate t hat juvenile sea lions (<4 years old) consume smaller walleye pollock than adult sea lions (Pitcher, 1981; Frost and Lowry, 1986; Merrick and Calkins, 1996). Juvenile walleye pol- lock are often found at shallow depths in bays and near shore habitat (Smith, 1981). Likewise, Loughlin et al. (2003) reported that juvenile Steller sea lions are typi- cally shallow divers and frequently make short range foraging trips (<15 km). Additional scat collections on summer haul-out sites are necessary to determine more conclusively prey-size selectivity for juvenile Steller sea lions. Annual changes in the size-frequency distribution of Atka mackerel consumed by Steller sea lions followed changes in the size distribution of Atka mackerel re- sulting from exceptionally strong year classes. Merrick and Calkins (1996) also showed that the size of prey consumed by Steller sea lions can reflect the size dis- tribution of the fish population. From the mid-1990s on, only 1999 was a strong recruitment year for walleye pollock in the Gulf of Alaska (Dorn et al., 2001), but we did not find a significantly greater proportion of juvenile fish eaten by Steller sea lions in 2000 than in 1999 or 1998 perhaps because sufficient numbers of larger size fish were available in regions where walleye pollock were consumed. Historical studies of Steller sea lion prey size have primarily been based on measurements of walleye pol- lock otoliths found in stomach samples but often with- out application of correction factors for erosion (Pitcher, 1981; Merrick and Calkins, 1996; Calkins. 1998). Prey- size estimates based on stomach contents will likely differ from estimates derived from scats because of differences in digestion rates and breakage ( Jobling and Breiby, 1986 i. However, results of studies examining the variability in prey size with sample type are vari- able. Sinclair et al. (1996) suggested that in northern fur seals (Callorhinus ursinus), another otariid, small otoliths tend to flush through the digestive system more quickly than larger ones, resulting in a possible bias in scats towards smaller otoliths. In contrast, experiments with captive sea lions have shown that smaller otoliths are recovered in lower relative frequencies than are larger ones (Tollit et al.. 1997). Frost and Lowry (1980) found no significant difference between the size of oto- liths obtained from stomach and intestines of ribbon seals. Overall, we believe useful comparisons of prey size consumed by Steller sea lions can be made between our study and earlier studies. Steller sea lions have been reported to consume a wide size range of walleye pollock. However, in most prior studies a larger proportion of juvenile fish were found than what we estimated from scats. Otoliths from stomach samples collected from 1975 to 1978 in the Gulf of Alaska contained primarily juvenile age pollock (mean FL=29.8cm; SD= 11.6; Pitcher, 1981). Undigested otoliths from stomach samples collected between 1975 and 1981 in the Bering Sea also contained mostly juve- nile fish (mean FL=29.3 cm) but had a distinct mode of adult-size pollock (48 cm FL: Frost and Lowry. 1986). Likewise, 43 stomach samples collected between 1985 Zeppelin et al.: Sizes of walleye pollock and Atka mackerel consumed by Eumetopias jubatus 519 and 1986 in the central Gulf of Alaska contained pri- marily juveniles (mean FL=25.4 cm; SD = 12.4) and had a weak mode of adult-size fish (39-43 cm; Merrick and Calkins, 1996; Calkins and Goodwin1). Mostly adult-size fish were found in stomachs recovered from Steller sea lions caught in trawl nets in the central Gulf of Alaska 11983-84; Loughlin and Nelson, 1986) and in stomach samples collected from 1994 to 1995 in Japanese waters (Goto and Shimazaki, 1998). However, in both these studies the samples of prey size may have been biased by the selectivity of the fishing gear for larger fish. Using identical methods to those of our study, Tollit et al. (2004a, this issue) estimated the size of wall- eye pollock consumed by the eastern stock of Steller sea lions between 1994 and 1999. The average size of walleye pollock consumed, estimated from all grade- corrected structures (mean=42.4 cm; SD = 11.6), was similar to the average size found in our study of the western stock of Steller sea lions. Furthermore, Tollit et al. (2004a, this issue) also found a greater occur- rence of adult pollock in scats collected on rookery sites than from scats collected on haul-out sites. However, Steller sea lions from the western stock consumed a greater proportion of juvenile and adolescent fish and less adult fish than those from the eastern stock dur- ing summer (June-July) and similar-size fish were consumed on haul-out sites in winter (March) in both regions. Neither study indicated the high occurrence of juvenile walleye pollock reported in the 1970s and 1980s. The greater occurrence of juvenile walleye pol- lock in historical studies may be a result of prey avail- ability or differences in sampling methods. By examining the relative size-frequency distributions of prey selected by Steller sea lions and those taken in the commercial trawl fishery, we found considerable overlap (689r walleye pollock and 53% Atka mackerel). Likewise, high levels of potential overlap in size were found between walleye pollock selected by Steller sea lions from the eastern stock and caught by the small commercial fishery bordering Southeast Alaska (Tollit et al., 2004a, this issue). The extent of overlap through- out the range of Steller sea lions between the size of prey consumed by sea lions and the size of fish targeted and taken by the pollock and Atka mackerel trawl fish- eries could result in competition between fisheries and foraging sea lions if resources are limited. Acknowledgments Fish specimens for the regression equations were pro- vided by the National Marine Fisheries Service, the University of Victoria, and the University of British Columbia bone reference collections. 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Randomization, bootstrap and Monte Carlo methods in biology, 2nd ed., 399 p. Chapman and Hall, London: New York, NY. McDermott, S. F, and S. A. Lowe. 1997. The reproductive cycle and sexual maturity of Atka mackerel, Pleurogrammus monopterygius, in Alaska waters. Fish. Bull. 95:231-333. Merrick, R. L., and D. G. Calkins. 1996. Importance of juvenile walleye pollock in the diet of Gulf of Alaska Steller sea lions. In Ecology of juvenile walleye pollock, Theragra chalcogramma (R. D. Brodeur, P. A. Livingston, T. R. Loughlin, and A. B. Hollowed, eds. I, p. 153-166. NOAA Tech. Rep. NMFS 126. NMFS (National Marine Fisheries Service). 2003. Fisheries of the United States, 2002. Current Fishery Statistics No. 2002, 126 p. Fisheries Statistics and Economics Division, Natl. Mar. Fish. Serv., Silver Spring, MD. NRC (National Research Council). 1996. The Bering Sea ecosystem, 307 p. National Acad- emy Press. Washington, D.C. Orchard, T. J. 2001. The role of selected fish species in Aleut paleo- diet. M.A. thesis, 228 p. Univ. Victoria, Victoria. BC. Owen, J. F, and J. R. Merrick. 1994. Analysis of coastal middens in south-eastern Aus- tralia: Sizing offish remains in holocene deposits. J. of Archaeol. Sci. 21:3-10. Pierce, G J., and P. R. Boyle. 1991. A review of methods for diet analysis in piscivo- rous marine mammals. Oceanogr. Mar. Biol. Annu. Rev. 29:409-486. Pitcher, K. W. 1981. Prey of the Steller sea lion, Eumetopias jubatus, in the Gulf of Alaska. Fish. Bull. 79:467-472. Prime J. H., and P. S. Hammond. 1987. Quantitative assessment of gray seal diet from fecal analysis. In Approaches to marine mammal energetics (A. C. Huntley, D. P. Costa, G. A. J. Worthy and M. A. Castellini, eds. I p. 165-181. Allen Press, Lawrence, KS. Raum-Suryan, K. L.. K. W. Pitcher. D. G. Calkins, J. L. Sease, and T R. Loughlin. 2002. Dispersal, rookery fidelity, and metapopulation structure of Steller sea lions (Eumetopias jubatus) in an increasing and a decreasing population in Alaska. J. Mammal. 18(3):746-764. Ringrose, T. J. 1993. Bone counts and statistics: a critique. J. Archaeol. Sci. 20:121-157. Sinclair, E. H., and T K. Zeppelin. 2002. Seasonal and spatial differences in diet in the west- ern stock of Steller sea lions (Eumetopias jubatus). J. Mammal. 83(4):973-990. Sinclair, E. H., G. A. Antonelis, B. W. Robson, R. R. Ream, and T. R. Loughlin. 1996. Northern fur seal, Callorhinus ursinus, predation on juvenile walleye pollock, Theragra chalcogramma. In Ecology of walleye pollock, Theragra chalcogramma (R. D. Brodeur, P. A. Livingston, T. R. Loughlin, and A. B. Hollowed, eds.) p. 167-178. NOAA Tech. Rep.. NMFS-126. Smith, G. B. 1981. The biology of walleye pollock. In The eastern Bering Sea shelf: oceanography and resources, vol. 1 (D. W. Hood and J. A. Calder, eds.) p. 527-552. Univ. Wash. Press, Seattle, WA. Templemann, W., and H. J. Squires. 1956. Relationship of otolith lengths and weights in the haddock, Melanogrammus aeglefinus (L.) to the rate of growth of the fish. J. Fish. Res. Board Can. 13: 467-487. Tollit, D. J.. S. G. Heaslip. and A. W. Trites. 2004a. Sizes of walleye pollock ( Theragra chalcogramma) consumed by the eastern stock of Steller sea lions (Eumetopias jubatus) in Southeast Alaska from 1994 to 1999. Fish. Bull. 102:522-532. Tollit, D. J.. S. G. Heaslip, T K. Zeppelin, R. Joy, K. A. Call, and A. W. Trites. 2004b. A method to improve size estimates of walleye pollock (Theragra chalcogramma) and Atka mackerel (.Pleurogrammus monopterygius) consumed by pinnipeds: digestion correction factors applied to bones and otoliths recovered in scats. Fish. Bull. 102:498-508. Tollit D. J., M. J. Steward, P. M. Thompson. G J. Pierce, M. B. Santos, and S. Hughes. 1997. Species and size differences in the digestion of otoliths and beaks: implications for estimates of pin- niped diet composition. Can. J. Fish Aquat. Sci. 54:105-119. Zeppelin et al.: Sizes of walleye pollock and Atka mackerel consumed by Eumetopias jubatus 521 Tollit, D. J., M. Wong, A. J. Winship, D. A. S. Rosen, and A. W. Trites. 2003. Quantifying errors associated with using prey skeletal structures from fecal samples to determine the diet of the Steller sea lion (Eumetopias jubatus). Mar. Mamm. Sci. 19(4):724-744. Walline, P. D. 1983. Growth of larval and juvenile walleye pollock re- lated to year-class strength. Ph.D. diss., 144 p. Univ. Washington, Seattle, WA. Wise, M. H. 1980. The use offish vertebrae in scats for estimating prey size of otters and mink. J. Zool., Lond. 192:25-31. Zolotov, O. G. 1993. Notes on the reproductive biology of Pleurogram- mus monopterygius in Kamchatkan waters. J. Ichthyol. 33(41:25-37. 522 Abstract— Lengths of walleye pollock i Theragra chalcogramma I consumed by Steller sea lions (Eumetopias jubatus) were estimated by using allometric regressions applied to seven diagnostic cranial structures recovered from 531 scats collected in Southeast Alaska between 1994 and 1999. Only elements in good and fair condition were selected. Selected structural measurements were cor- rected for loss of size due to erosion by using experimentally derived condition-specific digestion correc- tion factors. Correcting for digestion increased the estimated length of fish consumed by 23% , and the aver- age mass offish consumed by 88%. Mean corrected fork length (FLl of pollock consumed was 42.4 ±11.6 cm (range = 10. 0-78.1 cm, n=909). Adult pollock (FL>45.0 cm) occurred more frequently in scats collected from rookeries along the open ocean coast- line of Southeast Alaska during June and July (74% adults, mean FL = 48.4 cm l than they did in scats from haul- outs located in inside waters between October and May (51% adults, mean FL = 38.4 cm). Overall, the contribu- tion of juvenile pollock (<20 cm) to the sea lion diet was insignificant; whereas adults contributed 44% to the diet by number and 74% by mass. On average, larger pollock were eaten in summer at rookeries throughout Southeast Alaska than at rookeries in the Gulf of Alaska and the Bering Sea. Overall it appears that Steller sea lions are capable of consuming a wide size range of pollock, and the bulk offish fall between 20 and 60 cm. The use of cranial hard parts other than otoliths and the application of digestion correction factors are fun- damental to correctly estimating the sizes of prey consumed by sea lions and determining the extent that these sizes overlap with the sizes of pollock caught by commercial fisheries. Sizes of walleye pollock (Theragra chalcogramma) consumed by the eastern stock of Steller sea lions (Eumetopias jubatus) in Southeast Alaska from 1994 to 1999 Dominic J. Tollit Susan G. Heaslip Andrew W. Trites Marine Mammal Research Unit, Fisheries Centre University ol British Columbia Room 18, Hut B-3, 6248 Biological Sciences Road Vancouver, British Columbia, Canada, V6T 1Z4 E-mail (for D J Tollit). tollitia'zoology ubc ca Manuscript submitted 28 April 2003 to Scientific Editor's Office. Manuscript approved for publication 25 March 2004 by the Scientific Editor. Fish. Bull. 102:522-532(2004). The dramatic decline of the western population of Steller sea lions (Eume- topias jubatus) in the 1980s (Loughlin et al., 1992; Trites and Larkin, 1996) prompted a number of studies to deter- mine what they eat and the extent of overlap of the fish consumed by Steller sea lions and fish caught by commer- cial fisheries. The eastern population of sea lions (east of longitude 144°) located mainly in Southeast Alaska and British Columbia gradually increased as the western population declined (e.g.. Calkins et al., 1999), permitting insightful comparative studies to be undertaken (e.g., Mer- rick et al., 1995; Milette and Trites, 2003). Possible explanations for the different population trends include ocean climate, competition with fish- eries, predation, and the amount or the sizes of pollock in the diets of sea lions in the two regions (Loughlin and York, 2000; Benson and Trites, 2002; NRC, 2003; Trites and Don- nelly, 2003; Calkins and Goodwin1; Loughlin and Merrick-). Reliable estimates of prey size are important not only to investigate prey selectivity and the extent of overlap in size of prey with size of the same species caught by commercial fisher- ies and by other marine piscivores but are also vital for accurately as- sessing prey numbers, biomass, and total consumption (Beverton, 1985; Ringrose, 1993; Laake et al., 2002). One means of estimating prey size is to measure hard parts recovered from fecal remains and to apply allometric regressions relating fork length to the size of otoliths (Frost and Lowry, 1981) and other bones (Zeppelin et al., 2004, this issue). However, the extent of digestion incurred by both otoliths and bones as they pass through the digestive tract must be accounted for to ensure that prey size is not un- derestimated (Tollit et al., 2004, this issue). Application of these two steps is integral to correctly estimate the size of prey consumed by Steller sea lions and other pinnipeds. The goal of our study was to esti- mate the size of walleye pollock (Ther- agra chalcogramma) consumed by Steller sea lions in Southeast Alaska between 1994 and 1999 by using new methods outlined by Tollit et al. (2004, this issue) and Zeppelin et al. (2004, this issue). Previous size esti- mates for this region of Alaska are based on the analysis of only eight stomachs collected in 1986 i Calkins 1 Calkins, D. G., and E. Goodwin. 1988. Unpubl. report. Investigation of the declining sea lion population in the Gulf of Alaska, 76 p. Alaska Department of Fish and Game, 333 Raspberry Rd, Anchorage, AK 99518. 2 Loughlin, T. R., and R.L. Merrick. 1989. Comparison of commercial har- vest of walleye pollock and northern sea lion abundance in the Bering Sea and Gulf of Alaska. In Proceedings of the international symposium on the biology and management of w-alleye pollock. Nov. 14-16, 1988, Anchorage, AK, p. 679-700. Alaska Sua Grant KVp Sil- 01. LIniv. Alaska Fairbanks, Fairbanks. AK To Hit et al.: Sizes of walleye pollock consumed by Eumetopias /ubatus 523 58 N 56 A 50 Petersburg " 14- 15 fh. r 50 Kilometers Pollock trawl 19 ' Dixon ,lsherV Entrance 138 W 134 130 Figure t Location of Steller sea lion (Eumetopias jubatus) haul-outs and rookeries visited during 1994-99 to collect scats containing pollock hard remains. Symbols: haul-outs in inside waters (•), haul-outs in outside waters (©), haul-outs where scats were not collected or sites at which no pollock hard remains were found (O), rookeries ■ and cities (*i. and Goodwin1). We sought to compare the sizes of pol- lock consumed in the 1990s with these earlier samples, as well as with the sizes consumed by the declining population of sea lions in the Gulf of Alaska and Ber- ing Sea during the 1970s and 1980s (e.g., Pitcher, 1981; Merrick and Calkins, 1996) and between 1998 and 2000 (Zeppelin et al., 2004, this issue). We also wanted to evaluate the use of digestion correction factors (DCFs) and skeletal structures other than otoliths to estimate prey size, and to compare the different size estimates for fish consumed by sea lions in Southeast Alaska with sizes of fish caught by a nearby commercial trawl fishery. Materials and methods Estimating sizes of pollock consumed Scats that contained pollock hard remains were collected from four rookeries and 16 haul-outs from both inside and outside waters of Southeast Alaska between 1994 and 1999 (Fig. 1 and Table 1). Scats from three haul- outs and four rookeries in outside waters were collected from May through October 1994-99, but most were collected from June and July. Scats from inside waters were collected at 13 haul-outs located in the straits and sounds between Juneau and Petersburg, Alaska (56.8-58.6°N, 132. 8-134. 9°W) (Fig. 1). The majority of these "inside" scats were collected from Frederick Sound (Fig. 1) between October 1995 and February 1997. Most were collected in the winter and spring, but some were collected in the summer of 1999 (Trites et al.3). In gen- eral, the haul-out sites visited to collect scats were those with relatively high numbers of animals across South- east Alaska (Calkins et al., 1999; Sease et al., 2001). Scats were washed and sieved (0.5 mm) and hard remains were identified by Pacific IDentifications Inc. (Univ. of Victoria, Victoria, B.C.). Seven commonly 3 Trites, A. W., D. G. Calkins, and A. J. Winship. 2003. Unpubl. data. Marine Mammal Research Unit, Fisheries Centre, University of British Columbia, Room 18, Hut B-3. 6248 Bio- logical Sciences Rd., Vancouver, B.C., Canada, V6T 1Z4. 524 Fishery Bulletin 102(3) Table 1 Steller sea lion scat collection sites in Southeast Alaska, as illustrated in Figure 1, giving details of the type (HO=haul-outl. fish element sample size irir), and the estimated corrected mean fork length Imean FL, cm) of walleye pollock based on seven cranial structures found in scats at each site. Region Site no. Site name Type "/ Mean FL SD Inside waters 1 Benjamin Island HO 11 39.7 13.9 2 Dorothy Island HO 3 38.6 13.6 3 Circle Point HO 31 45.1 13.7 4 Point League HO 37 42.9 9.9 5 Sunset Point HO 196 37.4 10.3 6 Sail Island HO 36 35.5 10.0 7a W Brother Island HO 8 40.9 14.7 7b SW Brother Island HO 152 37.1 9.9 8 Turnabout Island HO 34 47.8 11.0 9 Yasha Island HO 19 44.8 5.3 10 Sukoi Islets HO 14 26.9 7.8 11 Horn Cliffs HO 19 44.3 12.7 12 Liesnoi Island HO 7 31.8 10.2 Outside waters 13 Cape Cross HO 7 45.3 3.0 14 Timbered Island HO 5 39.3 8.7 15 Point Addington HO 1 53.5 — 16 Graves Rock Rookery 49 42.9 7.7 17 White Sisters Rookery 33 43.4 9.6 18 Hazy Islands Rookery 54 45.4 8.7 19 Forrester Islands Rookery 193 51.4 10.0 occurring, robust, and diagnostic pollock structures were removed from all scats containing pollock (see Tollit et al., 2004, this issue). All were from the cra- nium region (see Zeppelin et al., 2004, this issue) and included the sagittal otolith (OTO), as well as the inter- hyal (INTE), hypobranchial 3 (HYPO), pharyngobran- chial 2 (PHAR), angular (ANGU), quadrate (QUAD), and the dentary (DENT). Each individual fish element was assigned one of three condition categories (good, fair, or poor) and was measured three times (±0.01 mm) at a specific location to calculate a mean estimate (see Tollit et al., 2004, this issue). Fork lengths of pollock eaten by Steller sea lions in Southeast Alaska were first estimated by applying al- lometric regressions (Zeppelin et al., 2004, this issue) to otolith lengths (OTOL) without correcting for par- tial digestion (see Pitcher, 1981; Merrick and Calkins, 1996). We also measured and substituted otolith width (OTOW) when otoliths were broken lengthwise. We then applied appropriate DCFs and regression formulae to otoliths assigned in good and fair condition (Tollit et al., 2004, this issue). Finally, we applied allometric regressions (Zeppelin et al., 2004, this issue) to all ele- ments of the remaining six cranial structures (bones) assigned to good or fair condition categories to provide estimates of fish size across structures both with and without applying the appropriate DCFs (Tollit et al., 2004, this issue). Structures in poor condition were excluded because of large intraspecific size variation noted from feeding experiments with captive sea lions (see also Sinclair et al.. 1994; Tollit et al., 1997; Tollit et al., 2004, this issue). To incorporate the major sources of error in our method, we calculated confidence intervals (95^r) for fork-length estimates. First, we applied a random bootstrapped regression equation, followed by a boot- strapped correction factor applicable to each selected structure (see Tollit et al., 2004, this issue). For the five structures (INTE, HYPO. PHAR, ANGU, and OTO) in good condition for which Tollit et al. (this issue) recommended a DCF of 1.0 (no correction), we drew bootstrapped values from a discrete declining triangu- lar probability distribution (hj ranging from 1.0 to 1.05 (to simulate a limited degree of digestion). Finally, we bootstrapped individual scats at random, by selecting n scats with replacement from the original sample size n (to account for resampling variability across scats) and included only selected elements within those ran- domly bootstrapped scats. Bootstrap randomizations for these steps were done 1000 times and 959! confidence intervals were taken as the 25th and 975,h values of the sorted bootstrapped values. Finally, consideration was also given to whether an individual fish might be represented by different structures within a single scat. We therefore compared length estimates using all structures with those esti- To Hit et al.: Sizes of walleye pollock consumed by Eumetopias jubatus 525 mated with the minimum number of individuals (MNI) technique (Ringrose, 1993; Browne et al., 2002). This technique is used to select structures within each scat that preclude pseudoreplication or double counting of fish. Within each scat, the structure with the great- est MNI was selected, and right-sided structures were selected over left-sided structures if both sides were found in equal number because right-sided structures are used in regression formulae. If two structures had the same MNI estimate, then selection was made on the structure with the larger regression determination coefficient, r2 (OTO-W>OTO-L>QUAD>DENT>HYPO> INTER>ANGU>PHAR ). Geographical and temporal variation in sizes of prey consumed All elements from the seven cranial structures in good or fair condition were used to compare size of pollock con- sumed by Steller sea lions in Southeast Alaska between regions (inside haul-outs versus outside rookeries), across years and across rookeries (with rookery data collected in June and July), and across months (with data col- lected from inside haul-outs). Biologically meaningful differences in FL of pollock were assessed by grouping corrected lengths into stage-class categories (juvenile or 1-year-old fish FL<20 cm; adolescent 2045 cm) (Smith, 1981; Walline, 1983; Dorn et al., 2001). Adults were considered to be mature fish >5 years old and targeted by fisheries (Smith, 1981). Subadults were likely 3 or 4 years old, of which only a proportion had matured or were targeted by the fishery. To avoid the possibility of pseudoreplication in our chi-squared comparisons, we used only the presence or absence of structures of each stage class in a scat because individual fish eaten by a sea lion may have come from an age-specific school and were therefore not independent (Hunt et al., 1996). Pres- ence-absence data was chosen over MNI data because the former greatly reduces potential concerns regarding size-dependent recovery of cranial structures (Tollit et al., 1997). With the exception of our regional comparison, data from juvenile and adolescent stage-classes were pooled because of the low sample sizes of juvenile fish. A Fisher's exact test was used as an alternative test to chi- square comparisons when counts for a stage-class group- ing were <5 (S-PLUS 2000, Mathsoft Inc., Seattle, WA). Overlap of prey size with size of fish caught by fisheries To assess the impact of using the new methods described and to compare the size of pollock consumed by sea lions with the size of pollock typically caught by fisheries, we obtained randomly subsampled size-frequency landing data from the Canadian commercial pollock fishery in Dixon Entrance (1993-1999) (Saunders4). This area is 4 Saunders, M. 2002. Unpubl. data. Fisheries and Oceans Canada, 3190 Hammond Bay Road, Nanaimo, B.C., Canada, V9T 6N7. 115-135 km SE of the Forrester Island rookery on the southern border of Southeast Alaska (Fig. 1). Results Sizes of pollock consumed The traditional method of estimating prey size from otoliths alone was not satisfactory because most otoliths were in poor condition (86%, ra=247) or were broken lengthwise (>89%) (or were both broken and in poor condition). Cranial bones, on the other hand, occurred in higher numbers than otoliths and were therefore more useful for estimating prey size (Table 2). Sixty-one percent of scats (1215 of 1987) collected from Southeast Alaska (1994-99) contained pollock remains, with an average MNI of 1.57 ±1.66 individual pollock per scat (range: 1-37 individuals). Many scats contained hard parts that were not useful for estimat- ing prey size (e.g., gill rakers), leaving 531 scats (26%) with measurable selected structures. Of these, 303 scats contained 1746 elements in good (n = 225), fair (n = 684). and poor condition (« = 837). Applying digestion correction factors had a consider- able effect on the estimated length and mass of fish consumed, and on the proportion that were deemed to be adults (Fig. 2). The estimated lengths of pollock calculated from all structures graded in good or fair condition (without accounting for digestion) was 34.4 ±9.7 cm (ra=909, modal range: 32-40) (Table 2, Fig. 2). Lengths increased by 23% on average when appropri- ate DCFs were applied to each structure to account for the observed degree of digestion (mean FL = 42.4 ±11.6 cm, modal range: 44-52, 95% CI = 41. 0-43.9) (paired t-test, i908=67.1, P<0.001). A DCF of 1.0 (no correction required to account for digestion) was applied to 62 ele- ments in good condition, resulting in a mean fork length of 39.6 ±11.9 cm estimated from those bones. The size-frequency distribution of pollock consumed by sea lions also varied significantly following the applica- tion of DCFs (Kolmogorov-Smirnov, KS = 176.2, P<0.001) and led to an increase in the proportion of fish thought to have been adult (>45 cm FL) from 16% to 44%. This result in turn reduced the proportion of fish thought to have been subadults (29%), adolescents (25%), and juveniles (<2%, <20 cm FL) (Fig. 2). The size range of pollock eaten ranged widely regardless of whether DCFs were applied (10-78 cm) or not (10-64 cm). When we calculated fork lengths using only elements selected according to MNI criteria, the means increased by just 0.5 cm for corrected and by just 0.3 cm for uncorrected lengths, with near identical standard deviations and distributions (Fig. 2) (Kolmogorov-Smirnov, uncorrected KS = 0.33, P=0.89, corrected KS = 0.032, P=0.91). The use of all otoliths regardless of digestion state resulted in a mean fork length that was only about half of that derived by using all structures corrected for digestion (Table 2). Excluding otoliths in poor con- dition significantly reduced sample size (Table 2) but 526 Fishery Bulletin 102(3) Fable 2 Estimated mean fork length mean FL, cmi of walleye pollock consumed by Stellei sea lions. Values were determined by using selected cranial structui es w ith or without the application of condition-specific di gestion correction factors DCFs . Data sets exclude all struc ures gr aded in poor condition (with the exception of data sets marked w th an asterisk l. Fi ih element sample sizes (n,- are given along with propo 'tion of elements assign* d condition category good ing). When DCFs were applied 95ri confi- dence intervals (95<~t CI i were estimated by using bootstrap resampling I see "Materials and methods"). Structure code DCF n ng Mean FL SD Range 95% CI INTE No 37 0.35 44.0 8.0 28.0-54.5 Yes 37 0.35 48.0 9.3 31.9-62.2 45.0-52.2 HYPO No 47 0.19 35.3 8.9 19.0-52.0 — Yes 47 0.19 39.8 10.1 19.0-60.4 36.7-43.6 PHAR No 20 0.25 38.1 8.5 20.4-50.3 — Yes 20 0.25 43.7 9.5 20.4-56.1 39.9-48.4 ANGU No 207 0.16 34.0 10.2 10.0-62.8 — Yes 207 0.16 39.4 11.4 10.0-63.2 37.4-41.5 QUAD No 238 0.36 33.1 10.4 14.0-63.8 — Yes 238 0.36 41.9 13.2 17.3-78.1 39.5-44.7 DENT No 326 0.24 34.9 8.1 11.0-63.0 — Yes 326 0.24 45.1 9.8 14.7-75.3 43.3-46.8 OTOL No 10 0.10 30.6 13.8 14.2-54.8 — Yes 10 0.10 36.6 17.6 16.7-67.2 27.0-51.1 OTOL or OTOW No 34 0.03 27.2 16.1 10.8-54.8 — Yes 34 0.03 33.7 12.8 13.3-67.2 29.5-39.5 All structures No 909 0.25 34.4 9.7 9.8-63.8 — Yes 909 0.25 42.4 11.6 10.0-78.1 41.0-43.9 OTOL* No 27 0.04 23.3 11.9 7.9-54.8 — OTOL or OTOW" No 247 <0.01 20.2 9.7 5.0-58.0 — increased our estimate of fork length by approximately .'!.'!'< Applying grade-specific DCFs increased these es- timates by another 19% (to 36.6 cm) for otolith length and by 24% (to 33.7 cm) for a combination of otolith length and width (Table 2). All six remaining struc- tures in good or fair condition provided larger corrected mean length estimates than did otoliths alone, but 95% confidence intervals derived from otoliths did overlap with other structures (Table 2). The smaller estimate provided by otoliths may reflect that >83% of grade corrected otoliths (n = 34) came from the inside haul- out sites, where animals seem to eat smaller fish (see following section). On the other hand, large pollock otoliths were observed to have been regurgitated in feeding studies on captive sea lions and also may be more easily crushed by rocks often found in the stom- achs of Steller sea lions (Tollit et ah, 2003). Regres- sion formulae used in our study to predict pollock FL from otolith length were similar to those of Frost and Lowry (1981) for juvenile fish (<10 mm otolith length) but led to smaller fish size estimates (—1—1.7 cm) over the range of 30-50 cm. The lengths of the biggest fish (corrected mean FL = 48.0 cm) were estimated from measurements of INTE (Table 2), the structure with the lowest DCF. Dentary bones (the most abundant structure recov- ered) predicted mean (Table 2) and modes (FL 44- 50 cm) similar to those predicted from all structures. Applying DCFs increased our length estimates by be- tween 9% (INTE) and 29% (DENT) (paired r- tests, all P<0.001). Overall, corrected fork length estimates from elements in good condition were similar to those from elements in fair condition (Mann-Whitney U, P=0.47), but multiple comparisons indicated a significant differ- ence (P<0.05> between condition categories for INTE and DENT (lengths were estimated to be longer from elements graded in fair condition). Repeat measurements of individual elements were all within 0.04 mm of the mean, and 88.9-99.5% were within 0.02 mm. The highest variability was associated with ANGU, HYPO, and PHAR with 88.9%, 91.7%, and 94.9% of their respective measurements falling within 0.02 mm. A 0.02-mm measurement difference corre- sponded to only a 0.1-0.2 cm difference in fish length, depending on the structure used. Small differences in estimates of fork lengths can have large effects on estimated body mass (given the exponential mass-length relationship, see Tollit et al., 2004, this issue) and can increase the mean mass of fish by more than sixfold depending on which method is used to estimate body length (all otoliths and no cor- rection versus condition-corrected structures). The ap- "To Hit et al.: Sizes of walleye pollock consumed by Eumetopias jubatus 527 All structures MNI 8 ■ ■ Otoliths n d Other bones r r n = 909 I n = 478 6 " n. lU 4 ■ ill J No DCFs Percent relative frequency ^ a> 03 o i\3 _A .. . Irun f ^n Ii k n- f Ik DCFs applied 2 " o ■ __jLi... kn>,„ r, rlR. L II II k„ h — r~ i i i i i i i i i i 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 Estimated fork length (cm) Figure 2 Relative frequency histograms of the estimated fork length of walleye pollock (Theragra ehalcogramma) predicted from seven cranial structures in good and fair condition. Otoliths (black bars) are stacked beneath all other selected structures (gray barsl. Comparisons were made with the application of digestion correction factors (DCFs) to take account of partial digestion and with the use of a selection technique (MNI) versus using all structures present in scats. plication of our DCFs to all structures in good and fair condition increased the estimated mean mass of pollock consumed by 88% (from a mean of 388 g to 731 g). Thus, although we estimate that 44% of the number of pollock eaten by Steller sea lions were adults, based on length, their contribution, based on weight, increased to 74%. In contrast, the contribution of juvenile fish (in terms of mass) dropped to <0.1% (compared to <2% by number). Geographical and temporal variation in sizes of pollock consumed More pollock elements in good and fair condition were recovered from inside haul-outs (?? = 567) than from sites on the outside coastline (n = 342) (Table 1). Upon investi- gation, we found the size of pollock consumed by Steller sea lions varied over time and across regions (Fig. 3). In particular, the frequency of occurrence of pollock stage classes differed significantly in the scats of sea lions resting at rookeries on the outside coastline of Southeast Alaska in summer (mean FL=48.4 cm, n = 328, modal range: 44-50 cm, 95% CI = 46.5-50.2, ns=126 scats) compared to those collected between October and May at haul-outs in the waters of the inside passages (mean FL = 38.4 cm, /; = 499, modal range; 30-34 cm, 95% CI=36.9-40.3, ns=168 scats) (^=45. 2, P<0.001). Scats from these inside haul-outs contained a greater diversity of stage classes, and there was an equal prob- ability of any given scat containing adults (51.2%), sub- adults (47.6%). and adolescents (53.0%), but a far less probability of containing juveniles (6.5%). In contrast, the pollock found in scats from the outside rookeries contained mostly adults (73.8%) and fewer occurrences of the remaining three stage classes (38.1%, 9.5%, and 3.2%. respectively). Notably, the stage-class compar- ison of summer 1999 with scats from inside versus outside waters was not significant (Fishers exact test, P=0.11). Similar proportions of each pollock stage class were found in scats collected between years (Forrester, Fisher's exact test, P=0.54; Hazy, Fisher's exact test, P=0.16), and between rookeries (1994 only, Fisher's ex- act test, P=0.57; all years, Fisher's exact test, P=0.22). Scats from inside haul-outs collected in spring 1996 contained comparatively smaller fish than in other months and years examined (Fig. 3). However, there were no significant monthly differences in the pro- portions of age classes from October 1995 to Febru- ary 1997 (*2 = 16.52, P=0.28) or when all monthly data (June and July 1999) were included from inside haul- outs (/=23.4, P=0.10). 528 Fishery Bulletin 102(3) Rookeries (outside) Haul-outs (inside) 80- F H W G 94 95 96 97 98 99 94 98 99 94 94 o 0 95 96 97 99 Oct Dec Mar Apr May Nov Feb Jun Jul 60- E o> c 0 £ 40- o "O CD I ( > 8 1 V -- 0 I o o | o oo oo h o Adult Subadult 1 1 o Correc |\J o i i 1 o ° 1 ■. ,8 r 8 . Adolescent § o 0 o Juvenile ty=42 29 35 59 9 18 17 18 19 33 49 n,=108 56 76 62 69 75 50 35 33 0- ns=19 13 10 26 3 10 9 10 9 6 11 ns=33 22 20 19 28 22 23 10 14 Figure 3 Box plots of corrected fork length of walleye pollock iTheragra chalcogramma) across months and years for haul-outs and rookeries in Southeast Alaska (Forrester Island. F; Hazy, H; White Sisters, W; Graves Rock. G). Box widths correspond to relative sample sizes for numbers offish elements in,) and numbers of scats (ns) are also provided, and stage-class categories are illustrated. Gray areas represent 95% confidence intervals. the whiskers bound 1.5x the interquartile range I boxes), the circles denote outliers, and the stars (*) denote extremes. Overlap of size of pollock consumed by Steller sea lions with size of pollock caught by the fisheries The Canadian commercial pollock trawl fishery in Dixon Entrance between 1993 and 1999 landed mostly (93%) adult fish (mean FL = 52.2 ±5.9 cm, «=2103, modal range: 48-54 cm). The majority (79%) of scats containing pol- lock from the Forrester Island rookery in June and July also contained remains of adult pollock (mean corrected FL=51.4 ±10 cm, a? = 192, modal range: 46-52 cm, ns=81 scats). Percentage overlap based on a comparison of size-frequency distributions totaled 75.1% for those fish eaten around Forrester Island and 52.1% for all fish eaten. However, the estimated overlap would have been assumed incorrectly to be half these values if DCFs had not been applied to the selected digested otoliths and bones (i.e., 36.7% overlap at Forrester and 24.1% for all areas combined). Clearly overlap levels would have been further underestimated if structures in poor condition had been included in our analyses. Discussion Only 57% of the scats (303 of 531) that contained suit- able pollock remains had structures that were in good enough condition to be measured reliably. Numbers of elements in good or fair condition (rc = 909) averaged three per scat, and a very small fraction of these con- sisted of otoliths (<4%). The most numerous structures were DENT, QUAD, and ANGU (Table 1). This finding is inconsistent with feeding trials with captive Steller sea lions where otoliths were found to be the most com- monly occurring structure (Cottrell and Trites, 2002; Tollit et al., 2003). Different structures yielded somewhat different mean sizes of pollock, although 95% confidence intervals gen- erally overlapped, ranging between 37 and 52 cm for bones (Table 2). Such discrepancy is not surprising given that different bones originate from different scats and possibly different fish (even within a single scat). Our comparison of estimates with all structures versus MNI selections indicates that the potential effect of double counting (and measuring) fish within a single scat is likely negligible with large sample sizes (Fig. 2). Although the use of all-structure data to estimate fish length results in a greatly increased sample size, there remains an underlying assumption that all structures are affected equally by digestion. Tollit et al. (2004, this issue) found no significant difference in the degree of erosion across the three size ranges (28.5-45.0 cm FL) for each structure within each condition category. Tollit et al.: Sizes of walleye pollock consumed by Eumetopias /ubatus 529 They also found that the relative shape, structure, and proportion of the morphological features used to es- timate erosion were consistent for both smaller and larger fish. We therefore assumed that the DCFs in that study could be used reliably for the fish in our study outside of the experimental size range in which they were considered. Applying DCFs increased mean fork length estimates by 23% (from 34.4 to 42.4 cm) on average and resulted in adult fish contributing 44% to the sea lion diet by number and 74% by mass. The contribution of juvenile fish was insignificant. Applying valid correction factors clearly provides better insights into prey-size selection and consequently niche overlap. It should also lead to more precise estimates of mass of prey consumed and the number of prey within a scat (Ringrose, 1993; Tollit et al., 1997; Laake et al., 2002). Over 61 species of prey were identified in the diet of Steller sea lions in Southeast Alaska from 1993 to 1999 (Trites et al.3). The most common prey were walleye pol- lock, Pacific herring (Clupea pallasi). Pacific sand lance (Ammodytes hexapterus), salmon iOncorhynchus spp.), arrowtooth flounder (Remhardtius stomias), rockfish (Se- bastes spp.), skates {Raja spp.), and cephalopods. During summer, gadids (most of which were pollock) made up 27% of the diet, and increased to 49-62% of the diet at other times of the year (Trites et al.3), confirming that pollock are a significant component of the diet. Steller sea lions consumed a wide size range of pollock in Southeast Alaska; the bulk of fish fell between 20 and 60 cm and peaked between 44 and 52 cm (Fig. 2). The contribution of juvenile fish (<20 cm) was insignificant. The only historical data to compare with these results are those from the stomach samples of eight Steller sea lions collected from Southeast Alaska in 1986 (Calkins and Goodwin1). Pollock lengths backcalculated from all otoliths found in the stomachs were generally shorter (mean FL = 25.5 ±10.4 cm, range; 4. 8-55. 7cm, n = 80) than our estimates from multiple structures found in scats collected during the 1990s (mean FL = 42.4 ±11.6 cm, range: 10.0-78.1 cm, n = 909). It should be noted that we derived our estimates after removing heavily eroded structures and applying DCFs, whereas Calkins and Goodwin1 did not account for partial digestion. However our estimates of pollock length would have been similar to those of Calkins and Goodwin1 if we had used only otoliths and had not corrected for diges- tion (Table 2). Although Frost and Lowry (1980) found no significant difference between the size of otoliths obtained from stomachs and intestines of ribbon seals, underestimates of fish size determined from otoliths from stomach samples will depend on the time since ingestion (i.e., on the extent of digestion). One possible explanation for the virtual absence of ju- venile pollock in the scats we examined is that the rela- tively smaller structures of smaller fish were more likely to be completely digested, and were therefore underrep- resented in the scats (Tollit et al., 1997; Bowen, 2000). However, juvenile pollock otoliths and bones were found in large numbers in a number of scats collected from the western stock (Zeppelin et al., 2004, this issue). Clearly, the potential for underestimating smaller fish depends heavily on the balance between relative re- covery rates and the number of different size fish con- sumed in a meal. For example, if an animal needs to eat 5 kg a day, then it would have to consume 195 15.5- cm pollock, but less than ten 41-cm pollock. Given that large pollock bones are at least three times more likely than small bones to pass through the digestive tract (Tollit et al., 2003; D. J. Tollit, unpubl. data), the sheer numbers of small pollock in this example would lead to a conclusion that smaller fish were more important numerically, when in fact they were equally important. Conversely, the relative proportion of large fish is likely to be overestimated if ten large and ten small pollock are consumed together. The generally low number of structures per scat provides little information to assess this balance. Hence we must assume that our results are representative and unbiased. Steller sea lions in Southeast Alaska did not seem to eat fish over 65 cm. Whether or not sea lions do not tar- get large fish, or whether large fish are harder to catch and handle, or are encountered at a lower rate is not known. However, large fish could be under-represented in scats if large fish cannot be swallowed whole, and head skeletal parts are lost while the fish is torn apart on the surface (Olesiuk et al., 1990; Wazenbock, 1995) or if bone regurgitation is size specific. Regional, geographical, and temporal variation in sizes of pollock consumed Stomach samples collected in 1975-78 and 1985-86 in the Gulf of Alaska contained substantial numbers of juvenile pollock, as well as larger fish (mode: 39-43 cm). In 1985, the distribution of sizes consumed by sea lions around Kodiak Island appeared to mimic that of the pol- lock population (Merrick and Calkins. 1996). However, juvenile sea lions ate significantly smaller and relatively more juvenile pollock than adult sea lions. Stomachs from the Gulf of Alaska contained an average of 49 pol- lock (1975-78) and 72 pollock (1985) compared with 1.6 pollock per scat in Southeast Alaska. In the Bering Sea, 90 stomachs were examined between 1975 and 1981 by using only non-eroded otoliths, and these also contained mainly (76%) juvenile pollock (mean FL=29.3 cm), but also some adult fish (Frost and Lowry, 1986). Between 1998 and 2000, Steller sea lions across the range of the western population in Alaska consumed pollock averaging 39.3 ±14.3 cm (range: 3.7-70.8 cm, Zeppelin et al., 2004, this issue). This finding suggests that sea lions may have been less reliant on juvenile pollock than they were during the 1970s and 1980s. Apparent differences may reflect differences in pollock year-class strength, and thus differences in the domi- nant size classes that were available to be consumed. However, Zeppelin et al. (2004, this issue) reported that the size distribution of walleye pollock consumed by Steller sea lions between 1998 and 2000 did not ap- pear to fluctuate with year-class strength, unlike the 530 Fishery Bulletin 102(3) sizes of Atka mackerel {Pleurogrammus monopterygius) consumed in western Alaska. Comparing samples collected at rookeries from the eastern and western populations reveals that sea lions in the western stock ate significantly greater numbers of smaller pollock and fewer adults in summer than sea lions in Southeast Alaska (Zeppelin et al., 2004, this is- sue; and our study). However, both eastern and western stock sea lions using haul-outs in March (winter) ate similar size pollock. Adult pollock occurred more fre- quently in scats collected from rookeries along the open ocean coastline of Southeast Alaska during June and July (74% adults) than they did in scats from haul-outs located in inside waters between October and May (51% adults). Scats collected at rookeries can be considered to be from adult female sea lions and to a lesser extent from adult males, whereas those collected at haul-outs during other times of the year contain a more diverse mix of age groups, including greater numbers of younger sea lions. Thus it is uncertain whether observed size differences in pollock between these two groups are seasonal or due more to size preferences of different aged animals. Lim- ited support for the former comes from the similar size pollock observed in the scats between the two groups in June and July of 1999. Overall, however, it is unknown whether the consumption patterns observed are a result of an actual size selection of prey or if they result from co- incidental distributions of sea lions and prey-size classes. Some pinnipeds may select prey of particular sizes (Sin- clair et al., 1994) and may encounter difficulties if they cannot switch to other sizes or species if the abundance of preferred prey is reduced. Fine-scale studies are now being undertaken to address such uncertainties. There are few assessments of pollock stock size for the 1990s in Southeast Alaska (Martin, 1997). However the biomass is believed to have been low compared to other regions of Alaska. Juvenile pollock are known to congregate in the shallow inside waters of Southeast Alaska during winter (Sigler0) but are also known to occur in significant numbers in the summer in waters shallower than 200 meters on the outer coastline (Mar- tin, 1997). Recruitment of 1-year-old fish was found to be high during acoustic studies in 1994 and 1999 in the Gulf of Alaska (Guttormsen et al., 2003). Steller sea lions using rookeries in Southeast Alaska consumed mainly adult pollock between 1994 and 1999 and showed no evidence of tracking any abundant age class of pollock. However, the trend in increasing length estimates for inside haul-outs after 1995 (Fig. 3 1 does suggest that sea lions might be tracking a particu- lar age class of prey. Certainly a greater range of age classes were consumed at these haul-outs (Fig. 3). Scientific trawls in 1996 indicated that the larger pol- lock on the outside coastline occurred generally in wa- ters 201-300 m deep during daylight hours (Martin, B Sigler, M. F. 2003. Unpubl. data. Auke Bay Lab, National Marine Fisheries Service. 1 L305 Glacier Highway, Juneau. AK 99801. 1997) and that smaller pollock were present in shallower depths. Larger pollock tend to disperse and move to shallow waters to feed at night (Smith, 1981). Thus, the observed crepuscular and nighttime foraging by lactating Steller sea lions (Higgins et al., 1988; Trites and Porter, 2002) would be a logical foraging strategy to capture adult pollock. Other important factors, in addition to depth, that likely influence size selection include prey density and spatial distribution in relation to rookeries and haul-outs. Given both the greater mass and energy content of adults compared with juveniles (Perez, 1994; Anthony et al., 2000), the selection of adults would be an energy efficient strategy — all other things being equal. Overlap in sizes of pollock consumed by Steller sea lions and sizes of pollock caught by fisheries There was no commercial fishery for pollock in South- east Alaska during the 1990s. However, a small fishery occurred in nearby Dixon Entrance, B.C., that might indicate sizes that could have been caught in Southeast Alaska if a fishery had occurred. Overlap in sizes of pollock caught by the B.C. fishery with those taken by Steller sea lions further north (our study) more than doubled after applying digestion correction factors (from 24% to 52%). Similarly, high levels of overlap were also found between the sizes of pollock consumed by the western stock (1998-2000) and those caught in the same region by fisheries (after our DCFs were applied to structures recovered from scats — Zeppelin et al., 2004. this issue). A high degree of overlap in size highlights a potential conflict between fisheries and sea lions, but this overlap cannot be considered indicative of competi- tion unless the resource that fisheries and sea lions seek is limited across the space and time in question (Krebs and Davies, 1991). Conclusions Our study provides the first substantial description of the size of pollock eaten by Steller sea lions in South- east Alaska. It also shows the benefits of using bones other than otoliths to estimate the sizes of prey eaten by Steller sea lions, and the importance of correcting for degree of digestion. Accurately reconstructing the sizes of bones and otoliths recovered from scats has a significant bearing, in turn, on accurately determining the mass of prey consumed, and on the extent of overlap of sizes of prey consumed and sizes of the same resource caught in commercial fisheries. We found that Steller sea lions in Southeast Alaska consumed a large proportion of adult pollock and few juveniles between 1994 and 1999. Although greater proportions of juvenile and adolescent pollock were con- sumed over the same period, during the summer in the Gulf of Alaska and Bering Sea, larger size fish still were the most abundant prey item in the diet of sea lions. A comparison of these estimates with the lengths of pol- lock consumed during the 1970s and 1980s shows that "To Hit et al.: Sizes of walleye pollock consumed by Eumetopias jubatus 531 Steller sea lions can consume a wide range of different size pollock (4-78 cm). Whether or not these differences in sizes of pollock consumed between regions and de- cades reflect differences in availability, size preferences, or year-class strength is not known and requires further study primarily with fine-scale data from scientific sur- veys and concurrent scat collections. Acknowledgments Funding was provided to the North Pacific Universities Marine Mammal Research Consortium by the National Oceanographic Atmospheric Administration and the North Pacific Marine Science Foundation. We would like to thank the contribution of personnel of the UBC Marine Mammal Research Unit, ADF&G, T. K. Zep- pelin, K. A. Call, A. J. Winship, E. H. Sinclair, and two anonymous reviewers. We are also grateful to J. L. Laake and R. Jov for statistical advice. Literature cited Anthony, J. A.. D. D. Roby, and K. R. Turco. 2000. Lipid content and energy density of forage fishes from the northern Gulf of Alaska. J. Exp. Mar. Biol. Ecol. 248:53-78. Benson, A. J., and A. W. Trites. 2002. 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Bone counts and statistics: a critique. J. Archaeol. Sci. 20:121-157. Sease, J. L., W. P. Taylor, T R. Loughlin, and K. W. Pitcher. 2001. Aerial and land-based surveys of Steller sea lions (Eumetopias jubatus) in Alaska, June and July 1999 and 2000. NOAA Tech. Mem. NMFS-ASFC-122, 52 p. Sinclair, E. H., T. Loughlin, and W. Pearcy. 1994. Prey selection by northern fur seals (Callorhinus ursinus) in the eastern Bering Sea. Fish. Bull. 92: 144-156. Smith, G. B. 1981. The biology of walleye pollock. In The eastern Bering Sea Shelf: oceanography and resources (D. W. Hood and J. A. Calder eds.), vol. 1, p. 527-551. Univ. Washington Press, Seattle, WA. Tollit, D. J., S. G. Heaslip, T. K. Zeppelin, R. Joy, K. A. Call, and A. W. Trites. 2004. A method to improve size estimates of walleye pollock (Theragra ehaleogramma) and Atka mackerel (Pleurogrammus monopterygius) consumed by pinnipeds: digestion correction factors applied to bones and otoliths recovered in scats. Fish. Bull. 102:498-508. Tollit, D. J., M. J. Steward, P. M. Thompson, G. J. Pierce, M. B. Santos, and S. Hughes. 1997. Species and size differences in the digestion of otoliths and beaks: implications for estimates of pin- niped diet composition. Can. J. Fish. Aquat. Sci. 54:105-119. Tollit, D. J., M. Wong, A. J. Winship, D. A. S. Rosen, and A. W. Trites. 2003. Quantifying errors associated with using prey skeletal structures from fecal samples to determine the diet of the Steller sea lion (Eumetopias jubatus). Mar. Mamm. Sci. 19(41:724-744. Trites, A. W., and L. P. Donnelly. 2003. The decline of Steller sea lions in Alaska: a re- view of the nutritional stress hypothesis. Mamm. Rev. 33:3-28. Trites, A. W., and P. A. Larkin. 1996. Changes in the abundance of Steller sea lions {Eumetopias jubatus) in Alaska from 1956 to 1992: how many were there? Aquat. Mamm. 22:153-166. Trites, A. W., and B. T. Porter. 2002. Attendance patterns of Steller sea lions (Eumeto- pias jubatus) and their young during winter. J. Zool. (Lond.l 256:547-556. Walline, P. D. 1983. Growth of larval and juvenile walleye pollock related to year-class strength. Ph.D. diss., 144 p. Univ. Washington, Seattle, WA. Wazenbock, J. 1995. Changing handling times during feeding and con- sequences for prey size selection of 0+ zooplanktivorous fish. Oecologia (Heidelb.l 104:372-586. Zeppelin, T. K., D. J. Tollit, K. A. Call. T. J. Orchard, and C. J. Gudmundson. 2004. Sizes of walleye pollock (Theragra ehaleogramma) and Atka mackerel (Pleurogrammus monopterygius) con- sumed by the western stock of Steller sea lions (Eume- topias jubatus) in Alaska from 1998 to 2000. Fish. Bull. 102:509-521. 533 Abstract— We examined movement patterns of sportfish that were tagged in the northern Indian River Lagoon, Florida, between 1990 and 1999 to assess the degree of fish exchange between an estuarine no-take zone iNTZ) and surrounding waters. The tagged fish were from seven spe- cies: red drum (Sciaenops ocella- tus); black drum (Pogonias cromis); sheepshead (Archosargus probato- cephalus); common snook iCentropo- mus undecimalis); spotted seatrout (Cynoscion nebulosus); bull shark {Carcharhinus leucas); and crevalle jack (Caranx hippos). A total of 403 tagged fish were recaptured during the study period, including 65 indi- viduals that emigrated from the NTZ and 16 individuals that immigrated into the NTZ from surrounding waters of the lagoon. Migration distances between the original tagging location and the sites where emigrating fish were recaptured were from 0 to 150 km, and these migration distances appeared to be influenced by the prox- imity of the NTZ to spawning areas or other habitats that are important to specific life-history stages of indi- vidual species. Fish that immigrated into the NTZ moved distances rang- ing from approximately 10 to 75 km. Recapture rates for sportfish species that migrated across the NTZ bound- ary suggested that more individuals may move into the protected habitats than move out. These data demon- strated that although this estuarine no-take reserve can protect species from fishing, it may also serve to extract exploitable individuals from surrounding fisheries; therefore, if the no-take reserve does function to replenish surrounding fisheries, then increased egg production and larval export may be more important mechanisms of replenishment than the spillover of excess adults from the reserve into fishable areas. Multidirectional movements of sportfish species between an estuarine no-take zone and surrounding waters of the Indian River Lagoon, Florida Derek M. Tremain Christopher W. Harnden Douglas H. Adams Florida Fish and Wildlife Conservation Commission Florida Marine Research Institute 1220 Prospect Avenue, Suite 285 Melbourne, Florida 32901 Email Derek Tremainia fwcstate-fl. us Manuscript submitted 12 May 2003 to Scientific Editor's Office. Manuscript approved for publication 20 January 2004 by the Scientific Editor. Fish. Bull 102:533-544 (2004). Fishery reserves or no-take sanctu- aries, defined as areas where all fishing activities are prohibited, are increasingly proposed as an addi- tional measure to traditional fishery management practices for protecting fish populations from overexploita- tion (PDT, 1990; Bohnsack and Ault, 1996). The American Fisheries Soci- ety recently issued a policy statement on the protection of marine fish stocks at risk of extinction and supported the development of large marine reserves to protect and rebuild vulnerable popu- lations (Musick et al., 2000). Although reserves have been established pri- marily in reef or coastal marine habi- tats, the potential to apply similar management strategies in estuarine systems may also be possible (Johnson et al., 1999; Roberts et al., 2001). Reserves in estuarine areas may help protect exploitable fishery spe- cies. Increases in species' sizes and densities within these reserves may also enhance adjacent fisheries by two separate mechanisms. Johnson et al. (1999) found that an existing estua- rine no-take sanctuary on Florida's central east coast protected popula- tions of larger, spawning-age sport- fish species. As a result, they sug- gested that protection of populations in no-take sanctuaries could also lead to the replenishment of surrounding fisheries through increased egg pro- duction, larval export, and juvenile recruitment. Additionally, mark-re- capture data have demonstrated that large juvenile and adult fishes emi- grate from estuarine protected ar- eas to surrounding waters (Bryant et al., 1989; Funicelli et al., 1989; Johnson et al., 1999; Roberts et al., 2001; Stevens and Sulak, 2001) and these data have been used to suggest that spillover of excess adult fish from estuarine reserve areas can di- rectly supplement nearby fisheries. Roberts et al. (2001) concluded that the abundance of International Game Fish Association based on line-class- record catches in the vicinity of the estuarine no-take sanctuary on Flor- ida's east coast resulted indirectly from protection and spillover of large adults to outlying waters. It has also been suggested that re- serves protect areas of undisturbed habitat (PDT, 1990), either by design or through cessation of destructive practices, and reserves are common- ly established in areas of pristine, productive, or otherwise important habitats required by the species be- ing protected (e.g., Russ. 1985). Fur- thermore, studies have shown that protecting fishery species can indi- rectly change the overall community structure (Cole and Keuskamp, 1998) and, under certain circumstances, can increase primary and secondary productivity (Sala and Zabala. 1996; Babcock et al., 1999). The influence of habitat quality on fish movements in relation to protected areas has not been investigated; however, reserve habitats that offer potential advan- tages in the form of improved habitat quality (Chapman and Kramer, 1999) 534 Fishery Bulletin 102(3) or increased food and habitat availability could be ex- pected to attract, or at least retain, individuals that immigrate to the reserves from surrounding unpro- tected habitats. Reserve areas that attract and retain exploitable individuals from surrounding habitats at higher rates than they replenish the surrounding habi- tats could be considered to be sinks in terms of their ability to directly supplement adjacent fisheries through spillover of exploitable-size individuals. Fish emigration from reserve habitats and the replenishment of nearby fisheries is a commonly predicted benefit of harvest re- serves (see reviews in Roberts and Polunin, 1991. and Rowley, 1994). However, there are currently no studies that simultaneously examine emigration and immigra- tion in relation to estuarine reserves or that document the extent to which reserve areas may also function to withdraw individuals from surrounding fisheries. With- out an assessment of net exchange, the interpretation of reserve benefits with respect to replenishment cannot be properly evaluated. The National Aeronautics and Space Administration (NASA) closed a portion of the Indian River Lagoon at the Merritt Island National Wildlife Refuge (MINWRi on Florida's east coast for security purposes in 1962. A direct result of this closure was the effective creation of an estuarine no-take zone that remains to the present time. The proximity of this no-take zone to productive estuarine fisheries provided an opportunity to examine sportfish movements in the area with mark-recapture methods. Johnson et al. (1999) first documented sport- fish migrations out of this no-take sanctuary, and in a related study, Stevens and Sulak (2001) provided more complete descriptions of movement patterns of indi- vidual species; each of these studies provided evidence that the restricted habitats protected fish populations and that adult sportfish egressed into surrounding wa- ters open to fishing. However, because all tagged fish originated from within restricted habitats, in neither of these studies was it possible to consider the potential for the movements of fish into protected areas from surrounding waters. Therefore, we (sponsored by tin- Florida Fish and Wildlife Conservation Commission- Florida Marine Research Institute [hereafter referred to as FMRII Fisheries-Independent Monitoring Program) tagged fish species throughout the northern Indian River Lagoon system, including both the MINWR no- take zone and the surrounding lagoon waters, from 1990 to 1999. We investigated the relationship between sportfish egress and ingress in relation to the MINWR no-take zone and offer a quantitative foundation for the discussion of net fish movements into or away from protected estuarine habitats. central east coast of Florida between Ponce de Leon Inlet in Volusia County and Jupiter Inlet in Palm Beach County. The lagoon is composed of three relatively iso- lated basins: Mosquito Lagoon, the Indian River proper, and the Banana River i Fig. 1). These three basins main- tain hydrological connections with each other through narrow man-made channels at Haulover Canal and the Merritt Island Barge Canal (shown on Fig. 2) and through a natural channel at the southern end of the Banana River. Hydrodynamie exchange and fish passage between the lagoon and the Atlantic Ocean occur pri- marily through five inlets, which are concentrated in the southern half of the system. The hydraulic lock system located at Port Canaveral provides only an intermittent opportunity for exchange between the IRL and Atlantic Ocean. Gilmore et al. (1981) and Mulligan and Snelson (1983) have provided detailed descriptions of the lagoon and its habitats. The no-take zone (NTZ) created by NASA and MIN- WR is located at the northern terminus of the Banana River basin of the lagoon. An earthen causeway defines the southern boundary of this no-access security area and contains only two openings that permit fish to mi- grate to and from adjacent waters. Much of the natural shoreline and saltmarsh habitats in the lagoon have been altered for mosquito control purposes. However, actual shoreline habitats surrounding MINWR — in- cluding the NTZ, the northern Banana River basin, the northern Indian River basin, and Mosquito La- goon— remain relatively undeveloped in comparison to the urban shoreline development in the southern IRL. Detailed descriptions of the habitat composition within the NTZ and surrounding study area were provided by Johnson et al. (1999). Data collection Fish were tagged as part of several related FMRI proj- ects (stratified-random, fixed-station, and directed sam- pling designs) in the northern IRL between 1990 and 1999 (FMRI1). In most cases, tagging was conducted opportunistically on healthy fish following capture in multipanel monofilament gill nets, nylon trammel nets, nylon haul seines, or on hook and line. In other cases, projects were designed specifically to assess tag-recap- ture information (Murphy et al., 1998). Because of the focus of our sampling programs in this area, the major- ity of our tagging efforts occurred north of Sebastian Inlet within the Indian and Banana River basins of the lagoon. A small percentage of tags were placed in fish captured south of Sebastian Inlet or in Mosquito Lagoon. Overall, our sampling collections in the NTZ Materials and methods Study area The Indian River Lagoon (IRL) is a shallow barrier island estuarine system spanning 25.3 km along the 1 FMRI (Florida Fish and Wildlife Conservation Commis- sion). 1999. Florida Marine Research Institute. Fisheries- independent monitoring program, 1999 annual data summary report In-house Report. Florida Fish and Wildlife Conser- vation Commission. Florida Marine Research Institute. LOO Eighth Ave. S.E., St. Petersburg, Florida, 33701. Tremain et ai.: Sportfish species movements in relation to an estuarine no-take zone 535 \ 80°30'W . Ponce Jt Leon Inlet mi 00' \\ \1I\WR No-take Zone (NTZ) upper Banana River I BR i Atlantic Ocean GulfoJ l/l'Wl 0 29° 00' N- 28 JO' N- 28 OO'N- Sebasritjn Inlet Ft. Pierce Inlet 27 in \ Florida Atlantu Ocean Si Lucielnlet 27"00'N Palm Bead County Figure 1 Map of Florida and the Indian River Lagoon study area. accounted for approximately 20% of our total sampling efforts and averaged approximately 1-2 days/month over the study period. Fish were tagged by inserting 50-mm, 70-mm. or 100-mm Hallprint dart tags (Halprint Ltd., Victor Har- bor, South Australia) into the dorsal musculature; the plastic dart was lodged beneath the pterygiophores of the dorsal fin. Each tag contained a visible exter- nal streamer with a unique alphanumeric code and instructions for anglers to contact us with recapture information in order to collect a reward (five dollars or equivalent). Information recorded at the time of ini- tial tagging included the tag number, species tagged, date, location (latitude and longitude i, and fish length (standard, fork, and total lengths as appropriate for the species). Recapture information on tagged fish was collected through August 2000 from angler reports and from fish recaptured during FMRI sampling ac- tivities. Because of public-access prohibitions, recap- ture information from inside the MINWR NTZ was gathered exclusively through FMRI sampling efforts. Data requested for recaptured fish included the same information as that recorded at initial tagging; however, in several cases, length or precise location information returned from anglers was considered to be unreliable, which prevented accurate statistical comparisons of relationships involving recapture lengths or distances traveled. Therefore, reported length data are limited to initial tagging information only (total length; TL). To prevent problems with pseudoreplication for individuals recaptured on multiple occasions, we included only the initial tag recovery data in our calculations of recapture percentages. Overall patterns offish migrations, including general recapture locations and direction of movements into or away from the NTZ, were described by using data 536 Fishery Bulletin 102(3) A B !';;:;"" rb Egress Catul s Ingress \ " R B TR Merritt Island B* ^-^. National Wildlife f \ 1 No-take ] Reflige /^^ ^\ | No-take j V zone / Kr R V J R y zone J b Rft; V y r R :r Cape R R Cape Canaveral B Canaveral Cocoa Sr i} • Cocoa R 8B Por, Barge Cona/ Canaveral H H R 2R R Co D 3 P6 R 2R • • Melbourne Melbourne R % also one "S" and one "J" -. approximately 75 km south in the St. Lucie River \s 3RH / /« Sebastian Inlet Figure 2 (A) Recapture locations of tagged fish that migrated out of the Merritt Island National Wildlife Refuge no-take zone. (B) Original tagging locations offish that migrated into the Merritt Island National Wildlife Refuge no-take zone. R = red drum, B = black drum, S = common snook, H = sheepshead, T = spotted seatrout, J = crevalle jack, K = bull shark. Numbers before species codes (letters) indicate the number of individuals of that species that were captured at that location. from all available recapture sources. In contrast, we calculated migration rates exclusively from the recap- ture data collected during FMRI sampling activities. Although this procedure excluded tag-return data from recreational anglers, it permitted a quantitative assess- ment of recapture rates based on standardized FMRI col lection gear, comparable sampling effort, and lOO'* tag reporting rates. We resolved potential problems related to differences in habitat characteristics and sampling intensity by including only data from the NTZ and a fishable area of a similar size and habitat type in the adjacent Banana River (BR, Fig. 1). This BR zone corresponded precisely to the sampling zone used for population comparisons in Johnson et al. (1999), denoted as "FBR" (fished Banana River) in that study. Species that did not contribute any FMRI recapture information in either of these two areas were excluded from our analyses. Tag recovery and migration rates were calculated separately for the NTZ and BR. For our purposes, "migration" was defined as a directional fish movement across the NTZ boundary from the original tagging location, and we made the assumption that the migration patterns of recaptured fish represented the migration patterns of the overall population. Rela- Tremain et al.: Sportfish species movements in relation to an estuarine no-take zone 537 Table 1 Summary of tagging and recapture data for seven of the most common sportfish species tagged by FMRI scientists ern Indian River Lagoon study area. Locations where tag and recapture data were collected are separated into the (NTZ) and the surrounding waters of the Indian River Lagoon (IRL). in the north- no-take zone Species No-take zone Indian River Lagoon Total no. offish tagged Total no. recaptured and percent recaptured Tagged inside NTZ Recapture location Tagged outside NTZ Recapture location NTZ IRL NTZ IRL Bull shark (Carcharhinus leucas) 1 1 24 1 25 2(8.0) Common snook (Centropomus undecimalis) 104 1 9 406 32 510 42(8.2) Crevalle jack ( Caranx hippos I 55 1 59 1 114 2(1.8) Sheepshead tArchosargus probatocephalus) 597 6 520 26 1117 32(2.9) Spotted seatrout (Cynoseion nebulosus) 193 2 171 1 3 364 6(1.6) Black drum (Pogonias cromis) 637 4 8 831 9 32 1468 53(3.6) Red drum (Seiaenops ocellatus) 720 30 40 1344 6 190 2064 266(12.9) Total 2307 37 65 3355 16 285 5662 403)7.1) tive migration rates were calculated as the percentage of recaptured fish that migrated from their original tagging location. These migration rates and their re- ciprocal (retention rates) were compared between the NTZ and the BR to determine the relative potential for sportfish movements into or away from protected habi- tats. Chi-square contingency tests for frequency data (with Yates's correction for small sample sizes) were used to test the hypothesis that recapture location was independent of the tagging location. Results A total of 5951 fish of 27 species were tagged during FMRI sampling within the IRL between September 1990 and December 1999. However, because 95% of these fish were represented by only seven species (Table 1), which included all fish that migrated across the reserve bound- aries, only these seven species were considered further in our analyses. Red drum (Seiaenops ocellatus) was the most commonly tagged species (n=2064), followed by black drum (Pogonias cromis, n = 1468), sheepshead (Archosargus probatocephalus, /? =1117 ), common snook, (Centropomus undecimalis, n = 510), spotted seatrout (Cynoscion nebulosus, /; = 364), crevalle jack (Caranx hippos, n=114), and bull shark (Carcharhinus leucas, n=25). Approximately 41% (n=2307) of these fish were tagged inside the boundaries of the NTZ. The remain- der (« = 3355> were tagged in the surrounding lagoon. Through August 2000, 403 tagged fish (7.1% of total) were recaptured and reported either by FMRI staff sampling in the lagoon or by the public. Overall recap- ture rates were highest for red drum (12.9%), followed by those for common snook (8.2%), bull shark, (8.0%), black drum (3.6%), and sheepshead (3.0%). Tagged fish were generally representative of the larg- er mobile members of the species and encompassed the legally exploitable size ranges for species with man- agement restrictions (Table 2). For species except the bull shark and red drum, mean lengths of fish tagged inside the NTZ exceeded those of fish tagged outside the NTZ. Approximately 25% (n = W2) of the 403 total recap- tured fish were fish originally tagged inside the NTZ (Table 1). Thirty-seven of these fish were also recovered inside the NTZ. including three red drum that were subsequently recaptured on multiple occasions in the protected area. The remaining 65 recaptured fish were caught after emigrating to outlying waters, including one red drum that was recaptured a second time outside the NTZ. Species that migrated out of the NTZ were red drum (n = 40, mean TL = 643 mm, SD = 135 mm), common snook (;? = 9, mean TL = 570 mm, SD = 97 mm), black drum (n = 8, mean TL = 845 mm, SD = 88 mm), sheepshead (n = 6, mean TL = 398 mm, SD = 38 mm), bull shark (n = l, TL=789 mm), and crevalle jack (n = l, TL = 628 mm). Recapture distances ranged from 0 km immediately outside the NTZ to approximately 150 km south in the St. Lucie River estuary, but recaptured fish were more abundant closer to the NTZ (Fig. 2A). Most of the recaptured fish were concentrated in areas of high fishing pressure, such as causeways, inlets, and waters near the boundary of the NTZ. Collectively, fish that emigrated from the NTZ did not appear to show a bias for any one direction of movement: recaptured fish were found both northward in the Indian River and southward throughout both the Indian River and Ba- nana River basins of the lagoon. For individual species, red drum that emigrated were distributed throughout the lagoon system and coastal habitats, whereas black drum were predominantly recaptured in the northern 538 Fishery Bulletin 102(3) Table 2 Total length ITL) and the outlying size ranges (in mm) and le ndian River Lagoon study gal size area. limits ( as of August 2000) foi tagged sportfish species from the no-take zone Species No-take zone Indian River Lagoon Legal size limits Mean(SD) min max MeanlSDl min max (mm TL) Bull shark 789( — i 789 789 974(135) 684 1180 None Common snook 570(106) 330 844 506(138) 227 944 660-8641+ lover) Crevalle jack 486(140) 305 720 443(113] 264 720 None Sheepshead 398(68) 235 614 365(76) 171 594 305 minimum Spotted seatrout 415(129) 185 754 335(111) 212 678 381-508 (+ 1 over) Black drum 786(129) 249 1156 742(240) 225 1135 356-610 ( + 1 over) Red drum 613(166) 308 1245 624(229) 203 1210 457-686 estuarine portion of the study area. Sheepshead and common snook were recaptured primarily to the south at inlets or in the adjacent Atlantic coastal waters out- side the lagoon. The remaining 75% (« = 301) of the total recaptured fish were from fish originally tagged outside the NTZ (Table 1). The majority of these (/!=285) were also re- covered in outlying waters, including 16 red drum and 1 sheepshead that were subsequently recaptured on multiple occasions. Sixteen fish were recaptured after they had immigrated into the reserve. These recaptured fish were from three sciaenid species: predominantly black drum in = 9, mean TL = 907 mm, SD = 66 mm) and red drum ln = 6, mean TL = 656 mm, SD = 170 mm), but also one spotted seatrout (TL=420 mmMFig. 2B). The longest migration distances into the NTZ were up to 75 km for red drum and spotted seatrout tagged in south- ern Mosquito Lagoon and the northern Indian River basins. All black drum that immigrated into the NTZ were tagged in the adjacent Banana River basin. A relatively large number of red drum, common snook, and sheepshead that were tagged inside the NTZ or in the outlying waters were recaptured in close proximity (0 to 2.75 km distance) to inlet habitats. Recaptured red drum from inlet habitats (n = 45, mean TL=647 mm, SD = 135 mm) peaked during September through No- vember. Recaptured common snook from inlet habitats (n=13, mean TL = 598 mm, SD = 111 mm) were distrib- uted throughout much of the year but peaked in late fall. Few common snook were recaptured from inlet spawning habitats during the peak summer spawning months (June-August) when their fishery was closed. Recaptured sheepshead from inlet habitats («=8, mean TL = 373 mm, SD = 53 mm) were concentrated in the winter and early spring. Estimated migration rates were calculated by using only those fish that were tagged and recovered from FMRI sampling in the NTZ and the immediately ad- jacent upper Banana River (BR). The number of fish tagged in the NTZ (n=1654) was approximately 1.7 times the number tagged in the BR (/(=965) (Table 3); however, the overall recapture rates of fish that were originally tagged in each of these two areas were equal (2.4%). Black drum and red drum made up the majority of tagged and recaptured fish in both areas and were the only species recaptured that had migrated both into and away from the NTZ in this comparison. For total sportfish (all species pooled), there was a significant relationship between the tagging location and the direc- tion offish movements (fh 005=13.8, P=0.0002). A total of 40 fish originating from the NTZ were recaptured, but that number included only 2 fish (one red drum and one black drum) that emigrated to the BR (5% overall migration rate). In contrast, 23 fish originat- ing in the BR were recaptured overall, including 12 that immigrated into the NTZ (52% overall migration rate). Species-specific migration rates were highest for black drum, and relative immigration rates (90%) were higher than emigration rates (25"7<). For this species, the frequency of immigration and emigration were sta- tistically independent of tagging location (xZi 005=0.01. P=0.9039), which is probably due to the low number of recaptures offish tagged inside the NTZ (Table 3). For red drum, relative immigration rates (27%) were also higher than emigration rates (3%), but in this case, there was a significant relationship between fish move- ments and tagging location (^ ,, (ia=20.58, P<0.0001). Common snook, spotted seatrout. and sheepshead were also recaptured by FMRI scientists in these compari- sons, but none of these recaptured fish represented evidence of migrations across the NTZ boundary from their original tagging location. Discussion This study demonstrated both the emigration and immi- gration of sportfish species across the boundaries of an estuarine no-take zone (NTZ). Legal-size large juveniles and adults of six of the recreationally valuable species tagged within NTZ boundaries — red drum, black drum, common snook, sheepshead, bull shark, and crevalle Tremain et al.: Sportfish species movements in relation to an estuarine no-take zone 539 Table 3 Summary of tag and recapture and the adjacent fished waters included in calculations of tota data from only the Florida Marine Research Institute sampl of the Banana River (BR). Species that did not contribute s or of migration percentages. ing efforts in the no-take zone ( NTZ ) any recapture information were not No-take zone Total Percent that migrated Banana River Total Percent that migrated No. of fish tagge No. fish recaptured No. of fish tagged No. fish recaptured i NTZ BR NTZ BR Red drum 720 32 1 33 3.3 176 3 8 11 27.3 Black drum 637 3 1 4 25.0 495 9 1 10 90.0 Common snook 104 1 0 1 0 62 0 1 1 0.0 Spotted seatrout 193 2 0 2 0 121 0 0 0 — Sheepshead 597 0 0 0 — 232 0 1 1 0.0 Totals 1654 38 2 40 5.0 965 12 11 23 52.2 Tag recovery (% ) 2.4 2.4 jack — were documented to migrate out of the protected area. Johnson et al. (1999) and Stevens and Sulak (2001) also observed many of these same species emigrat- ing from no-take zones within the same refuge system during the late 1980s, although the species with the highest recapture rates in their studies (common snook) differed from the current study (red drum). This differ- ence may reflect an increase in the popularity of the red drum fishery on Florida's east coast during the current study period. Since 1989, when the recreational red drum fishery reopened under strict management regula- tions, there has been a significant increase in both the total red drum landings on the Atlantic coast and in the estimated number of fishing trips made by anglers seek- ing or catching red drum each year (Murphy2). Tagging studies in estuarine areas of the Everglades National Park have previously documented emigrations of striped mullet (Mugil cephalus), gray snapper (Lutjanus gri- seus), and spotted seatrout away from protected habitats (Bryant et al., 1989; Funicelli et al., 1989). Recent stud- ies suggest that fish moving out of protected areas in the IRL may help to replenish nearby fisheries and may contribute to trophy fisheries in the surrounding system (Johnson et al., 1999; Roberts et al., 2001). In our study, overall emigration rates were low, but many of the fish that emigrated from the estuarine NTZ moved comparatively large distances. The egress pat- terns of exploitable species may affect both the species' potential for protection and the degree to which fisheries located adjacent to protected reserves will be enhanced (DeMartini, 1993). In coastal marine and tropical reef systems, where the large majority of reserves have been established, long-distance movements greater than a 2 Murphy, M. D. 2002. A stock assessment of red drum. Seiaenops ocellatus, in Florida: status of the stocks through 2000, 32 p. Florida Fish and Wildlife Conservation Com- mission Report, Melbourne, FL. few kilometers by demersal fishery species are limited to a very small percentage of individuals (Beaumar- iage, 1969; PDT, 1990 and references therein; Rowley, 1994), and the direct supplementation of nearby fisher- ies by exploitable species appears to be highly localized (Buxton and Allen, 1989; Russ and Alcala, 1996). The majority of fish that emigrated from the NTZ were recaptured between 10 and 75 km from the boundary, but fish were also recovered as far as 150 km from the NTZ boundary. Our observations on migration distances and recapture locations corresponded well with those reported from previous studies of fish movements out of this same reserve system (Johnson et al., 1999; Ste- vens and Sulak, 2001). although maximum recapture distances in earlier studies were even greater. Many of the fish that emigrated from the NTZ — such as red drum, common snook, and sheepshead — were recaptured at inlet locations or in the nearshore coastal waters at sizes that were large enough to include re- productively mature adults (Murphy and Taylor, 1990; Render and Wilson, 1992; Taylor et al., 2000). The seasonality of inlet-associated recaptures was consistent with the seasonality of documented spawning and move- ment patterns for these species. In Florida, red drum typically spawn in nearshore coastal waters during the fall (Murphy and Taylor, 1990), although spawning within the IRL has also been documented (Johnson and Funicelli, 1991). Spawning by common snook may occur year-round on Florida's east coast (Gilmore et al., 1983), but most spawning takes place between May and October in or near major inlets to the Atlantic Ocean (Taylor et al., 1998). The limited number of common snook recaptured from inlet spawning habitats dur- ing the peak summer spawning season (June-August) was likely due to the fishery being closed during those months. Sheepshead move offshore with the onset of cool weather in the late fall (Gunter, 1945; Kelly, 1965), and spawning likely occurs in offshore waters during 540 Fishery Bulletin 102(3) the spring (Springer and Woodburn, 1960; Jennings, 1985; Tucker and Barbera, 1987). In the northern por- tion of the IRL, where the NTZ is located, the closest access to the coastal environment is through two inlets located approximately 75 km (Sebastian Inlet) and 100 km (Ponce de Leon Inlet) swimming distance away or through an intermittent lock opening at Port Canaveral approximately 12 km to the south. In order to reach nearshore or tidal-pass spawning habitats, species must first migrate to these locations. The coincidence of tag recoveries from these areas during identified spawning or migration periods likely indicated that the relatively long movement distances we observed resulted from a combination of geographical, environmental, and bio- logical factors, including the proximity of the NTZ to habitats that are important for specific life-history re- quirements of individual species. From a management viewpoint, these relationships can affect the spatial extent of species' migrations in relation to protected habitats, as well as the degree of protection provided to individuals that are migratory, and should be consid- ered carefully in the design of estuarine reserves. This study documented the ingress of exploitable es- tuarine sportfish species into protected habitats and demonstrated that these movements can also cover sub- stantial distances. Species moving towards the NTZ traveled distances of at least 10-75 km. The original tagging locations of these fish were distributed through- out the northern Indian and Banana rivers and southern Mosquito Lagoon, which paralleled the primary region of our tagging efforts. Whether or not fish from more southerly locations in the IRL system would migrate into the NTZ is largely unknown because of the lack of tagging effort in those areas. However, for tropical species such as the common snook, permit (Trachinotus falcatus), gray snapper, and others whose abundances increase seasonally in the northern lagoon habitats dur- ing the warmer months (Tremain and Adams, 1995), it seems probable that seasonal movements could bring them into contact with the protected habitats. In such cases, these species would benefit only temporarily from fishing protection until their return migrations made them again vulnerable to capture. In contrast, species observed migrating into the NTZ that typically have a high degree of site fidelity during specific life-history stages, such as the red drum (Beaumariage, 1969; Ad- ams and Tremain. 2000), black drum (Murphy et al., 1998), and spotted seatrout (Moffett, 1961), should de- rive greater long-term benefits from reserve protection following immigration into protected areas. Tagging studies that examine the transfer of fishery species between reserve and outlying habitats are rare, and we have found only one recent study on any fishery species, the American lobster (Homarus americanus\, that investigated the effects that multidirectional spe- cies migrations may have upon protective reserve func- tions (Rowe, 2001). Studies in which fish movements have been examined, in both estuarine and marine protected areas, have focused exclusively on fish egress from reserve habitats (Bryant et al., 1989; Buxton and Allen, 1989; Funicelli et al., 1989; Holland et al., 1996; Zeller and Russ, 1998; Johnson et al., 1999. Stevens and Sulak, 2001) or on home ranges of species associ- ated with reserve habitats (Eristhee and Oxenford. 2001; Starr et al., 2002). In the present study, we simul- taneously examined both egress and ingress of sportfish in relation to a no-take reserve and the surrounding unprotected waters, and the results provide a starting point to quantitatively discuss the relationship between fish emigration and immigration, as well as the implica- tions of such movements to the resulting functions of replenishment to or withdrawal from nearby estuarine fisheries. When all recapture sources were considered, the ratio of migrating to nonmigrating individuals was much higher for fish tagged inside the NTZ (1.58) than for those tagged outside the NTZ (0.05); this ratio im- plies that there is a spillover effect from the reserve. However, this difference is less apparent when measured against the large disparity between recapture effort from inside the NTZ (12-24 FMRI sampling days/year + 12-24 angler days/year) and recapture effort from the surrounding lagoon waters of Brevard County (50-100 FMRI sampling days/year + 114,000-181,000 angler days/year [FMRI. unpubl. data]). Furthermore, this direct comparison assumes that recapture potential was the same in protected and unprotected areas, which is unlikely given the differences between the primary re- capture gear used in scientific research activities inside the reserve (nets) and the gear used in recreational an- gling outside the reserve (hook and line). There were no reliable estimates of sportfish species landings available for the limited study region that could have enabled us to intercalibrate for these differences; therefore. we limited further comparisons to only data recovered through FMRI sampling activities in the northern Ba- nana River basin. This limitation came at the expense of important tag-recovery data collected by anglers or collected from more outlying areas of the lagoon but permitted a more quantitative comparison of migra- tion potential that focused comparisons on immediately adjacent areas where the effects of spillover would most likely be realized (Buxton and Allen, 1989; Russ and Alcala, 1996). In these comparisons, a disproportionate number of fish were tagged inside the NTZ, but overall tag-recovery rates for fish originating in both the NTZ and the adjacent Banana River were equivalent. This finding indicated that tagged individuals from both areas were equally susceptible to recapture. However, there were substantial differences in the migration patterns of fish between the two areas. In the vicinity of the NTZ, the relative potential for overall sportfish migrations (primarily for red drum and black drum, which provided the greatest quantity of tag recovery data) towards the NTZ from unprotected habitats (52%) was greater than the potential for migrations out of the NTZ (5%). Two potential limitations must be considered when comparing these migration rates. First, it is possible that recreational fishing in the upper Banana River could have reduced the number of tags available to FM- Tremain et al.: Sportfish species movements in relation to an estuarine no-take zone 541 RI sampling activities outside the NTZ, leading to lower tag recovery rates from this area. However, several fish from the Banana River study area were recaptured on multiple occasions — a common occurrence in this region where fish are caught and released in fishing practices. Although there is some postrelease cryptic mortality associated with catch-and-release practices, these re- leases likely limited the effects of local fishing on our analyses. Second, our assumption that the migration patterns of recaptured fish represented the migration patterns of the overall population may not be valid if the respective length frequencies were not also equally represented. The use of multiple gear types and sam- pling strategies to collect fish for tagging increased the likelihood that the length frequencies of species in our collections represented the available population. Report- ed recapture length frequencies closely approximated the population length frequencies in our collections for red drum, black drum, and sheepshead but over-repre- sented the frequency of larger individuals for common snook and spotted seatrout. Because red drum and black drum were the principal species that displayed multidirectional migration patterns, we considered the potential for size bias to be minimal in our comparisons of estimated ingress and egress rates. Ultimately, a determination of the net result of these migration patterns, in terms of replenishment to or withdrawal from adjacent fisheries, would require ac- curate assessments of species population abundances that were beyond the scope of this study. If there are large enough differences in population densities across the NTZ boundary, either as a result of increased pro- duction inside the reserve or high fishing mortality outside, then the relatively low emigration rates that we observed could still result in a net export of exploitable individuals to fished populations in surrounding waters. In trammel-net collections from this same reserve dur- ing the late 1980's, Johnson et al. (1999) estimated that in the protected habitats, relative abundances of red drum populations were 6.3 times greater and of black drum were 12.8 times greater than the relative abundances of these populations in adjacent unpro- tected areas. More recent shoreline haul-seine data from 1997-2000 show that these abundances were only 1.8 times greater for red drum and 1.5 times greater for black drum (FMRI, unpubl. data). To what extent the difference in abundance estimates between these two temporally separate studies is related to fish move- ments, to stringent changes in management regulations that have occurred, or to the difference in sampling methods used is undetermined. However, if we consider the more recent population level differences between the NTZ and adjacent waters, then the emigration and im- migration rates observed in the present study indicate that there is a potential for more substantial move- ments by these species towards protected habitats than away from them. One limitation of tag-recapture data is that such data provide only a snapshot view of overall fish movements, and the whereabouts of tagged individuals between the time of tagging and recapture are unknown. It is possible that the movements we observed for red drum and black drum in the vicinity of the NTZ were simply instantaneous views of a more complex series of movements between the NTZ and adjacent waters. One possibility is that these movements could be related to daily or seasonal home ranges that extend across reserve boundaries. Studies that attempt to quantify home ranges for these species at any temporal scale are limited. Carr and Chaney 1 1976 1 followed a single red drum, which was fitted with an ultrasonic trans- mitter, for up to two days after releasing it into the Intracoastal Waterway near St. Augustine, Florida. During that time, fish movements were oriented against the direction of tidal flow but remained within 2 km of the release point. Adams and Tremain (2000) found that large juvenile red drum repeatedly used or were continually associated with a 2-km section of a northern IRL tidal creek for periods of up to 18 months. Tag- ging studies from estuarine waters generally indicate that the majority of red drum and black drum do not make substantial movements from their release sites, although some individuals are capable of migrating up to several hundred kilometers (Beaumariage, 1969: Osburn et al. 1982; Music and Pafford, 1984; Murphy et al., 1998i. During the present study, 20 red drum were recaptured on multiple occasions; however, none of these fish exhibited movements that could provide evidence for home ranges that overlapped the NTZ boundar- ies. Another possibility for the movement patterns we observed is that they are related to population equilib- rium adjustments that occur when the relative attri- butes of the NTZ and surrounding areas change with respect to each other. For example, beginning in 1990 and coinciding with the onset of the present study, the Banana River adjacent to the NTZ (including much of our BR study area) was closed to motorized boat traffic. Although the area remained open to fishing, it became considerably more difficult to access by fishermen. If this limitation resulted in lower fishing pressure (i.e., predation) and fewer habitat disturbances, then the relative habitat value and rates of migration into this area may have increased during that time. There are no quantifiable estimates of migration rates prior to this study for comparison, but our results do not dem- onstrate an equilibrium adjustment toward potentially higher quality BR habitats during our study period. If species movements are not equilibrium adjustments, but rather are driven by an attraction to or retention within habitats that offer protective benefits, then ul- timately reserve habitats should become saturated. Predicted equilibrium population sizes for queen conch iStrombus gigas) and spiny lobster (Panulirus argus) were achieved in just three years after the effective creation of a Caribbean reef harvest refuge, but models suggested that relatively minor changes in refuge area and boundary condition (i.e., permeability) could result in major population-level responses by exploited species, depending upon dispersal dynamics and habitat avail- ability (Acosta, 2002). The estuarine no-take zone at 542 Fishery Bulletin 102(3) MINWR has been in effect for approximately 40 years, presumably long enough for fish populations to reach equilibrium levels, yet we observed a net movement of fish into protected habitats over the past decade. A wide range of factors interact to determine the distributions of large mobile fish in the IRL, where physical environmental conditions (salinity, inlet dis- tance, temperature, etc.) have a primary influence on the species' distributions over a lagoon-wide scale, and where species responses to biological variables (sea- grass cover, depth, seasonality, etc.) act secondarily to influence distributions at smaller scales (Kupschus and Tremain, 2001). The specific mechanisms that lead to the greater ingress rates into the NTZ for red drum and black drum in the present study cannot be determined from our data. Possibilities include a behavioral attrac- tion to the NTZ due to the interrelated influences of habitat preference, spawning, and social structure, or due to potentially higher retention rates after migra- tion into the reserve. Red drum and black drum were routinely observed foraging in large schools within both the NTZ and surrounding waters, which suggested that food resources were available in each of these habitats; however, there are few studies that have attempted to quantify differences in resource availability between these areas. Johnson et al. (1999) described the habitat characteristics of their study areas within the same re- serve system but found that protection from fishing, and not habitat difference, was the primary factor contribut- ing to differences in the abundance of sportfish species between fished and unfished areas. The availability of suitable spawning habitats within the NTZ may also attract red drum and black drum to the reserve habi- tats. We observed indications of reproductive behavior by both of these species inside the NTZ that is common among members of the drum family, including concen- trations of drumming fish (Mok and Gilmore, 1983) and repeated side-to-side contact among individual fish (Tabb, 1966) in the presence of ripe and running males. Although we did not directly observe these behaviors for either species outside of the NTZ, black drum and red drum are documented to spawn elsewhere within the IRL system (Mok and Gilmore, 1983; Johnson and Funicelli. 1991) and we cannot automatically presume that suitable spawning habitats do not also occur in the surrounding waters. If there is a behavioral attraction to protected habitats, then the subsequent retention of individuals that have immigrated into these areas may be prolonged by the limited boundary permeability of this reserve, which contains only two potential egress pathways back into the adjacent waters. In order to ful- ly understand the protective functions of this estuarine reserve and others, it will be important to identify the biological, behavioral, and physical mechanisms that influence species movements in relation to the reserve boundaries. The opportunistic nature of our tagging efforts within the design of a larger sampling program precluded sta- tistically valid sample replication, and only one reserve and adjacent fished area were examined; therefore, the results of this study should not be generalized to other areas. Still, the IRL is typical of other bar-built estuaries where access by estuarine fishes to coastal waters through passes or inlets may be limited, and it is reasonable to expect that the geographical, en- vironmental, and biological processes that influence species movements in the IRL would also be important in other estuaries of similar structure. Studies show- that no-take areas in estuarine systems can have an effect on species' abundances and size distributions within these protected areas and may indicate that these areas protect species from the effects of fishing pressure (Johnson et al., 1999; FMRI unpubl. datai. Whether or not these areas will actually increase fish abundance in adjacent waters or benefit surrounding fisheries through direct supplemental replenishment of exploitable species is less evident. Certainly, some indi- viduals will migrate out of protected areas in response to environmental, biological, or physiological stimuli, and these individuals may contribute to trophy fisheries in surrounding waters (Roberts et al.. 2001); however, our data indicated that within estuaries, reciprocal movements over relatively large distances into protected areas also occur and have the potential to extract ex- ploitable individuals from surrounding fisheries. The overall impact of such withdrawals on these fisheries will depend on the degree of retention following migra- tions into protected areas. If retention rates are high, then increased egg production, larval export, and juve- nile recruitment may be more important mechanisms for replenishment of nearby fisheries than spillover of exploitable species, but production and export will be limited unless reserves encompass spawning or nursery- habitats (or both) that will support long-term protection and population growth. For estuarine-dependent coastal species that support estuarine fisheries, the benefits obtained within protected areas will be determined, in part, by their specific life-history characteristics, movement patterns, and the reserve design. Although the establishment and study of reserves in marine or coastal systems has increased in recent years, research on the effects of protected no-take reserves in estuarine habitats is still in its infancy. Information on the daily, seasonal, or annual movement patterns of estuarine- resident or estuarine-dependent coastal species is neces- sary for understanding and designing effective reserve areas in these habitats. Acknowledgments We wish to thank the crewmembers and volunteers at FMRI's Indian River Field Laboratory for collecting data and assisting in this study and the many fishermen who willingly provided us with recapture information. We are grateful to U.S. Fish and Wildlife Service personnel for providing access to sampling areas within restricted areas of the Merritt Island National Wildlife Refuge. This paper benefitted from reviews by R. Cody. J. Col- vocoresses, L. French, J. Leiby, R. Paperno, J. Quinn, Tremain et al.: Sportfish species movements in relation to an estuarine no-take zone 543 T. Tuckey, and F. Vose, and two anonymous reviewers. This work was supported in part by funding from the Department of Interior, U. S. 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Seriola dumerili were col- lected off Galveston, Texas, from May to July over a two-year period (2000 and 20011 in both inshore (<15 nauti- cal miles [nmijl and offshore zones (15-70 nmi). Relative abundance of YOY S. dumerili (32-210 mm stan- dard length) from purse-seine col- lections peaked in May and June, and abundance was highest in the offshore zone. Ages of S. dumerili ranged from 39 to 150 days and hatch- ing-date analysis indicated that the majority of spawning events occurred from February to April. Average daily growth rates of YOY S. dumerili for 2000 and 2001 were 1.65 mm/d and 2.00 mm/d. respectively. Intra-annual differences in growth were observed; the late-season I April I cohort expe- rienced the fastest growth in both years. In addition, growth was signifi- cantly higher for S. dumerili collected from the offshore zone. Mortality was approximated by using catch-curve analysis, and the predicted instan- taneous mortality rate (Z) of YOY S. dumerili was 0.0045 (0.45%/d). Distribution, age, and growth of young-of-the-year greater amberjack {Seriola dumerili) associated with pelagic Sargassum R. J. David Wells Jay R. Rooker Texas A&M University Department ol Marine Biology 5007 Avenue U Galveston, Texas 77551 Present address (lor R. J, D Wells): Coastal Fisheries Institute Louisiana State University Baton Rouge, Louisiana 70803 E-mail address (for R J D Wells) rwells4@lsu.edu Manuscript submitted 9 December 2002 to Scientific Editor's Office. Manuscript approved for publication 2 March 2004 by the Scientific Editor. Fish. Bull. 102:545-554 (2004). Recruitment of marine fishes is highly variable and closely linked to early life events (Houde, 1996; Cole, 1999). Early life survival is dependent upon several biological and environmen- tal factors including spawning time, prey availability, predation pres- sure, growth, and physical transport mechanisms (Bricelj, 1993; Schnack et al., 1998). Recruitment success is commonly assessed by examining patterns of relative abundance (Sano, 1997), whereas estimates of growth and mortality are commonly used to index recruitment potential (Rilling and Houde, 1999; Rooker et al., 1999). Early life growth and mortality are linked because fishes with high growth rates often exhibit decreased size-spe- cific predator vulnerability (Meekan and Fortier, 1996). As a result, esti- mates of juvenile abundance, growth, and mortality provide insight into patterns of nursery habitat quality and thus may be used to delineate essential fish habitat (EFH) (Pihl et al., 2000; Sullivan et al, 2000). Greater amberjack ( Seriola dumer- ili) is a reef-associated species with a circumglobal distribution in sub- tropical and temperate waters (Ma- nooch and Potts, 1997a). In the Gulf of Mexico, S. dumerili is the largest carangid and supports important recreational and commercial fisher- ies (Thompson et al., 1999). Owing to increased fishing effort and land- ings, S. dumerili in the Gulf are cur- rently assessed as overfished (NOAA, 2000). Consequently, detailed life his- tory information is needed to effec- tively guide fishery management of this valuable resource. To date, avail- able life history data on S. dumerili have almost entirely been based on assessments of subadults and adults (Manooch and Potts, 1997a, 1997b; Thompson et al.1). Despite the impor- tance of early life processes, data on juvenile or young-of-the-year (YOY) S. dumerili are limited to qualitative surveys of pelagic Sar-gassum (Bor- tone et al., 1977; Settle, 1993). The National Marine Fisheries Ser- vice has recently designated Sargas- sum as essential fish habitat (EFH) of several coastal migratory species including S. dumerili (NOAA, 1996). In response, the goal of this study was to examine the distribution and growth of S. dumerili associated with pelagic Sargassum mats in the NW Gulf of Mexico. Specifically, objectives of this research were to quantify spa- tial and temporal patterns of habitat use by S. dumerili and to determine age, hatching-date, growth, and mor- tality of S. dumerili by using otolith- based techniques. Thompson, B. A., C. A. Wilson, J. H. Render, M. Beasley, and C. Cauthron. 1992. Age, growth, and reproductive biology of greater amberjack and cobia from Louisiana waters. Final report NA90AA-H-MF722, 77 p. Marine Fish- eries Initiative (MARFIN) program. National Marine Fisheries Service, NOAA, St. Petersburg, FL. 546 Fishery Bulletin 102(3) 96 00' W 95 i i 50' W 94 : 40'W i 94 00'W 93-20'W i 30 00' N" / ^STUDY * \ I "7 SITE ;v \ Louisiana 29 20' N" Galveston. TX Inshore zone y Offshore ,'T---- zone /" 28c 40' NJ ^)r^ >"■-- _ .. ~ t~ '; /' 28 00' N- 20 m depth contour t N 25 km Figure 1 Map of sampling locations along the Texas Gulf coast for S. dumerili. Inshore (<15 nautical miles) and offshore (>15 nautical miles I zones off Galveston, TX, are shown. Materials and methods Field collections Seriola dumerili associated with pelagic Sar-gassum mats were collected off Galveston, Texas, from May to July over a two-year period (2000 and 2001) (Fig. 1). Inshore (<15 nautical miles |nmi]) and offshore (15-70 nmil zones were sampled to evaluate the potential importance of physiochemical conditions because inshore waters off the coast of Texas are heavily influenced by estuarine processes (Smith, 1980; Sahl et al., 1993). Replicate samples (3-5 per trip) in both the inshore and offshore zones were collected monthly by using a larval purse seine (20 m long x 3.3 m deep, 1000-pm mesh). The purse seine was deployed into the water as the boat encircled a randomly chosen mat. The seine was pursed, the Sargas- sum was discarded, and fishes were tunneled into the codend, collected, and frozen on dry ice. Distribution and abundance were expressed as relative abundance, and catch per unit of effort (CPUE) represented the number of fishes per purse-seine collection. In addition, a small number of YOY S. dumerili were collected with hook- and-line for age and growth information only. Standard lengths (SL) were measured to the nearest 0.1 mm, and weights to the nearest 0.1 g before otolith extraction. GPS locations and mat volume (lengthx widthxdepthi were recorded at each sample location. Environmental parameters measured included sea surface temperature, salinity, and dissolved oxygen. Daily sea surface tem- perature data were also taken from NOAA buoy 42035, 22 nmi offshore of Galveston, TX. Otolith procedures Sagittal otoliths were extracted from S. dumerili. Oto- liths were measured to the nearest 0.001 mm and weighed to the nearest 0.0001 g. Left or right sagittae were randomly selected and mounted in epoxy resin (Spurr, 1969). Once mounted, a Buehler isomet low- speed saw equipped with a diamond wafering blade was used to transversely cut embedded otoliths. Otolith sections were then attached to petrographic slides with Crystalbond thermoplastic cement. Type A alumina powder 1 0.3 fim) and 400- and 600-grit sandpaper were used to grind both sides of the otolith, and a polishing cloth was used for final preparations. Age was determined by counting growth increments along the sulcus from the core to the outer margin by using a Nikon Labophot-2 light microscope and Opti- mas 6.2 image analysis software (Media Cybernetics, Silver Spring, MD). Because of the difficulty of enu- merating some inner increments near the otolith core, a relationship between age and otolith radius of several clear specimens was used to predict the number of increments within the unclear region. Age was deter- mined by adding the correction factor to the increment count from the first identifiable increment to the otolith margin (Rooker and Holt, 1997). Correction factors consisting of mure than five days were applied to 499r Wells and Rooker: Distribution, age, and growth of young-of-the-year Senola dumerili 547 of the fishes and the average correction accounted for 9.5% of the actual age estimate. Otolith readings with correction factors accounting for more than 20% of the predicted age were not used for estimates of growth. The following correction factor was used Age (d) = 2.88 x otolith radius (jUm) - 0.096 (r2 = 0.88, n=20). Additionally, all otolith counts were repeated twice to ensure adequate precision. Differences in readings of more than 20% were not incorporated into growth estimates. Daily deposition of growth increments on sagit- tal otoliths was validated by using wild S. dumerili (re=14, 136-193 mm SL). Fishes caught in the wild were brought into the laboratory and placed in a cir- cular holding tank (1.71 m diameterx0.75 m depth) for 48 hours. Fishes were then placed in a separate tank containing 80 liters of seawater with 100 mg/L of alizarin complexone for two hours (Thomas et al., 1995) and returned to the circular holding tank. Individuals were fed approximately 10% of their body weight daily. Fishes marked with alizarin were removed from the tank after 5 (;? = 5), 10 (n=5), and 15 (n=4) days. The number of otolith increments between the alizarin mark and outer edge were then counted for daily increment verification. Otolith slides were coded so that all read- ings were blind. Hatching dates were determined for all individuals by subtracting daily age from date of capture. An age- specific mortality adjustment was made for individuals because larger S. dumerili have spent more time in the early life stages and hence individuals from these cohorts have experienced greater cumulative mortality. Because of the limited number of individuals in 2001, the mortality correction was calculated only for year 2000 collections and applied to hatching-date distribu- tions in 2000 and 2001. Age-specific mortality adjust- ments were made according to the method described by Rooker and Holt (1997). Growth and mortality of S. dumerili were estimated by using otolith-derived ages. Daily growth rates were estimated by using the linear growth equation SL - slope (age) + y-intercept and were reported as mm/d. Length-at-age data were also fitted with curvilinear growth models (von Ber- talanffy, Laird-Gompertz). Percent variation in length explained by age for both curvilinear models was slightly better at times than the percent variation in length explained by age for the linear model; however, certain model parameters (i.e. LJ were biologically unrealistic and thus the linear model was deemed more appropri- ate. Moreover, when possible, L_ values were used to model length-at-age data and the nonlinear models were essentially linear over the limited size range examined. Mortality estimates for year 2000 S. dumerili were determined by using a regression on the decline in log(>- transformed abundance on age. A regression coefficient (slope) was used to predict the instantaneous mortality rate: \r\N, = ln7V0 - Zt, where Nt = abundance at age t (expressed in days); N0 = an estimate of abundance at hatching; and Z (slope) = the instantaneous mortality coefficient. Mortality estimates were based upon 10-day cohort groupings. Individuals <40 days old were not included in the mortality regression because of an ascending catch curve and because there were too few individuals >139 days old in our sample — probably owing to gear avoidance or emigration (or both). Therefore, only S. dumerili between 40 and 139 days (45-192 mm) were used to estimate mortality. Data analysis Effects of location and date on CPUE and size estimates were examined by using a two-way analysis of vari- ance (ANOVA). Levene's test and residual examination established if the homogeneity of variance assumption was met. Normality was evaluated by plotting residuals versus expected values. Abundance data were log (.v+1) transformed when necessary to normalize data and reduce heteroscedasticity. Tukey's honestly significant difference ( HSD ) test was used to determine a posteriori differences among means. Comparisons of spatial and temporal variation in growth were performed by using analysis of covariance (ANCOVA). Prior to ANCOVA testing, the homogeneity of slopes assumption was exam- ined using an interaction regression (Ott, 1993). If no significant interaction was detected, ANCOVA models were used to test for differences in length-at-age (y- intercepts) (Ott, 1993). Statistical analysis was car- ried out by using SYSTAT 8.0 (SYSTAT Software Inc., Richmond, CA), and significance was set at the alpha level of 0.05. Results Environmental conditions Average temperatures from May to July ranged from 27.9 to 30.1°C in 2000 and from 24.5 to 30.4°C in 2001 (Fig. 2). Mean temperatures over the sampling period were 29.2°C and 27.9°C for 2000 and 2001, respectively. Zonal differences occurred: the inshore zone averaged 28.7°C (±0.3) in 2000 and 28.1°C (±0.9) in 2001, and the offshore zone averaged 29.8°C (±0.3) in 2000 and 27.6°C (±0.9) in 2001. Similar to temperature trends, mean salinity was higher in 2000 (34.6%< ) than in 2001 (31.9%o) (Fig. 2). Average salinity values gradually increased from an average of 31.5%o in May to 37.2%r in July of 2000. A large drop in salinity occurred during 548 Fishery Bulletin 102(3) mid-summer of 2001, from 37.6r/ic in May to 25.7'7~-~~^'^ O Salinity a — - — ■ - 35 - 30 25 - ^*<^*~~ - 25 20 - 15 - ^^^^^ - 20 £ 2T - 15 10 - - 10 o 5- - 5 U i i i i i i \j uj 2? Jan Feb Mar Apr May June July = CO ^ CD if 40 - A Temperature n ^UUl "40 i H 35 - A Buoy temperature ^v - 35 30 - O Salinity \. -^^L - 30 25 - Jr-—***^^ - 25 20 - ^^~ - 20 15 - tr-——*^^^ ~ 15 10 - - 10 5 - - 5 0 0 ■ i i i i i Jan Feb Mar Apr May June July Month Figure 2 Environmental conditions from January to July of 2000 and 2001. Average temperature (°C) and salinity ('«) values. Open triangles represent temperature data from NOAA buoy 42035, located 22 nautical miles offshore of Galveston, TX. Hatching-date distribution Hatching-date distributions for S. dumerili were pro- tracted in both 2000 and 2001. Fishes collected in 2000 exhibited hatching-dates from 29 January to 25 May (117 days), whereas those collected in 2001 hatched from 11 January to 30 May (139 days) (Fig. 4). In 2000, over 80% of the fishes appeared to result from spawning events in March and early April. The adjusted distri- butions from the age-specific mortality correction for both 2000 and 2001 were indistinguishable from those without the correction. Age and growth Results of the age-validation exercise indicated that juvenile S. dumerili deposit otolith increments on a daily basis (Fig. 5). Average increment counts at day 5, 10, and 15 were 4.8 (±0.2 SD), 9.2 (±0.4), and 14.0 (±0.7), respectively. A relationship between the observed versus expected increments was described by the follow- ing equation: Observed increments - 0.92 (expected increments) + 0.14 (r2=0.95) where days after staining represent expected increment count. Wells and Rooker: Distribution, age, and growth of young-of-the-year Senola dumenli 549 Validation of daily growth increments has been ob- served in a similar study involving juvenile (0-60 days) Seriola quinqueradiata (Sakakura and Tsuka- moto, 1997). Age of S. dumerili was similar between years; es- timated ages ranged from 41 to 150 days (35 to 210 mm SL) in 2000 and from 35 to 120 days (33 to 198 mm SL) in 2001 (Fig. 6). Interannual differences in growth were observed: 2000 (1.65 mm/d). 2001 (2.00 mm/d) (ANCOVA, slope, P<0.001) (Fig. 7). A signifi- cant cohort effect was also observed; the late-season (April) cohort experienced the fastest growth (ANCO- VA, slopes, P<0.001) (Fig. 8). Average cohort-specific growth rates of S. dumerili spawned in February, March, and April of 2000 were 0.85 mm/d, 1.15 mm/d, and 2.76 mm/d, respectively. In addition, a signifi- cant difference in growth was observed for S. du- merili collected from inshore (1.55 mm/d) and offshore (1.65 mm/d) zones of 2000 (ANCOVA, slope, P<0.001) (Fig. 9). Again, the lack of individuals within the in- shore zone in 2001 precluded a comparison between zones for that year. Mortality Owing to the limited number of S. dumerili collected in 2001, a single catch curve was developed for the 2000 year class, and the mortality coefficient (Z) was 0.0045 (0.45%/d) for individuals between 40 and 139 days (Fig. 10). Cumulative mortality was estimated for the 100-day period (40-139 days), resulting in an overall mortality of 36%. 20 15 10 5 0 1 A □ 2000 ■ 2001 MAY JUNE JULY 40 " UJ 30 - B □ Offshore ■ Inshore z> CL O 20- T 10 - o- 1 MAY JUNE JULY o - 8 - 6 - c 4 J 2 " U □ Offshore ■ Inshore r^i^ MAY JUNE Months JULY Figure 3 Relative abundance (number per purse seine) (±1 SE) of S. dumerili collected in association with Sargassum mats: (A) 2000 and 2001: (B) 2000 by zones; (C) 2001 bv zones. Discussion The size range of S. dumerili collected in association with Sargassum ranged from approximately 30 to 210 mm (SL), and these sizes are similar to those reported in other studies investigating fish assemblages associated with pelagic Sargassum. Bortone et al. (1977) collected several small S. dumerili (12-72 mm SL) in the eastern Gulf, whereas individuals collected in the western Atlan- tic by Dooley (1972) ranged from 13 to 108 mm (SL). Cho et al. (2001) found juvenile S. dumerili (35-120 mm TL) associated with drifting Sargassum in the western Pacific. Additionally, Sakakura and Tsukamoto (1997) collected over 200 juvenile Japanese amberjack (S. quin- queradiata) (18-114 mm TL) associated with pelagic Sargassum in the East China Sea. Results of the present study and others indicate that pelagic Sargassum mats in the NW Gulf of Mexico serve as nursery habitat for S. dumerili. The limited size range of S. dumerili associated with pelagic Sargassum indicates that a shift in habitat use may occur at approximately 5-6 months of age. Indi- viduals greater than 210 mm (SL) have not been found in association with pelagic Sargassum, and larger S. dumerili (ca. 300 mm TL) are relatively common in the recreational headboat fishery in the Gulf of Mexico (Ma- nooch and Potts, 1997a). As a consequence, S. dumerili may transition from a pelagic to a demersal existence at the late juvenile stage (between 200 mm SL and 300 mm TL). Pipitone and Andaloro (1995) found a shift in the diet of S. dumerili, from a diet predominately consisting of crustaceans toward one of fish >200 mm (SL), further supporting this hypothesis. Seriola dumerili abundance was greater in the off- shore zone than the inshore zone throughout the sam- pling period. These patterns of habitat use are consis- tent with earlier information that indicates S. dumerili is an offshore species (Hildebrand and Cable, 1930). The proximity to spawning grounds may contribute to the observed spatial patterns because S. dumerili are known to spawn in offshore areas (Fahay, 1975). Physi- ological preferences may also contribute to the domi- nance of S. dumerili in the offshore zone. In our study, salinity values were higher in the offshore zone but more variable within the inshore zone, suggesting that freshwater inflow influences conditions within the in- shore zone. Chen et al. (1997) determined that optimum salinity conditions for S. dumerili larvae were between 32% l- i- CM 2001 T- T- C\J t- r> ■* *- CO Rl <- CM t- *- CN CO CO CO Hatching date Figure 4 Hatch-date distributions of S. dumerili associated with Sargassum mats in 2000 and 2001. 16 i £ 14 - / & f* / 12 - / 0 CD S/ E 10 - tft/ cd '/rV CJ ''/ £ 8 - '/ ° o CD c a ° - ty E &y 3 Z 4 - / Increments (expected) 2 - / O Increments (observed) u I I I I I I 1 1 0 2 4 6 8 10 12 14 16 Days after staining Figure 5 Linear regression of age verification for S. dumerili. Circles and solid line represent the number of daily increments observed after staining, and dotted line represents the number of increments expected. attributed the distribution of fishes from nearshore to offshore stations to environmental factors, season, and life history strategies. Furthermore, the combination of available resources (i.e. food and habitat), seasons, and physiochemical tolerances may account for the observed spatial patterns of habitat use. Temporal patterns of size-specific habitat use showed similar trends between years and appeared to be relat- ed to spawning season. Relative abundance of small S. dumerili was highest early in the season (May), declined in June, and further increased late into the season (July) for both 2000 and 2001. Nevertheless, small juve- niles were collected during the entire collection period, which suggests that S. dumerili spawning in the NW Gulf is protracted. Previous studies have found that S. dumerili spawn throughout the spring and summer months ( March- July) (Marino et al., 1995; Cummings and McClellan, 1996). In addition, Fahay (1975) sug- gested, on the basis of larval collections in the western Atlantic, that spawning occurs in the winter. Despite the limited duration of our collection efforts, our results are consistent with these findings with 63% of year- 2000 S. dumerili and 36f> of year-2001 fish resulting from spring spawning events. The remaining individu- als were spawned January through early March. Growth estimates indicated that S. dumerili have rapid growth throughout early life stages. Based on linear growth models, average growth of S. dumerili was 1.45 mm/d — an estimate similar to that of Manooch and Potts's (1997b) study in the Gulf (average growth of 1.17 mm/d for age-1 individuals). However, growth com- parisons may be invalid because their study estimated growth based on counts of annuli and no temperature Wells and Rooker: Distribution, age, and growth of young-of-the-year Senolo dumerili 551 data were presented. Because of the lack of studies investigating growth of YOY S. dumerili, we compared our estimates to those in Sakakura and Tsukamoto's (1997) study of YOY S. quinqueradiata where growth rates were estimated at 1.3 mm/d. Average temperature in their study was 21.2°C, which was considerably lower than the average during our study (28.6°C) and may account for their slower growth rates. Variation in growth of S. dumerili was observed and rates were significantly higher in the offshore zone and greater for the late season cohort. Differences in water temperature may be partly responsible for ob- served differences in growth. Planes et al. (1999) sug- gested that spatial differences in growth of juvenile sparid fishes were a result of water temperature and currents. The proximity between zones in this study may have masked differences in hydrography; however, temperatures were higher in the offshore zone (29.8°C, CV=0.03) than in the inshore zone (28.7°C, CV=0.04), and warmer temperatures were likely contributing to faster growth rates in offshore waters. Intra-annual (cohort-specific) growth patterns indicated that the late- season cohort had the fastest growth. Similar to trends between zones, temperature was lowest for the slowest growing cohort (early season) and highest for the fast- est growing cohort (late season). Although temperature may affect early life growth of S. dumerili, differences in growth may be attributed to other factors such as prey availability and predator activity (Houde, 1987; Paperno et al., 2000; Plaganyi et al., 2000). Moreover, a clear distinction exists in the size classes of YOY S. dumerili in comparisons of growth rates and these dif- ferences likely contribute to the observed results. The mortality rate of YOY S. dumerili associated with pelagic Sargassum was estimated at 0.45 %/d for fishes 4-1 2- 0- 8" 6" 2000 4- 2 r ...M„. - f " .-d-Lo-n 2001 mm ^ w n n Age class (days) Figure 6 Age-frequency distribution of S. dumerili collected in association with Sargassum in 2000 and 2001. collected in 2000. These findings are well below similar studies investigating mortality of YOY individuals. Nelson (1998) calculated a mortality estimate of 2.1-2.3^/d for pinfish in three different bay areas in the eastern Gulf of Mexico. In addition, Deegan (1990) estimated YOY men- haden mortality between 1.7 and 2.1'S/d in the northern 250" ,' a 200" A t&y^u D E A°£a$ $ffi^° ~X 150 - a Lt^PSjaKi- CT J^r 0> ■Vx D "D /y ffl 100 - /? "D C ns CO *P D 50 - /Z& fSS -B- 2000 SL= 1.65Mge)-15.33 r2=0.86 -A- 2001 Si. = 2.00(/lc;e)-37.32 r = 0 95 0 1 ' ' ' ' 0 20 40 60 80 1 00 Age (days) Figure 7 120 140 160 Age-length relationship of S. dumerili determined with linear growth curves for inters nnual comparison. 552 Fishery Bulletin 102(3) 250 " Standard length (mm) O en O o o o s.*£ o a/ -&- February SU 0 85(4ge)+65.12 MT° i2 = 0.34 50 - Djfl Van ~°~ March SL = 1 1 5(4ge)+40 69 r = 0.55 -a— April SL=2 76(Age)-80 48 ^=0 95 1 ' I t i 0 20 40 60 80 100 120 140 160 Age (days) Figure 8 Age-length relationship of S. dumerili determined with linear growth curves for a com parison of cohorts of different hatching dates in 2000. 250 " 200 - E P s' / y / a _□ 0 n & B}flO Standard length ( en o oi o o o ^JSsS —*- Inshore SL = 1 55{Age)-\2 11 'rffi' f = 0.74 -a- Offshore SL = 1.65(/*ge)-15.33 o ■ ^ = 086 0 20 40 60 80 100 120 140 160 Age (days) Figure 9 Age-length relationship of S. dumerili determined with linear growth curves for a comparison of zones in 2000. Gulf. These studies included estuarine-dependent spe- cies and consisted of smaller individuals. Because our estimates were limited to age 40-139 d individuals, the lack of smaller fishes precluded any mortality estimates of younger S. dumerili. These estimates provide baseline information on mortality of YOY S. dumerili; however, more detailed studies will be needed to adequately de- termine mortality rates of YOY S. dumerili. Based on observed patterns of distribution and growth in the NW Gulf of Mexico, early life survival of S. dumerili may depend on pelagic Sargassum. Results of this study suggest that S. dumerili are associated with this habitat over a limited size range and exhibit rapid growth during the first six months. Addition- ally, S. dumerili were more abundant and exhibited higher growth in offshore areas where potential spawn- ing may occur. Thus, Sargassum appears to provide nursery habitat for YOY S. dumerili, and may influ- ence the recruitment potential of this valuable fishery- species. Wells and Rooker: Distribution, age, and growth of young-of-the-year Senola dumerili 553 Log.W = 1 506-0 0045(4ge) x r =0.315 "i i.o - CD ■D C CD * 0.5 - en O _l ♦ ♦ O OO OOOOOOOO o O OOO i-c\jcr>"^-mcDr--.oocr)oi-CMco-^-ir) Age (days) Figure 10 Mortality curve of S. dumerili based upon regression plot of loge abundance on age of individuals collected in 2000. Acknowledgments We thank J. Harper, M. Lowe, B. Geary, J. Turner, and J. Wells for their assistance in the field. Fund- ing for this project was provided by The Aquarium at Moody Gardens (grant 479005 to JRR). Top Hatt char- ters provided boat time offshore, and Kirk Winemiller and Jaime Alvarado offered constructive criticism and suggestions. Literature cited Bortone, S. A.. P. A. Hastings, and S. B. Collard. 1977. 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Stud. 36:146-158. 555 Identification of formalin-preserved eggs of red sea bream iPagrus major) (Pisces: Sparidae) using monoclonal antibodies Shingo Hiroishi Yasutaka Yuki Eriko Yuruzume Faculty of Biotechnology Fukui Prefectural University 1-1 Gakuen-cho Obama City, 917-0003 Fukui, Japan E-mail address (for S Hiroishi), hiroishi@fpu.ac.|p Yosuke Onishi Tomoji Ikeda Hironobu Komaki Kansai Environmental Engineer Center 1-3-5 Azuchi-cho, Chuo-ku Osaka City, 541-0052 Osaka, Japan Muneo Okiyama Ocean Research Institute University of Tokyo 1-15-1 Minamidai, Nakano-ku, Tokyo, Japan Catches of important commercial fish such as red sea bream, fiat fish, and yellowtail are decreasing in Japan. In order to sustain these species it is especially important that their distri- bution and biomass at all life stages are known. However, information on the early life stages of these species is limited because identifying the eggs and larvae of such fish is sometimes extremely difficult. Mito (1960, 1979) and Ikeda and Mito (1988) developed methods for identifying pelagic fish eggs based on morphological features. However, their methods have limitations because many unidentified eggs have similar features. In addition, eggs are usu- ally fixed in formaldehyde solution just after collection in the field. This procedure may alter several egg char- acteristics and therefore prevent iden- tification (Ikeda and Mito, 1988), or make identification difficult when the egg diameter measures 0.8-1.0 mm because so many kinds of eggs fall in that range. Thus, an alternative iden- tification method would be useful. Effective genetic analyses for iden- tifying fish eggs or larvae (or both) have been developed by Graves et al. (1989), Daniel and Graves (1994), and Shao et al. (2002). However, their methods may have limitations if samples are preserved in formal- dehyde for several years or if DNA must be extracted from numerous samples. In addition, we are lacking the DNA sequences for many species sequences that are necessary for iden- tifying eggs in the field. We have successfully produced monoclonal antibodies to differenti- ate harmful marine phytoplankton species from morphologically simi- lar harmless species (Hiroishi et al., 1988; Nagasaki et al., 1991b; Sako et al., 1993; Vrieling et al., 1993; Hiroishi et al., 2002) as well as Microcystis, a toxic fresh water bloom-forming cyanobacteria (Kondo et al., 1998). These antibodies were obtained from a culture supernatant solution of hybridoma cells that was produced by a cell fusion procedure between myeloma cells and antibody- producing spleen cells. The specific antibodies described above could be used to detect and quantify harmful bloom-forming microorganisms that react with the monoclonal antibod- ies and that secondarily react with fluorescein isothiocyanate conjugated goat anti-mouse Ig(G+M) antibody. With fluorescence microscopy with B-exciting light, yellowish fluorescein coronas around the cells of the toxic species were observed, confirming a positive reaction. These antibodies can recognize different molecules distributed on the cell surface, even when the organisms have similar morphological features. One of the molecules distributed on Chattonella was determined to be glycoprotein (Nagasaki et al., 1991a). This method would help us to differentiate small marine organisms like fish eggs. Red sea bream (Pagrus major) (Table 1) eggs can easily be distin- guished from those of other sparids also found in Japan, such as Acan- thopagrus latus, by differences in egg size and spawning seasons, and from those of Evynnis japonica by differ- ences in spawning seasons (Ikeda and Mito, 1988; Kinoshita, 1988; Hayashi. 2000). However, eggs of some sparids, such as Aeanthopagrus schlegeli, Sparus sarba, and Dentex tumifrons are extremely difficult to distinguish from eggs of P. major. Therefore, we developed monoclonal antibodies that allow P. major eggs to be clearly identified by immuno- staining, thus differentiating them from other similar sparids. This technique may be a useful new tool for identifying fish eggs. Here, we report a method for identi- fying P. major eggs using monoclonal antibodies developed to react specifi- cally with the eggs. Materials and methods Eggs of P. major were obtained from adult female fish that had spawned in Manuscript submitted 4 April 2003 to Scientific Editor's Office. Manuscript approved for publication 2 March 2004 by the Scientific Editor. Fish. Bull. 102:555-560 (20041. 556 Fishery Bulletin 102(3) Table 1 Characteristics of Sparidae distributed in Japan. Egg oil globule Suborder Species Distribution Spawning season Egg size (mmi size (mm) Pagrinae Pagrus major South of Hokkaido I Coastal) Mar-May 0.90-1.03 0.19-0.25 Evynnis jappon ica South of Hokkaido (Coastal) Oct-Dec 0.89-0.98 0.19-0.21 Sparinae Acanthopagrus schlegi li South of Hokkaido (Coastal) Mar-Jun 0.83-0.91 0.20-0.22 Acanthopagrus latus South Japan (Coastal) Oct-Nov 0.76-0.81 0.2 Sparus sarba South Japan (Coastal) Apr-Jun 0.88-0.92 0.19-0.22 Denticinae Dentex tumifrons South Japan (Oceanic) May-Jun 0.90-0.93 0.19 isolation tanks at several sea farming centers described in Table 2. Immediately after collection, fish eggs were fixed in a solution of 57c formaldehyde to sea water solu- tion and stored. Before use, the eggs were thoroughly washed with distilled water and suspended in phosphate buffered saline (PBS) solution. Monoclonal antibodies were developed according to the methods of Kdhler and Milstein (1975), Garfre and Milstein (1981), and Hiroishi et al. (1984, 1988): 0.5 mL of egg suspension (200 eggs/PBS solution from Fukui Prefectural Sea Farming Center, Obama City, Fukui Prefecture) was mixed with 0.5 mL Freund's complete adjuvant (Nacalai Tesque, Inc., Kyoto, Japan). The mix- ture were then injected subcutaneously into BALB/c female mice (4 weeks of age). The female mice received second and third injections at 2-week intervals. For the final immunization, P. major eggs collected in the sea farming center of Kansai Environmental Engineering Center Co. (Miyazu City, Kyoto, Japan) were injected into the mouse after being emulsified with Freund's incomplete adjuvant (Nacalai Tesque, Inc.). Three days after the final immunization, the spleens of the mice were removed and passed through a mesh (mesh size: 100 urn). The spleen cells obtained by this procedure were fused with the myeloma cell line X63-AG8.653 at a ratio of 10:1 with 50% polyethylene glycol. After cell fusion, hybrid cells were incubated in a selective hypoxanthine-aminopterin-thymidine medium (Kohler, 1979; Garfre and Milstein, 1981). The reactivity of the antibodies produced by the hy- bridomas was then determined. Eggs fixed with 57c formaldehyde in seawater were washed with PBS solu- tion in a 96-well plate. Throughout the experiments, the principal eggs used were from the Fukui Prefec- tural Sea Farming Center. Normal horse serum solution (200 jUL), diluted 100-fold with PBS, was added to the wells to prevent any nonspecific reactions. After incuba- tion at room temperature for 20 minutes, the eggs were washed with 200 pL of PBS. After removing the PBS, 200 fih of the hybridoma culture supernatant solution was added to the wells and incubated at room tempera- ture for 30 minutes. After washing with PBS (100 /jL), biotinylated horse anti-mouse IgG (100 pL) was added to the wells and incubated at room temperature for 20 minutes. After the incubation, VECTASTAINR ABC re- agent (avidin DH + biotinylated horseradish peroxidase/ PBS, 100 jjL) was added according to the direction of VECTASTAINR Elite ABC kit (ABC Mouse IgG Kit, Fu- nakoshi Co., Tokyo, Japan). After immunostaining the eggs were observed by stereoscopic microscopy ( SMZ-2T, Nikon Co., Tokyo, Japan). In a positive reaction, the surface of the fish egg was stained brown as a result of the oxidation of 3,3'-diaminobenzidine (substrate) by horseradish peroxidase bound to the egg surface by the antibody. Unidentified pelagic fish eggs from open water were collected by using a plankton net (MTD net, NGG54 with mesh size of 0.344 mm, Rigo Co., Tokyo, Japan) from Wakasa Bay (Fukui Prefecture, Japan) in May 1997. They were fixed with 59c formaldehyde in sea water, either immediately or after incubation in seawa- ter in finger bowls at 20°C for 24 hours, and identified by careful observation as described by Ikeda and Mito (1988) and Ikeda et al. (1991). The fixed eggs were transferred to net wells (mesh size 200 ^m, diameter 24 mm, Corning Incorporated, Corning, NY) and washed with 10 mL of distilled water three times. Then the eggs in the netwells were immersed in 100 mL of PBS in a polystyrene tray (Corning Incorporated, Corning, NY) for 5 minutes. The egg suspension was placed into the wells of a six-well plate and incubated with 10 mL of normal horse serum solution for 20 minutes. After incubation, the eggs were incubated with 10 mL of MT-1 antibody solution (hybridoma culture supernatant) and then incubated with 10 mL of biotinylated horse anti- mouse IgG. The subsequent procedure was performed as described above. The immunoglobulin subclass of monoclonal antibod- ies was determined according to the directions of the mouse monoclonal antibody isotyping kit (Amersham Pharmacia Biotech Co., Uppsala, Sweden) as follows: 3 mL of monoclonal antibodies solution (hybridoma supernatant solution) obtained in this study was added to 0.3 mL of horseradish peroxidase-conjugated anti- mouse IgG in the kit. An isotyping stick in the kit was incubated with the above solution at room tern- NOTE Hiroishi et al.: Identification of Pagus ma/or eggs using monoclonal antibodies 557 perature for 15 minutes. Then the stick was washed with 0.1% Tween 20/PBS, and incubated with 4-chloro-l-naphthol solution (substrate of horseradish peroxi- dase in the kit) containing 0.1% H202 at room temperature for 15 minutes. The immunoglobu- lin subclass of the monoclonal antibodies was determined by observing the positions of bands that appeared on the stick. Results and discussion After cell fusion, hybridomas were grown in 42 wells of 96- well plates. Supernatant solu- tions of the cultures were used for the immunostaining assay to select hybridomas producing antibodies reactive to P. major eggs. After the assay, positive reactions were observed in six wells. These hybridomas were cloned by the limiting dilution method, and finally three clones producing monoclonal antibod- ies reactive with P. major were obtained. Those antibodies were named MT-1, MT-2, and MT-3. The subclass of all antibodies was IgGj. Specificity of the anti- bodies was examined by using the eggs shown in Table 2. As a result, the antibodies were reac- tive with all the P. major eggs in both the early and late stages (before or after tail-bud stage), but not with eggs of other species (Table 3, Fig. 1). Thus, it becomes possible to identify P. major eggs. The immunostaining assay took 2.5 hours. The oldest eggs of P. major (20 April, 1995) could react with the antibodies obtained as clearly as the recently collected eggs of P. major, indicating that egg samples preserved for up to 7 years could be analyzed by this method. The method was also success- ful with 102 eggs collected from Wakasa Bay (Table 4), which had been immediately fixed with 5% formaldehyde in seawater. Among them, only 11 eggs were identified as Callionymoidei spp. P1 CO m co OS CO C35 CO 1a en CD 05 CO en CD cn cn cn jn cn bo c k O) P5 >. S-. -. >i >. >> 05 >* >. > 3 3 CO 34 >! 3 CO >-3 k. en >, CO s "o. a. < < c 3 c 3 CO CO -. c 3 CO -. CO co C 3 cO CO 0 2 ■-3 jn 4^3 a, < CO 0. < e o ■-3 >-5 ,_, ^_( ■-3 ^_, ,_( cn !-J 0) -. ,_( 1 u 0 ~ i-H CO CO CO cm 4J bo bo 01 _ffl £ TJ c C — c <4_ o B CO 0 S ; B CO 0 Co _>, >, _>. _>> _>> _>. ^ _>> _>> B B _>> >. c bo CO "k CD -4-i "C CO 4J c ■~ ■~ - ^ CO 4-> - T^ CO 4-3 ^ CO 4J -i! 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CO s §■ ? 3 CO -g ^ CO X "~ 3 3 CO k c c k. C3 k. bo cs cs CS cr k bo 53 0 co D. CO e a, < 1: ay Q a, K3 6 c bo bo T—t CM CO ■* in CD i> CO 05 S 1— 1 CN CO -0* m ■r t- X cn r i—l fa CM CS 558 Fishery Bulletin 102(3) (type II). The remaining 91 unidentified eggs were di- vided into three groups (types I, III, and IV) based on diameters. Of these 91, 51 type-II eggs reacted with MT-1. This finding is compatible with the possibility that the eggs were P. major, because the size was simi- lar to that of P. major and each contained a single oil globule of a similar size (Tables 1 and 4). Another 43 eggs were collected from another area of Wakasa Bay (Table 5). None of the eggs fixed just after collection Table 3 Reactivity of monoclonal antibodies to fish eggs. + repre- sents positive reaction; - represents negative reaction. Reactivity Egg no. Species MT-1 MT-2 MT-3 1 Pagrus major + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 Acanthopagrus schlegeli - 10 - 11 - 12 - 13 - 14 Acanthopagrus latus - 15 Sparus sarba - 16 Dentex tumifrons - 17 Paralichthys olivaceus - 18 - 19 - 20 - 21 Engraulis japonica - were morphologically identifiable. But, after incuba- tion at 20°C for 24 hours until the late stage, all six eggs identified as P. major were reactive with the an- tibody MT-1, whereas the others were not. These find- ings strongly suggest that the method developed in this study is useful for identifying P. major eggs in seawater. Although only late stage eggs were used in this experi- ment, early stage eggs are also detectable because the antibody recognized both stages of P. major eggs from several sea farming centers (Table 2). Compared to genetic analysis of fish eggs, this method has the advantage of being able to assay many eggs simultaneously without the need to separate individual eggs in tubes and without extracting DNA from the in- dividual egg in each tube. Further, this method works with formalin-fixed eggs, whereas extraction of DNA from formalin-fixed material is problematic. Plankton samples from field studies are typically fixed in forma- lin-seawater solution. There was no problem obtaining a large amount of the monoclonal antibody required when identifying P. major eggs. The antibody can be easily obtained by large-scale cultures of hybridoma cells. About 50 mL of antibody solution was obtained after two weeks of cultivation. There was no technical problem assaying 43 or 102 eggs from natural waters. However, one assay of a field sample cost about 20 U.S. dollars. To keep costs down an assay kit cheaper than the VECTASTAINR Elite ABC kit is needed when a large number of field samples are analyzed. Acknowledgments We would like to thank the following sea farming centers and universities for providing the fish eggs used in this study: Fukui Prefectural Sea Farming Center; Kyoto Prefectural Sea Farming Center; Faculty of Agriculture, Kyushu University; Osaka Prefectural Fisheries Station; Sea Farming Center of the Japan Sea-Farming Associa- tion; Fisheries Laboratory of Kinki University. We also thank Jeffrey M. Leis, Australian Museum, Sydney, Australia, for his kind advice during the writing of this manuscript. Table 4 Reactivity of monoclonal antibody MT-1 to the pelagic eggs fixed with formaldehyde just after collection from Wakasa Bay. O.G. diameter = oil globule diameter Fish egg type Egg diameter imm) O.G. diameter imm) Reactivity (%) ( positive egg no./ total egg no.) I II III IV 0.72-0.79 0.75-0.82 0.81-1.02 1.07 0.16-0.19 no oil globule 0.19-0.28 0.21 0(0/2) 0(0/11) 58(51/88) 0(0/1) NOTE Hiroishi et al.: Identification of Pagus ma/or eggs using monoclonal antibodies 559 Figure 1 Reactivity of monoclonal antibody MT-1 to fish eggs detected by immunos- taining. (A) Pagrus major (positive reaction); (B) Pagrus major (negative control reaction without primary antibody); (C) Acanthopagrus shlegeli; (D) Sparus sarba; (E) ParaUchthys olivaceus; (F) Engraulis japonica. Bar represents 1 mm. Only the P. major eggs stained brown and showed a positive reaction. Table 5 Reactivity of monoclonal antibody MT-1 to the pel agic eggs reared for 24 hours after collection from Wakasa Bay. Egg Species Reactivity (%) (positive egg no. /total egg no.) Fish Pagrus major Acanthopagrus shlegeli ParaUchthys olivaceus Triglidae sp. Konosirus punctutus Soleoidei sp. Englauris japonicus 100(6/6) 0(0/8) 0(0/1) 0(0/1) 0(0/2) 0(0/7) 0(0/13) Decapod Enploteuthidae sp. 0(0/5) 560 Fishery Bulletin 102(3) Literature cited Daniel III, L. B„ and J. E. Graves. 1994. Morphometric and genetic identification of eggs of spring-spawning sciaenids in lower Chesapeake Bay. Fish. Bull. 92:254-261. Garfre, G., and C. Milstein. 1981. Preparation of monoclonal antibodies: strategies and procedures. In Methods in enzymology (J. J. Lan- gone and H. V. Vunakis, eds.), vol. 73, p. 3-46. Academic press, New York, NY. Graves, J. E. M. J. Curtis, P. S. Oeth, and R. S. Waples. 1989. Biochemical genetics of southern California basses of the genus Paralabrax: specific identification of fresh and ethanol-preserved individual eggs and early larvae. Fish. Bull. 88:59-66. Hayashi, K. 2000. Sparidae. In Fishes of Japan with pictorial keys to the species (second ed.), T. Nakabo (ed.), p. 857-858. Tokai University Press, Tokyo. [In Japanese.] Hiroishi, S., S. Matsuyama, T. Kaneko, Y Nishimura, and J. Arita. 1984. Inhibition of cytotoxicity for screening a monoclo- nal antibody to HLA antigen. Preparation of a highly specific monoclonal antibody to HLA antigen. Tissue Antigens 24:307-312. Hiroishi, S., R. Nakai, H. Seto,, T Yoshida, and I. Imai. 2002. Identification of Heterocapsa circularisquama by means of antibody. Fisheries Sci. 68:627-628. Hiroishi, S., A. Uchida, K. Nagasaki, and Y. Ishida. 1988. A new method for identification of inter- and intra-species of the red tide algae Chattonella antiqua and Chattonella marina (Raphidophyceae) by means of monoclonal antibodies. J. Phycol. 24:442-443. Ikeda, T, S. Chuma, and M. Okiyama. 1991. Identification of pelagic eggs of marine fishes by rearing method. Jap. J. Ichthyol. 38:199-206. [In Japanese.] Ikeda, T, and S. Mito. 1988. Identification of eggs and hatched larvae. In An atlas of the early stage fishes in Japan (M. Okiyama, ed.), p. 999-1119. Tokai Univ. Press, Tokyo. [In Japanese.] Kinoshita, I. 1988. Sparidae. In An atlas of the early stage fishes in Japan (M. Okiyama, ed.), p. 527-536. Tokai University Press, Tokyo. ]In Japanese.] Kohler, G 1979. Fusion of lymphocytes. In Immunological methods (I. Lefkovits and B. Pernis, eds.), p. 391-395. Academic press, New York. Kohler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting anti- body of predefined specificity. Nature (Lond.) 256: 495-497. Kondo, R., G. Kagiya, Y Yuki, S. Hiroishi, sand M. Watanabe. 1998. Taxonomy of a bloom-forming cyanobacterial genus Microcystis. Nippon Suisan Gakkaishi 64:291-292. [In Japanese.] Mito, S. 1960. Keys to the pelagic fish eggs and hatched larvae found in the adjacent waters of Japan. The Science Bulletin of the Faculty of Kyushu University 18:71-94, pis. 2-17. [In Japanese.] 1979. Fish egg. Kaiyokagaku 11:126-130. [In Japanese.] Nagasaki, K, A. Uchida, S. Hiroishi, and Y. Ishida. 1991a. An epitope recognized by the monoclonal antibody MR-21 which is reactive with the cell surface of Chat- tonella marina type II. Fish. Sci. 57:885-890. Nagasaki, K, A. Uchida, and Y. Ishida. 1991b. A monoclonal antibody which recognizes the cell sur- face of red tide alga Gymnodinium nagasakiense. Fish. Sci. 57:1211-1214. Sako, Y, M. Adachi, Y Ishida, C. Scholin, and D. M. Anderson. 1993. Preparation and characterization of monoclonal antibodies to Alexandrium species. In Toxic phytoplank- ton blooms in the sea (T. J. Smayda and Y. Shimizu, eds.) p. 87-93. Elsevier, New York, NY. Shao, K.-T, K.-C. Chen, and J.-H. Wu. 2002. Identification of marine fish eggs in Taiwan using light microscopy, scanning electric microscopy and mt DNA sequencing. Mar. Freshw. Res. 53:355-365. Vrieling, E., A. Draaijer, L. Van Zeiljl, W. Gieskes, and M. Veenhuis. 1993. The effect of labeling intensity, estimated by realtime confocal laser scanning microscopy, on flow cytometric appearance and identification of immuno- chemical labeled marine dinoflagellates. J. Phycol. 29:180-188. Fishery Bulletin 102(3) 561 Superintendent of Documents Publications Order Form *5178 YES, please send me the following publications: Subscriptions to Fishery Bulletin for $55.00 per year ($68.75 foreign) The total cost of my order is $ . . Prices include regular domestic postage and handling and are subject to change. 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Addiliona! copies may be purchased in lots of 100 when tin author receives page proofs. tftf^oi U.S. Department of Commerce Volume 102 Number 4 October 2004 — c o ■~- 3 S «> «• & c «■ 2o ° S. ■«=»■ C3 CD -> CSJ O* M H- CJ ■ o —■» Fishery Bulletin U.S. Department of Commerce Donald L. Evans Secretary National Oceanic and Atmospheric Administration Vice Admiral Conrad C. Lautenbacher Jr., USN (ret.) Under Secretary for Oceans and Atmosphere National Marine Fisheries Service William T. Hogarth Assistant Administrator for Fisheries Sr4T£S 0* The Fishery Bulletin I ISSN 0090-0656) is published quarterly by the Scientific Publications Office, National Marine Fish- eries Service, NOAA, 7600 Sand Point Way NE, BIN C15700, Seattle. WA98115-0070. Periodicals postage is paid at Seattle. WA, and at additional mailil POST- MASTER: Send address changes tor sub- source is appreciated. The Secretary ol e has deter- mined that the publication of this pi ri odical is necessary according to law for the tr:i: of this f funds lor printing of this periodical has been approved h\ the i ol the Office of Management and Budget. For sale by the Superintendent of Documi ment Printing Office, Washington, DC 20402. Sub lion price per year: $55.00 domes! i foreign. I ingle issue: $28.00 domestic and $35.00 foreign. See back for order form. Scientific Editor Norman Bartoo, PhD Associate Editor Sarah Shoffler National Marine Fisheries Service, NOAA 8604 La Jolla Shores Drive La Jolla, California 92037 Managing Editor Sharyn Matriotti National Marine Fisheries Service Scientific Publications Office 7600 Sand Point Way NE, BIN C15700 Seattle, Washington 981 15-0070 Editorial Committee Harlyn O. Halvorson, PhD Ronald W. Hardy, PhD Richard D. Methot, PhD Theodore W. Pietsch, PhD Joseph E. Powers, PhD Harald Rosenthal, PhD Fredric M. Serchuk, PhD George Waiters, PhD University of Massachusetts, Boston University of Idaho, Hagerman National Marine Fisheries Service University of Washington, Seattle National Marine Fisheries Service Universitat Kiel, Germany National Marine Fisheries Service National Marine Fisheries Service Fishery Bulletin web site: www.fishbull.noaa.gov The Fishery Bulletin carries original research reports and technical notes on investigations in fishery science, engineering, and economics. It began as the Bulletin of the United States Fish Commission in 1881; it became the Bulletin of the Bureau of Fisheries in 1904 and the Fishery Bulletin of the Fish and Wildlife Service in 194 1 Separates were issued as documents tl volume 46; the last document was No. 1103. Beginning with volume -17 m 1931 and continuing through volume 62 in 1963, each separate appeared as a numbered bulletin A new system began in 1963 with volume 63 in which papers are bound together in a single issue of the bulletin. Beginning with volume 70, number 1. January 1972, the Fishery Bulletin became a periodical, issued quarterly In this form, it is available by subscription from the Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402. It is also available free in limited numbers to libraries, research institutions, State and Federal agencies, and m exchange for other scientific publications. U.S. Department of Commerce Seattle, Washington Volume 102 Number 4 October 2004 Fishery Bulletin Contents Articles 563-580 Calambokidis, John, Gretchen H. Steiger, David K. Ellifrit, Barry L. Troutman, and C. Edward Bowlby Distribution and abundance of humpback whales (Megaptera novaeangliae) and other marine mammals off the northern Washington coast 581-592 Danilewicz, Daniel, Juan A. Claver, Alejo L. Perez Carrera, Eduardo R. Secchi, and Nelson F. Fontoura Reproductive biology of male franciscanas (Pontoporia bloinvillei) (Mammalia: Cetacea) from Rio Grande do Sul, southern Brazil 593-603 Fischer, Andrew J., M. Scott Baker Jr., and Charles A. Wilson Red snapper (Lut/anus campechanus) demographic structure in the northern Gulf of Mexico based on spatial patterns in growth rates and morphometries The conclusions and opinions expressed in Fishery Bulletin are solely those of the authors and do not represent the official position of the National Marine Fisher- ies Service 'NOAAi or any other agency or institution. The National Marine Fisheries Service NMFS i does not approve, recommend, or endorse any proprietary product or pro- prietary material mentioned in this pub- lication- No reference shall be made to NMFS, or to this publication furnished by NMFS. in any advertising or sales pro- motion which would indicate or imply that NMFS approves, recommends, or endorses any proprietary product or pro- prietary material mentioned herein, or which has as its purpose an intent to cause directly or indirectly the advertised product to be used or purchased because of this NMFS publication. 604-616 FitzGerald, Jennifer L, Simon R. Thorrold, Kevin M. Bailey, Annette L. Brown, and Kenneth P. Severin Elemental signatures in otoliths of larval walleye pollock (Theragra chalcogramma) from the northeast Pacific Ocean 617-633 Gaughan, Daniel J., Timothy I. Leary, Ronald W. Mitchel and Ian W. Wright A sudden collapse in distribution of Pacific sardine (Sardinops sagax) off southwestern Australia enables an obiective re-assessment of biomass estimates 634-647 Griffiths, Shane P., Ron J. West, Andy R. Davis, and Ken G. Russell Fish recolonization in temperate Australian rockpools: a quantitative experimental approach Fishery Bulletin 102(4) 648-660 Hesp, S. Alexander, Ian C. Potter, and Sonja R. M. Schubert Factors influencing the timing and frequency of spawning and fecundity of the goldlined seabream (Rhabdosargus sarba) (Spandae) in the lower reaches of an estuary 661-670 Maxwell, Michael R., Annette Henry, Christopher D. Elvidge, Jeffrey Safran, Vinita R. Hobson, Ingrid Nelson, Benjamin T. Tuttle, John B. Dietz, and John R. Hunter Fishery dynamics of the California market squid (Loligo opalescens), as measured by satellite remote sensing 671-681 Murray, Kimberly T. Magnitude and distribution of sea turtle bycatch in the sea scallop (Placopecten magellamcus) dredge fishery in two areas of the northwestern Atlantic Ocean, 2001-2002 682-692 Snover, Melissa L, and Aleta A. Hohn Validation and interpretation of annual skeletal marks in loggerhead (Caretta caretta) and Kemp's ridley (Lepidochelys kempii) sea turtles 693-710 Stehlik, Linda L, Robert A. Pikanowski, and Donald G. McMillan The Hudson-Raritan Estuary as a crossroads for distribution of blue (Callmectes sapidus), lady (Ovalipes ocellatus), and Atlantic rock (Cancer irroratus) crabs 711-722 Stevens, Melissa M., Allen H. Andrews, Gregor M. Cailliet, Kenneth H. Coale, and Craig C. Lundstrom Radiometric validation of age, growth, and longevity for the blackgill rockfish (Sebastes melanostomus) 723-732 Tolan, James M., and David A. Newstead Descriptions of larval, preiuvenile, and |uvenile finescale menhaden {Brevoortia gunteri) (family Clupeidae), and comparisons to gulf menhaden (6. patronus) 733-739 Uchikawa, Kazuhisa, John R. Bower, Yasuko Sato, and Yasunori Sakurai Diet of the minimal armhook squid (Berryteuthis anonychus) (Cephalopoda: Gonatidae) in the northeast Pacific during spring 740-749 Weinberg, Kenneth L, Robert S. Otto, and David A. Somerton Capture probability of a survey trawl for red king crab (Paralithodes camtschaticus) Notes 750-756 Kerstetter, David W., Jeffery J. Polovina, and John E. Graves Evidence of shark predation and scavenging on fishes equipped with pop-up satellite archival tags 757-759 Vladimir V. Laptikhovsky Survival rates of rays discarded by the bottom trawl squid fishery off the Falkland Islands 760 Acknowledgment of 2004 reviewers 761 2004 indexes 770 Subscription form 563 Abstract— We examined the summer distribution of marine mammals off the northern Washington coast based on six ship transect surveys conducted between 1995 and 2002, primarily from the NOAA ship McArthur. Additionally, small boat surveys were conducted in the same region between 1989 and 2002 to gather photographic identification data on humpback whales iMegap- tera novaeangliae) and killer whales (Orcinus orca) to examine movements and population structure. In the six years of ship survey effort. 706 sight- ings of 15 marine mammal species were made. Humpback whales were the most common large cetacean spe- cies and were seen every year and a total of 232 sightings of 402 animals were recorded during ship surveys. Highest numbers were observed in 2002, when there were 79 sightings of 139 whales. Line-transect estimates for humpback whales indicated that about 100 humpback whales inhab- ited these waters each year between 1995 and 2000; in 2002, however, the estimate was 562 (CV=0.21) whales. A total of 191 unique individuals were identified photographically and mark- recapture estimates also indicated that the number of animals increased from under 100 to over 200 from 1995 to 2002. There was only limited inter- change of humpback whales between this area and feeding areas off Oregon and California. Killer whales were also seen on every ship survey and represented all known ecotypes of the Pacific Northwest, including southern and northern residents, transients, and offshore-type killer whales. Dall's porpoise iPhocoenoides dalli) were the most frequently sighted small ceta- cean; abundance was estimated at 181-291 individuals, except for 2002 when we observed dramatically higher numbers (876, CV=0.30i. Northern fur seals (Callorhinus ursinus) and elephant seals iMirounga angustiros- tris) were the most common pinnipeds observed. There were clear habitat differences related to distance off- shore and water depth for different species. Manuscript submitted 25 September 2003 to the Scientific Editor's Office. Manuscript approved for publication 4 June 2004 by the Scientific Editor. Fish. Bull. 102:563-580 i2004). Distribution and abundance of humpback whales (Megoptera novaeangliae) and other marine mammals off the northern Washington coast John Calambokidis Gretchen H. Steiger David K. Ellifrit Cascadia Research Collective Waterstreet Building 218V2 West Fourth Ave. Olympia, Washington 98501 E-mail address (for J Calambokidis) calambokidis@cascadiaresearch.org Barry L. Troutman Washington Dept of Fish and Wildlife 600 Capitol Way Olympia, Washington 98501 C. Edward Bowlby Olympic Coast National Marine Sanctuary, NOAA 115 Railroad Ave E, Suite 301 Port Angeles, Washington 98362 Marine mammals have had an impor- tant role in the history of the Olympic Peninsula for centuries. Many species, including sea otters ( En hydra lutris). harbor seals iPhoca vitulina), hump- back whales (Megaptera novaean- gliae), and gray whales iEschrichtius robustus) were hunted by the Makah tribe (Swan, 1868; Huelsbeck, 1988). Much later, modern whalers targeted humpback whales in this region from stations at Bay City, Washington (1911-25, Scheffer and Slipp, 1948). and southern Vancouver Island, Brit- ish Columbia (1905-43, Gregr et al., 2000). A small aboriginal hunt for gray whales resumed in these waters in 1998, and the Makah killed one gray whale in May 1999. Since the end of commercial whaling, marine mammals have been afforded protec- tion under the Marine Mammal Pro- tection Act of 1972. In addition, the waters off the northern Washington coast were designated as the Olympic Coast National Marine Sanctuary in 1994. A number of studies have docu- mented marine mammals in this re- gion. Some surveys of broader areas have included the waters off north- ern Washington (Von Saunder and Barlow, 1999; Brueggeman1; Green et al.2). Species-specific studies also 1 Brueggeman, J. J. 1992. Oregon and Washington marine mammal and sea- bird surveys. Final report of OCS Study MMS" 91-0093 by Ebasco Envi- ronmental, Bellevue, Washington, and Ecological Consulting, Inc., Portland. Oregon, for the Minerals Management Service (MMS), 445 p. MMS, Pacific OCS Region, U.S. Dept. of Interior. 770 Paseo Camarillo, Camarillo. CA 93010. 2 Green, G. A., M. A. Smultea, C. E. Bowlby. and R. A. Rowlett. 1993. Delphinid aerial surveys in Oregon and Washing- ton offshore waters. Final report for contract 50ABNF200058 to the National Marine Mammal Laboratory, National Marine Fisheries Service, 100 p. Nat. Mar. Mamm. Lab., NMFS, 7600 Sand Point Way NE F/AKC3, Seattle, WA 98115.1 564 Fishery Bulletin 102(4) have been conducted on harbor porpoise iPhocoena pho- coena; Barlow et al., 1988; Osmek et al., 1996; Calam- bokidis et al.3) and, to a limited degree, on humpback whales ( Calambokidis et al., 1996, 2000) and gray whales (Darling, 1984; Green et al., 1995; Shelden et al., 2000; Calambokidis et al., 2002). Studies on pin- nipeds and sea otters have also been conducted in this region (Jeffries et al., 2003; Jameson et al., 1982, 1986; Kvitek et al. 1992, 1998; Bowlby et al.4). Information on humpback whales is of particular interest because they were the primary species hunted by whalers off Washington in the early 1900s. Since then, little has been known about their movements and distribution in this region. Photo-identification research has helped define the movements and stock structure of the humpback whales feeding off California (Calamboki- dis et al., 1990. 1996, 2000). Calambokidis et al. (1996) suggested that a demographic boundary exists between humpback whales that feed off the coasts of California, Oregon, and Washington and humpback whales feeding farther north off British Columbia and Alaska. The identity and degree of interchange of the whales that feed in this boundary area have been unclear. Similarly for killer whales, photo-identification stud- ies have revealed much about whale groups that fre- quent the inland waters of Washington and British Columbia (Bigg et al., 1990; Ford et al., 1994). Very little is known about their occurrence off the coast, in particular, about the "offshore" groups that are believed to be a distinct race (Ford et al., 1994) that are seen primarily offshore but occasionally also enter inland waterways. We report here on the summer distribution of marine mammals off the northern Washington coast based on six ship line-transect surveys conducted between 1995 and 2002. These surveys were initiated to understand marine mammal distribution and abundance in the newly designated Olympic Coast National Marine Sanc- tuary, as well as to collect information on seabirds, oceanographic conditions, and juvenile fish. Each ship survey was conducted between mid-June and late July. Density estimates were made for the two most common species: humpback whales and Dall's porpoise. In ad- dition, photo-identification data gathered during these ship surveys and from supplemental small boat surveys :i Calambokidis, J., J. C. Cubbage, J. R. Evenson, S. D. Osmek, J. L. Laake, P. J. Gearin, B. J. Turnock, S. J. Jeffries, and R. F. Brown. 1993. Abundance estimates of harbor porpoise in Washington and Oregon waters. Report to the National Marine Mammal Laboratory. National Marine Fisheries Ser- vice, 55 p. Nat. Mar. Mamm. Lab., NMFS, 7600 Sand Point Way NE F/AKC3, Seattle, WA 98115. 4 Bowlby, C. E., B. L. Troutman, and S. J. Jeffries. 1988. Sea otters in Washington: distribution, abundance, and activ- ity patterns. Final report to National Coastal Resources Research and Development Institute, Hatfield Marine Sci- ence Center, 2030 S. Marine Dr., Newport, Oregon 97365, 131 p. Cascadia Research Collective, Wash. State Dept. of Wildlife, Olympia, WA. within the same area between 1989 and 2002 provided information on humpback and killer whale movements and stock structure. Materials and methods Ship surveys Generally, ship surveys covered the area between the 20-m isobath and the landward margin of the continental shelf i200-m isobath) from the entrance to Strait of Juan de Fuca to the mouth of the Copalis River to include the boundaries of the Olympic Coast National Marine Sanc- tuary (Fig. 1). Although the northern extent of these waters is off southern British Columbia (Vancouver Island), the entire overlapping region will be referred to as northern Washington. Fourteen east-west tracklines were selected, follow- ing permanent tracklines established by the NOAA ship Miller Freeman in 1989. Tracklines were spaced at 5-nmi intervals and were surveyed each year ex- cept in 2002, when only ten lines were surveyed (four southernmost lines were not included). Extra ship time allowed for replicate surveys of the northern survey legs in 1995, a short offshore extension of two lines in 1996 and 2000 (up to 17 nmi in 1986), the addition of three short east-west lines off southern Vancouver Island around La Perouse Bank in 1997, and one ad- ditional line that was surveyed south of the study area in 2000 (Fig. 1). Ship surveys were conducted over a two-week period in late-June and July 1995, 1996, 1997. 1998, and 2000 (Table 1). In 2002, a shorter, one-week survey was done in mid-June. The marine mammal ship surveys were conducted by a single primary observer from the vessel's flying bridge (the sighting platform) with a viewing height of 10 m above the water level. All surveys were conducted from the NOAA ship Mc Arthur (55 m) except during 2000, when the naval ship Agate Passage (33 m) was used. From these platforms, the primary observer scanned a 180-degree arc encompassing the area ahead of the ship and abeam to either side. Observers used reticle binoculars when possible and obtained measure- ments of distance to a sighting derived from the angle below the horizon (measured with graded reticles in the binoculars) and the known platform height. For sight- ings where the species could not be determined by the observer, animals were identified to a general taxonomic level (e.g., unidentified pinniped). Photo-identification surveys In addition, photo-identification data were examined that had been gathered within the survey area. Research- ers took photographs directly from the survey ship, or from a Zodiac rigid-hulled inflatable that was launched when animals were sighted. In 1996, the last two days of vessel time on the McArthur were used to photograph whales for identification. Calambokidis et al.: Distribution and abundance of marine mammals off the northern Washington coast 565 1 26°0'0"W 1 125:'0'CrW I Vancouver Island Barkley Sound N 3 lines only in 1997 La Perouse Bank t Swiftsure Bank . Strait of Juan de Fuca Cape Flattery Olympic Peninsula Sanctuary Boundary Figure 1 On-effort ship survey tracklines (horizontal lines) off the northern coast of Wash- ington between 1995 and 2002. The Olympic Coast National Marine Sanctuary boundary is delineated and labeled. Dashed and dotted lines show three northern lines surveyed only in 1997, the western extension of two lines surveyed only in 1996, and the southern four lines missed in 2002. In addition, dedicated photo-identification surveys were conducted by Cascadia Research scientists us- ing a 5.3-m Novurania rigid-hulled inflatable that was launched from nearby ports and operated in areas where whales were concentrated. Photo-identification data in the present study includes data collected off the northern Washington coast between 1989 and 2002 (Table 2). It also includes photographs contributed by other researchers and boat operators taken in the area during this time (Table 2). Generally, photographs were taken with Nikon 8008 35-mm cameras equipped with 300-mm Nikkor telepho- to lenses. High-speed black-and-white film (Ilford HP 5+) was pushed IV2 stops so that exposure times were generally 1/1000 or 1/2000 of a second. Identification photographs were taken with standard procedures used 566 Fishery Bulletin 102(4) Table 1 Summa ry of ship survey effort off northern Washin gton (does not include small boat surveys). Dates of effort No. of nmi on Year Start End legs Effort (h) effort Ship Observers 1995 21 Jul 27 Jul 10 46 546 Mc Arthur Troutman. Ellifrit 1996 28 Jun 5 Jul 14 46 540 Mc Arthur Troutman, Ellifrit 1997 9 Jul 18 Jul 17 52 513 Mc Arthur Troutman, Ellifrit 1998 25 Jun 4 Jul 14 55 572 McArthur Troutman, Quan 2000 16 Jun 24 Jun 14 60 589 Agate Passage Rowlett, Nelson 2002 12 Jun 18 Jun 10 32 315 McArthur Troutman, Douglas All years 291 3075 Table 2 Photo-identification effort off the coast of northern Washington bet ween 1989 and 2002. These data include whales identified from the ship or small boats launched from the sh ip, dedicated small boat surveys, and opportunistic photographs taken by others. Unique = number of different animals. Days IDs obtained Humpback whales identified Other sources of photographs Year No First Last No. Unique No. of mothers No of calves 1989 1 lOct 1 Oct 1 1 0 0 1990 3 25 Aug 6 Sep 10 10 1 1 Balcomb/Bloedel' 1991 4 23 Aug 4 Sep 14 13 0 0 Balcomb/Bloedel' 1993 1 15 Jul 15 Jul 3 3 0 0 1994 3 25 Jun 15 Jul 20 16 0 0 G. Ellis.- R. Baird 1995 7 14 Jul 25 Jul 50 35 4 2 S. Mizroch3 1996 9 29 Jun 6 Oct 55 34 1 0 1997 9 13 Jul 18 Oct 25 23 2 0 1998 19 28Mav 16 Oct 71 48 1 1 V. Deeke, B. Gisborne 1999 28 20 May 20 Oct 103 60 2 0 B. Gisborne 2000 12 2 Jun 4 Oct 56 40 2 1 B. Gisborne 2001 15 8 Jun 5 Oct 59 41 2 1 SWFSC.J B. Gisborne 2002 9 13 Jun 5 Sep 41 32 0 0 Total 120 508 356 15 6 Unique 191 ; Center for Whale Research, P.O. Bo> 1577, Fn day Harboi , WA 98250. 2 Dept. of Fisheries and Oceans, Pacific Biological Station Nanaimo, BC , V9T 6N7, Canada. 3 National Marine Mammal Laboratory, NMFS 7600 Sand Point Way NE, Seattle, WA 98115. 4 Southwest Fisheries Science Center 8604 La lolla Shores Dr., La Jolla CA 92037. in past research (Calambokidis et al., 1990). For hump- back whales, photographs were taken of the ventral side of the tail flukes. For killer whales, the dorsal fin and surrounding saddle-patch area were photographed from both sides. Photographs of individuals were first compared to those identified in the same region. To analyze inter- change with other regions, we compared these individu- als with existing catalogs to obtain sighting histories. For humpback whales, a catalog was used of over 1000 humpback whales identified since 1986 along the West Coast. The regions used for comparison were Oregon, northern California (Oregon-California border to Pt. Arena i, northern central California (Pt. Arena to north of Monterey Bay), southern central California (north of Monterey Bay to Pt. Conception! and southern Cali- fornia (southern California Bight). For killer whales, whales were matched to existing catalogs (Bigg et al., 1987; Ford et al., 1994; Black et al., 1997). All iden- tifications and group determinations were confirmed by one of the authors tDKEi or Graeme Ellis (Dept. of Fisheries and Oceans. Nanaimo. British Columbia). Calambokidis et al.: Distribution and abundance of marine mammals off the northern Washington coast 567 Data analysis For ship surveys between 1995 and 2000, position and oceanographic data (including depth, sea surface tem- perature) logged by the ship's computer were later rec- onciled with the sighting and effort data recorded by the observers. Sighting positions were analyzed for each species for water depth, distance from shore, distance from shelf edge (200-m depth contour) and sea surface temperature. Data analysis and mapping were conducted by using a geographic information system (GIS) with Arclnfo software (ESRI, Redlands, CA). Data from the shorter 2002 ship survey were included in the summary of sightings but were not available for the analyses of sightings related to oceanographic features. Line-transect analysis to determine density and abun- dance was conducted for the two species with more than 30 sightings (humpback whales and Dall's porpoise). We used the program (Distance, version 3.5, Research Unit for Wildlife Population Assessment, University of St. Andrews, St. Andrews. UK) to conduct analyses. For these analyses, we used only effort and sightings from the regular east-west transect lines and did not include on-effort data from opportunistic lines or cross- tracks. We included sightings made by secondary as well as the primary observer. Although whales were reportedly seen out to 6 nmi, we truncated the sight- ings at 3 nmi for humpback whales and 2.5 nmi for Dall's porpoise. For humpback whales we included 16 sightings of unidentified whales (unidentified mainly because of distance). These were probably humpback whales because the only other large whales that were seen in the surveys were a few gray whales seen close to shore. Distance position data were incomplete for 13 of the 188 whale sightings and 14 of 82 Dall's porpoise sightings; for these the missing value was randomly selected from the observed measurements. The Distance program was used to select the best model for sighting probability in relation to distance off the transect. We allowed the program to select among models (half-normal, uniform, hazard-rate, and nega- tive exponential) and varying numbers of adjustment terms (cosine and simple polynomials) based on lowest Akaike's information criterion (AIC) score. All years were pooled for the model of sighting probability, but encounter rate and group size were calculated by year. An adjustment to group size was calculated if there was a significant group size bias with distance from the track line, which was not the case for humpback whales but was present in some years (1996 and 1997) for Dall's porpoise. Area was calculated for abundance estimation based on the zone covered by the regularly scheduled transect lines covered in most years (study area was considered to encompass waters 2.5 nmi north of the northernmost line and 2.5 nmi south of the southernmost line). The only annual adjustment for area was for humpback whales in 2002. Surveys in that year did not cover the southern end of the study area (because of limited ship time), an area with a typically lower abundance of whales. To avoid extrapolating the higher density of whales from the northern portion of the study area to this region, we excluded this missed area from the abundance estimates. Estimates of abundance for humpback whales were also calculated by using capture-recapture models (Se- ber, 1982; Hammond, 1986). We used identifications obtained in pairs of adjacent years taken from 1994 to 2002 to generate Petersen capture-recapture estimates. The Chapman modification of the Petersen estimate (Seber, 1982) was used because it was appropriate for sampling without replacement (Hammond, 1986). Results In total, there were 706 sightings of 2467 animals over the six ship surveys combined (Table 3). Fifteen differ- ent marine mammal species were seen: nine cetacean species, five pinniped species, and the sea otter were identified. Each year, 9 to 12 different species were seen, except in 2002 when only six species were observed. This 2002 survey, although shorter than those of the other years, showed a dramatic change in the species diversity and numbers of animals. We saw many more humpback and Dall's porpoise than in previous years. We also noted the absence of six regularly observed spe- cies: harbor porpoise, gray whales, Pacific white-sided dolphins (Lagenorhynchus obliquidens), Risso's dolphin (Grampus griseus), harbor seals, and California sea lions iZalophus californianus). Humpback whales Of the large cetaceans, humpback whales were the most common species seen; there were 232 sightings of 402 animals during ship surveys (Table 3). Largest numbers of humpback whales were seen in 2002, when there were 79 sightings of 139 individuals during the one- week survey. Group sizes ranged from 1 to 8 animals (mean=1.7, SD=1.1). Only six calves were recorded from the ship surveys — probably because it was difficult to identify calves at the distance at which most sightings were made. Of these six sightings of mothers with calves, four sightings were outside the primary areas where other humpback whale groups were seen. Sightings were concentrated in the northern part of the study area between Juan de Fuca Canyon and the outer edge of the continental shelf, an area known as "the Prairie" (Fig. 2). A small area east of the mouth of Barkley Canyon and north of the Nitnat Canyon where the water depth was 125-145 m had a high density of sightings in all years. A smaller number of humpback whales were also seen on Swiftsure Bank. Sightings in 2002 were not only more numerous but more broadly distributed; sightings were recorded in the areas de- scribed above and also farther south and closer to shore than those seen in previous years. 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CD *j a-g -R ^3^5 = '3 £ CD "C3 CD U w 3 " ~ CO ^S 3 CD c CD -a 3 ■a k o S D •§ a X cC z cs fcc; d 3 ac a O 0Q Z 00 D cn pq o £ H Calambokidis et al.: Distribution and abundance of marine mammals off the northern Washington coast 569 Figure 2 Locations (by year) for humpback whales (Megaptera novaeangliae) seen during ship surveys off the northern Washington coast between 1995 and 2002. 2000, Table 4, Fig. 3). The encounter rate of groups (0.046-0.053 sightings per nmi), density (0.034-0.050 whales per nmi2), and abundance (85-125 individuals) were similar among these years. These data indicate that about 100 humpback whales used the study area during this period. The sighting rate of humpback whales was dramati- cally higher in 2002 than in all previous years and was reflected in the line-transect estimates (Fig. 3). Estimated density (0.23 whales per nmi2) was more than four times higher than any previous year. Apply- ing this density to only the reduced area surveyed in 2002 (1953 instead of 2505 nmi2) still yielded much higher estimates of abundance (562, CV=0.21) than in any previous year. These higher abundance estimates could not have been an artifact of random variation; the lower bound of the 95% confidence interval for the 2002 estimates was well above the upper confidence interval of any of the previous years (Table 4). Of the humpback whales photographed during small boat surveys off the northern Washington-BC border between 1989 and 2002, 508 individuals were success- 570 Fishery Bulletin 102(4) Table 4 Results of line-transeet analysis for h umpback whales off northern Wash ngton. C n-effort sight ngs of b umpback and un- identified large whales made during regular transects ( not including deadheads [areas between transect lines and opportunistic sightings) within 3 nm of ship were used. Best detection model fit (AIC scores) was a negative exponential w th 1 cosine adjust- ment yielding /'l 0) = 1.0E . Effect ve strip width was 0.95 nmi w ith CV=0.09. Survey effort 953 Conf. int. Sightings Encounter Group Density Area Estimated Year /! lines nmi rate size (per nmi2) (nmi2) abundance CV lower upper 1995 23 58 438 0.053 1.48 0.041 2505 102 0.33 54 193 1996 24 59 474 0.051 1.54 0.041 2505 103 0.33 55 193 1997 26 92 493 0.053 1.62 0.045 2505 112 0.3 63 199 1998 20 62 432 0.046 1.40 0.034 2505 85 0.31 47 155 2000 23 70 504 0.046 2.09 0.050 2505 125 0.32 67 234 2002 72 43 305 0.236 1.81 0.224 1953 562 0.21 375 841 Total 188 384 2646 fully identified of which 191 were unique individuals i Table 2). Of these 191, 83 (44%) had been seen in this area in more than one year within this time period. The proportion of animals seen more than one year changed over the course of the study ( Fig. 4). The proportion of whales identified each year that had been seen in others years decreased annually (Fig. 4, regression r- = 0.63, P=0.002); the most dramatic drop occurred between 1998 and 1999. Photographs of humpback whales documented animal movements within the study area and provided some insight into possible reasons for the high sighting rates during the 2002 ship surveys. On two occasions, the same humpback whale was identified on different days in a slightly different area and represented a duplicate 600 -1 • •♦- ■ Line-transect ♦ — ■ — Capture-recapture 500 ■ ,' 400 - .' O § 300- c 200 - 100 - ■ m '-•♦---' ■ ' l<)')4 IW5 1996 1997 1998 1999 2000 2001 .' 2 2003 Year Figure 3 Line-transect Idashed line i and capture-recapture (solid line) estimates for humpback whale iM. noracannliae) abundance between 1995 and 2002. sighting of this animal from the ship survey. It is pos- sible that shifting humpback whale distribution during the course of the 2002 survey could have occurred in a manner that resulted in the same animals being encountered multiple times and that elevated the sight- ing rate and line-transect abundance estimate (Fig. 3). We cannot test this hypothesis because other animals may have shifted in a manner that they avoided being detected at all. Abundance of humpback whales from capture-re- capture models yielded estimates of 89 to 343 whales (Table 5, Fig. 3). These estimates tended to increase over the course of the study from a low of 89 whales for 1994-95 to a high of 343 for 2000-2001 and 230 for 2001-2002 (regression r2 = 0.60, P=0.02). There was fairly good agreement between the capture-re- capture and line-transect estimates until 2002 (Fig. 3). A total of 17 of the 191 (9%) whales that we identified off northern Washington had also been photographed off California and Oregon (Table 6). Interchange of whales seen off north- ern Washington and other feeding areas to the south decreased as distance among feeding ar- eas increased. About 10% (10 of 105) of the whales that were identified off Oregon were also photographed off northern Washington. This rate of matching dropped below 3% (8 of 313) off northern California and continued to decrease to no interchange seen for whales pho- tographed off southern California. The proportion of whales that were seen in areas to the south appeared to change over the course of the study. From 1989 to 1998, when resighting rates between years within our study area were highest, we also had a higher proportion of interchange with feeding areas to the south (13 of 109 whales or 12%). From 1999 to 2002, after resightings within our region decreased, there was also a decrease Calambokidis et al.: Distribution and abundance of marine mammals off the northern Washington coast 571 Table 5 Estimates of humpback Each estimate was base whale abundance d on the identificat Est ions ) off northern Washington obtained obtained (n) in each of two adjacent with the Petersen years. capt are-recapture model. Period Sample 1 Sc mple 2 Match Est. CV Year n Year n 1994- -95 1994 14 1995 35 5 89 0.27 1995- -96 1995 35 1996 34 11 104 0.19 1996- -97 1996 34 1997 21 7 95 0.24 1997- -98 1997 23 1998 48 6 167 0.28 1998- -99 1998 48 1999 60 13 213 0.19 1999- -2000 1999 60 2000 31 14 129 0.16 2000- -01 2000 40 2001 41 4 343 0.36 2001- -02 2001 41 2002 32 5 230 0.32 in the proportion of these whales that had also been seen off California and Oregon (7 of 136 whales or 5%). This difference falls just short of statistical significance (j2 = 3.71, P<0.10) but is in the reverse direction from what would be expected if immigration from the south were to increase over time. Between 1989 and 2002, 15 different mothers were seen with 16 calves (one mother seen with a calf in two different years). Mothers with calves represented 4.2% of the individual whales iden- tified each year ( 15 of 356 unique annual iden- tifications. Table 2). For each year only a small proportion of the calves were identified because calves raise their flukes less often. Killer whales One other large cetacean species (killer whales) was also seen every year; there were a total of 14 sightings of 124 animals from ship surveys (Table 3). Three of these sightings were of large groups between 20 and 35 animals, and the rest were in groups fewer than ten (14 sight- ings, mean = 8.9, SD = 11.2). Killer whales were widely distributed across different habitats; there were sightings of animals both close to and far from shore and in fairly shallow and deep water (Fig. 5). All three ecotypes of killer whales (namely, 1) southern and northern residents, 2) transients, and 3) offshore residents) were observed off the northern Washington coast. Of the 15 groups identified pho- tographically between 1989 and 2002, there were sightings of animals from the southern resident (2 groups), northern resident (3), transient (5) and off- shore (3) groupings (Table 7). Other sightings appeared to be northern residents (1) and offshore (1) animals but the quality of the photographs were too poor for ■B 80* V*^— -5 >* ** A* ** ** <** * Olympic 48'0'0"N- + \* 4 "* * • * Peninsula * AA± A • ** A A • A* * ** "1 » + * £* T * ** + * * \ • +++ t A 1 * * -H- ■H- + N Sanctuary Boundary 47 O'ff'N- 1 1 r- Figure 6 Locations of small cetaceans seen during ship surveys off the northern Washington coast between 1995 and 2002. cally yielding an estimated abundance of 876 porpoises (CV=0.30, Table 8). Confidence intervals for some of the annual estimates overlapped among years. of 28 occasions. All but one of these sightings were of a single animal. Elephant seals were seen in all years except 1998 and 2002. Pinnipeds Pinnipeds were not as frequently observed as cetaceans • Table 3, Fig. 7). The two most pelagic species observed in this region, northern fur seals and elephant seals, were the most commonly seen pinnipeds. Northern fur seals were observed every year except 2002 on a total Habitat differences A number of broad habitat patterns emerged for differ- ent groups of species based on their association with water depth and distance from shore during the ship surveys from 1995 to 2000 (Table 9, data were not avail- able for 2002). Five species were seen in shallow waters Calambokidis et at: Distribution and abundance of marine mammals off the northern Washington coast 575 125=0'0"W I Vancouver Island Barkley Sound Pinnipeds • Northern Fur Seal ■ Northern Sea Lion * Sea Otter a Elephant Seal + Harbor Seal ♦ California Sea Lion La Perouse Bank Strait of I Juan de Fuca Cape Flattery Olympic Peninsula 1 Sanctuary Boundary Figure 7 Locations of pinnipeds and sea otters {Enhydra lutris) seen during ship surveys off the northern Washington coast between 1995 and 2002. (<100 m). Gray whales and sea otters were seen in the shallowest water of all species with average water depths of just 20 and 22 m. respectively; they also were the only two species for which sightings averaged less than 10 km from shore. The three other species — harbor porpoise, California sea lions, and northern sea lions (JEumetopias jubatus) — were seen in slightly deeper waters (averag- ing 34 to 91 m) and farther from shore (averaging 11 to 23 km). The five species that were predominantly found at mid-shelf depths (mean depths at 100-200 m) were humpback whales, killer whales, Dall's porpoises, harbor seals, and minke whales (Balaenoptera acutorostrata). Species seen far from shore (>40 km) and also in deepest waters (>200 m) included Pacific white-sided dolphins, Risso's dolphins, elephant seals, and northern fur seals. All of these species are known to feed along the conti- nental slope or off the shelf. Distances from the shelf break for different species did not fall into as clear a pattern as water depth and distance from shore (Table 9). This disparity may be the result of the varied habitat (with canyons cutting through the study area) and the lack of much effort off 576 Fishery Bulletin 102(4) Table 9 Summary of habitat and oceanograph ic parameters for si jhtings of different species during ship surveys from 1995 to 2000. Distance fr om Distance from Sea surface Species Water depth iml shore (km I shelf l km) temp. (°C) n Mean SD n Mean SD 7f Mean SD n Mean SD Baleen whales Humpback whale 153 144 87 153 43.8 14.9 153 8.4 6.7 101 13.9 1.6 Gray whale 5 20 8 5 5.0 2.0 5 26.1 8.1 5 14.4 1.9 Minke whale 3 106 67 3 41.2 27.7 3 8.0 6.5 3 16.1 0.9 Unidentified large whale 21 189 280 21 40.5 18.4 21 8.0 7.3 18 15.4 1.3 Unidentified whale 1 197 — 1 36.3 — 1 0.1 — 1 13.0 — Odontocetes Dall's porpoise 90 167 118 90 40.1 14.9 90 5.6 5.5 72 14.3 1.7 Harbor porpoise 38 58 70 38 16.3 15.6 38 17.2 11.6 29 13.9 1.7 Pacific white-sided dolphin 24 689 505 24 65.6 25.7 24 8.3 8.7 20 15.0 0.8 Northern right-whale dolphin 1 259 — 1 16.2 — 1 0.7 — Risso's dolphin 9 552 310 9 55.4 21.4 9 4.9 5.2 8 14.4 1.3 Killer whale 12 148 58 12 28.8 15.0 12 5.9 4.7 7 14.1 1.1 Unidentified delphinid 19 219 253 19 37.4 17.4 19 5.7 6.7 19 14.5 1.5 Pinnipeds and otters Harbor seal 15 102 154 15 17.3 11.0 15 15.5 12.0 14 14.2 1.4 Elephant seal 20 466 370 20 46.2 18.5 20 3.8 5.0 16 14.7 1.8 California sea lion 4 91 74 4 22.8 15.2 4 9.3 14.2 1 13.9 — Steller sea lion 4 34 18 4 11.3 5.4 4 18.5 6.6 3 13.6 0.4 Northern fur seal 22 382 349 22 47.1 17.1 22 3.1 3.7 21 14.3 1.4 Sea otter 3 22 1 3 8.9 0.5 3 25.5 18.1 3 12.6 0.4 Unidentified pinniped 13 170 144 13 30.5 18.4 13 8.0 8.1 11 14.5 1.9 All sightings 457 205 251 457 39.1 20.1 457 8.4 8.4 352 14.3 1.6 the continental shelf. Despite most of our effort being on the continental shelf, the presence of several deep can- yons in addition to the shelf edge, resulted in all species being an average of less than 11 km from the 200 m depth contour. The average surface water temperature for species that were seen also varied and was likely both a function of distance from shore and association with upwelling areas (Table 9). Sea otters were seen in the coldest waters (12.6°C) where they are predominant- ly found. Among the more offshore species, humpback whales, tended to be seen in colder waters (13.9°C) than most other offshore species, probably because of their association with offshore upwelling areas. Discussion Although humpback whales were the most abundant large cetacean seen in our study, their numbers of a few hundred still appear to be substantially lower than numbers found prior to whaling. Commercial hunting of humpback whales occurred in the 1900s from coastal whaling stations in northern California, Washington, and British Columbia. In these areas, thousands of humpback whales were killed over a relatively short time period (less than 10 years) before catches dropped precipitously with the depletion of the population. At the south end of our study area, 1933 humpback whales were taken from a station at Bay City (in Grays Harbor), Washington, from 1911 to 1925 (Scheffer and Slipp, 1948). To the north, 5638 humpback whales were taken from British Columbia stations from 1908 to 1967, of which 60f'f (3393) were taken from 1908 to 1917 from the two southernmost whaling stations on Vancouver Island closest to our study area (Gregr et al., 2000; Nichol et al., 2002). Additionally, 1871 humpback whales were taken from two stations in northern California from 1919 to 1926 (Clapham et al., 1997). Although these hunts encompassed areas larger than our study area, the number killed in short periods dwarfs even the sum of our abundance estimates for Washington and British Columbia and the estimate of under 1000 whales esti- mated in the 1990s for California. Oregon, and Wash- ington (Calambokidis and Barlow, 2004). Moreover, humpback whales have not returned to some of the areas where they were once found prior to commercial Calambokidis et al.: Distribution and abundance of marine mammals off the northern Washington coast 577 whaling; humpback whales were commonly observed in the inside waters of Washington and British Columbia (Scheffer and Slipp, 1948; Webb, 1988) and have not returned to these areas in any numbers (Calambokidis and Steiger, 1990). The distribution of humpback whales within our study area was not uniform and indicated that some specific areas were important feeding habitat for this recovering species. The region between the Juan de Fuca Canyon and the shelf edge (the Prairie) — the mouth of Bark- ley Canyon and Swiftsure Bank — was the area where humpback whales were concentrated. In monthly aerial surveys in 1989-90 by Green et al.,5 there were only a total of 13 sightings of 25 humpback whales along the entire Washington coast between July and September. Over half of those sightings were in the Prairie area. Our line-transect estimates revealed that about 100 humpback whales inhabit the northern Washington coast waters each summer; substantially more (over 500), however, were present in 2002. Although this is a small number compared to estimates of just under 1000 humpback whales for California, Oregon, and Wash- ington (Calambokidis and Barlow, 2004), our study area encompasses a relatively small area and reflects a high density of animals. Additionally our line-transect estimates were not corrected for any missed animals; therefore they are probably biased slightly downward. Despite the relatively high density of humpback whales in this region, the photographic identification data indicated that a relatively small number of indi- viduals use the area consistently. Both the line-transect and the photographic identification data (increasing capture-recapture estimates, as well as decreased pro- portions of animals sighted multiple years) showed that the number of whales using this region has increased in recent years. The growing number of whales in this re- gion could be either the result of births or immigration into this area. Births alone could not account for this in- crease, especially because the proportion of whales that were mothers with calves seen in this region was not high. There did not appear to be a shift in distribution of animals from areas to the south because interchange with those areas dropped from 1999 to 2002. The most likely explanation for these changes is that there was a shift of animals from feeding areas from the north into this region beginning in the late 1990s. This interchange of humpback whales with feeding ar- eas to the south provides new insight into the structure of humpback whale feeding aggregations. In a study that examined interchange rates of humpback whales 6 Green, G. A., J. J. Brueggeman, R. A. Grotefendt, and C. E. Bowlby. 1992. Cetacean distribution and abundance off Oregon and Washington, 1989-1990. In Oregon and Washington marine mammal and seabird surveys (J. J. Brueggeman, ed. I, 100 p. Final report of OCS Study MMS 91-0093 by Ebasco Environmental, Bellevue, Washington, and Ecological Consulting, Inc., Portland, Oregon, for the Minerals Management Service, Pacific OCS Region, U.S. Dept. of Interior, 770 Paseo Camarillo, Camarillo, CA 93010. along the west coast, Calambokidis et al. (1996) iden- tified northern Washington as a demographic bound- ary between the whales feeding area along California, Oregon, and Washington and those to the north. The larger sample reported here shows the same general pattern of decreasing interchange with distance from a feeding area as that reported previously for whales off California (Calambokidis et al., 1996). The decreas- ing rate of interchange with distance among feeding areas does not allow for a clear demarcation between feeding areas, however, as suggested by Calambokidis et al. (1996). Although humpback whales demonstrate site fidelity to specific feeding locations, their feeding aggregations may not have clear boundaries and may occupy overlapping ranges. The commercial whaling data also tended to support the existence of somewhat discrete feeding areas off the west coast of the United States and British Columbia. Commercial whaling resulted in the depletion of hump- back whales off British Columbia by 1917, whereas the numbers taken off Washington and California did not decline until the mid-1920s (Scheffer and Slipp, 1948; Clapham et al., 1997; Gregr et al, 2000). The relatively small proportion of mothers with calves identified in our study is consistent with findings off California and Oregon (Steiger and Calambokidis, 2000). Steiger and Calambokidis reported reproductive rates along the California, Oregon, and Washington coasts that are lower than those reported for other re- gions in southeastern Alaska and the North Atlantic (Clapham and Mayo, 1987, 1990; Baker et al., 1992; von Ziegesar et al., 1994). In aerial transect surveys, no humpback whale calves were seen among the 68 humpbacks observed off the Oregon and Washington coasts in 1989-90 (Green et al.5). If geographic segre- gation is occurring by humpback mothers and calves, as was suggested by Steiger and Calambokidis (2000), this northern region is not the area where mothers and calves are congregated. It is interesting to note, how- ever, that mothers and calves were distributed around the periphery of the main feeding region — a finding that suggests that a more local segregation may be occurring. A bias in sampling would occur if large concentrations of whales are targeted and mother with calves feeding on the perimeter of these groups were underrepresented. In contrast to humpback whales, no other large ror- quals (blue, fin, or sei whales) were observed during any of our ship or small boat surveys. Likewise, these species were absent in other recent surveys of Wash- ington waters (Wahl, 1977; Von Saunder and Barlow. 1999; Shelden et al., 2000; Green et al.5), although they were seen in surveys farther offshore in surveys in July 1994 (Thomason et al.6). Fin whales were common ,; Thomason, J., M. Dahlheim, S. E. Moore, J. Braham, K. Stafford, and C. Fox. 1997. Acoustic investigations of large cetaceans off Oregon and Washington: NOAA ship Surveyor (21 July-1 August 1994), 27 p. Final report by the National Marine Mammal Laboratory, 7600 Sand Point Way NE F/AKC3, Seattle. WA 98115. 578 Fishery Bulletin 102(4) in Washington waters in the early 1900s when they were the second most commonly killed species by Bay City whalers (Scheffer and Slipp, 1948). Blue and sei whales were less common, although they were present historically (Scheffer and Slipp. 1948). Although Bay City whaling stations (in Grays Harbor, Washington) were closed after humpback whales were depleted, se- rial depletion of whale populations continued off British Columbia waters, beginning with humpback and blue whales, then with fin and sperm whales, and finally with sei whales (Gregr et al., 2000). No sperm whales or beaked whales were seen during our surveys, although our study area did not include the deeper waters where we would expect to find these spe- cies. Most of the sperm whales (90%) seen by Green et al.5 off Washington and Oregon were present in deeper offshore waters outside of our study area. The other cetacean species not seen in our surveys that have been reported to occur off Washington his- torically included northern right whale (Eubalaena japonica), pygmy sperm whale iKogia breviceps), false killer whale iPseudorea er-assidens), short-finned pilot whale (Globicephala macrorhynchus), and striped dol- phin (Stenella coeruleoalba) (Scheffer and Slipp, 1948). Sightings of northern right whales throughout the east- ern North Pacific are scarce; there have been only a small number of sightings since the 1960s (Brownell et al., 2001). Several of these sightings, however, have been off the northern Washington coast (Fiscus and Niggol, 1965; Osborne et al., 1988; Rowlett et al., 1994). The primary reason for the paucity of sightings in the eastern North Pacific in recent decades is due to the il- legal take of 372 right whales in the early to mid-1960s by the USSR (Brownell et al., 2001; Doroshenko7). Although some small cetacean species such as Pacific white-sided dolphins and Risso's dolphins were sighted frequently on our surveys, they were not as common as in some previous surveys (Green et al.5), probably because our coverage was concentrated in shallower waters inside the shelf break. In contrast to our find- ings of a number of species seen near the shelf edge. Wahl (1977) reported that most marine mammal species off central Washington tended to be in either inshore or in deeper offshore waters and only killer whales and Dall's porpoises regularly used the slope waters (13- 45 km offshore). It is difficult to make abundance estimates of Dall's porpoise because of their proclivity to approach ships (Buckland and Turnock, 1992). If they begin to ap- proach the ship before the observer sights them, the es- timate is biased upwards, which would be the case with our estimate. Our estimate would also have a downward bias because we did not attempt to adjust for animals missed even if they were on the track line. Doroshenko, N. V. 2000. Soviet whaling for blue, gray, bowhead and right whales in the North Pacific Ocean. 1961-1979. In Soviet whaling sata (1949-1979), p. 96-103. Center for Russian Environmental Policv. Vavilov St. 26, Moscow 117071, Russia. All three types of killer whales (residents [both northern and southern], transients, and offshore type) were identified in the waters off northern Washington. These sightings are interesting because of concerns about killer whale populations, especially the southern resident community that has declined in recent years. Although killer whales have been intensely studied in inside waters of the Pacific Northwest, little has been known about their use of outside waters, where they may spend large portions of their lives. Little is known about the offshore type of killer whales, which is be- lieved to be a distinct race of killer whale that has only recently been described. These whales are believed to be found usually in large groups along the continental shelf but also have been seen in inland waters (Ford et al., 1994; Dahlheim et al., 1997). All three sightings of the offshore form were just west of the Juan de Fuca canyon on the Prairie; the closest sighting to shore was 37 km (30 animals on 15 July 1997). Acknowledgments We are grateful to those who assisted with this study. This work was supported by the Olympic Coast National Marine Sanctuary and Southwest Fisheries Science Center (Jay Barlow, COTR). Many people contributed to this study. Jennifer Quan, Richard Rowlett, Anne Nelson, and Annie Douglas worked on the ship surveys. We thank the ship personnel on board the McArthur and Agate Passage. Researchers who helped with small boat work included Joe Evenson and Todd Chandler. Photo- graphs of whales from this area were also contributed by L. Baraff, R. Baird, P. Bloedel, V. Deeke. P. Ellifrit, G. Ellis, J. Evenson, B. Gisborne. B. Halliday, H. Hunt, S. Mizroch, K. Rasmussen, J. Wilson and SWFSC research- ers. Permission to survey in Canadian waters was given by the Dept. of Fisheries and Oceans. Lisa Schlender, Kristin Rasmussen, and Annie Douglas organized and conducted the photographic matching with the help of many interns at Cascadia Research. DKE and Graeme Ellis identified the killer whales; Oscar Torres assisted with the photographic matching. Data analyses and mapping were conducted with the help of Scot McQueen at ESRI and Tom Williams. Literature cited Baker, C. S., A. Perry, and L. M. Herman. 1992. 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Nat. 39:257-337. Seber, G. A. F. 1982. The estimation of animal abundance and related parameters, 2nd ed., 654 p. Griffin, London. Shelden, K. E. W., D. J. Rugh, J. L. Laake, J. M. Waite, P. J. Gearin, and T. R. Wahl. 2000. Winter observations of cetaceans off the northern Washington coast. Northwestern Nat. 81:54-59. Steiger, G. H., and J. Calambokidis. 2000. Reproductive rates of humpback whales off California. Mar. Mamm. Sci. 16:220-239. Swan, J. G. 1868. The Indians of Cape Flattery, 109 p. Shorey Pub- lications, Seattle, WA. Von Saunder, A., and J. Barlow. 1999. A report of the Oregon, California and Washington line-transect experiment lORCAWALE) conducted in West Coast waters during summer/fall 1996. NOAA Tech. Memo. NOAA-TM-NMFS-SWFSC-264, 49 p. von Ziegesar, O.. E. Miller, and M. E. Dahlheim. 1994. Impacts of humpback whales in Prince William Sound. In Marine mammals and the Exxon Valdez IT. R. Loughlin. ed.), p. 173-191. Academic Press, San Diego, CA. Wahl, T. R. 1977. Sight records of some marine mammals offshore from Westport, Washington. Murrelet 58:21-23. Webb, L. W. 1988. On the Northwest. Commercial whaling in the Pacific Northwest 1790-1967, 425 p. Univ. British Columbia Press. Vancouver, Canada. 581 Abstract — The reproductive biology of male franciseanas iPontoporia blainvillei), based on 121 individu- als collected in Rio Grande do Sul State, southern Brazil, was studied. Estimates on age, length, and weight at attainment of sexual maturity are presented. Data on the reproductive seasonality and on the relationship between some testicular characteris- tics and age. size, and maturity status are provided. Sexual maturity was assessed by histological examina- tion of the testes. Seasonality was determined by changes in relative and total testis weight, and in seminifer- ous tubule diameters. Testis weight, testicular index of maturity, and seminiferous tubule diameters were reliable indicators of sexual maturity, whereas testis length, age, length, and weight of the dolphin were not. Sexual maturity was estimated to be attained at 3.6 years (CI 957c = 2.7- 4.5) with the DeMaster method and 3.0 years with the logistic equation. Length and weight at attainment of sexual maturity were 128.2 cm (CI 95<£ = 125.3-131.1 cm) and 26.4 kg (CI 95^=24.7-28.1 kg), respectively. It could not be verified that there was any seasonal change in the testis weight and in the seminiferous tubule diameters in mature males. It is sug- gested that at least some mature males may remain reproductively active throughout the year. The extremely low relative testis weight indicates that sperm competition does not occur in the species. On the other hand, the absence of secondary sexual char- acteristics, the reversed sexual size dimorphism, and the small number of scars from intrassexual combats in males reinforce the hypothesis that male combats for female reproductive access may be rare for franciscana. It is hypothesized that P. blainvillei form temporary pairs (one male copu- lating with only one female) during the reproductive period. Reproductive biology of male franciseanas (Pontoporio blainvillei) (Mammalia: Cetacea) from Rio Grande do Sul, southern Brazil* Daniel Daniiewicz Grupo de Estudos de Mamiferos Aquaticos do Rio Grande do Sul (GEMARS) Rua Felipe Nen, 382/203 Porto Alegre 90440-150, Brazil Present address: Laboratono de Dinamica Populacional-Pontilicia Universidade Catolica do Rio Grande do Sul (PUCRS) Av. Ipiranga, 6681 Porto Alegre 90619-900, Brazil Email address (for D Daniiewicz): Daniel. Danilewicza1 terra com br Juan A. Claver Alejo L. Perez Camera Area Histologia y Embnologia Facultad de Ciencias Vetermarias Universidad de Buenos Aires Ar Chorroarin 280 C1427CWO Buenos Aires, Argentina Eduardo R. Secchi Laboratono de Mamiferos Mannhos, Museu Oceanografico "Prof. Eliezer C. Rios" Fundacao Universidade Federal do Rio Grande, Cx P. 379 Rio Grande 96200, Brazil Nelson F. Fontoura Laboratono de Dinamica Populacional-Pontificia Universidade Catolica do Rio Grande do Sul (PUCRS) Av. Ipiranga, 6681 Porto Alegre 90619-900, Brazil Manuscript submitted 4 October 2002 to the Scientific Editor's Office. Manuscript approved for publication 18 May 2004 by the Scientific Editor. Fish. Bull. 102:581-592 (2004). The franciscana (Pontoporia blainvil- lei) is a small dolphin endemic to the coastal waters of the southwestern Atlantic Ocean. The distribution of this species ranges from Golfo Nuevo (42°35'S; 64°48'W), Chubut Province, Argentina (Crespo et al., 1998) to Itaunas (18°25'S; 30°42'W), Espirito Santo, southeastern Brazil (Moreira and Siciliano, 1991) (Fig. 1). The franciseanas coastal habitat makes it vulnerable to being caught as incidental catch in gill nets and trammel nets throughout most of the species range (e.g., Praderi et al., 1989; Corcuera et al., 1994; Secchi et al., 2003). Because of its vulnerability as bycatch, the franciscana has been considered the most impacted small cetacean in the southwestern Atlantic Ocean (Secchi et al., 2002). In the Rio Grande do Sul coast, southern Brazil, this species has been subject to an intense bycatch in gill nets for at least three decades (Moreno et al., 1997; Secchi et al., 1997; Ott, 1998; Ott et al., 2002). The annual mor- tality of franciseanas in this region was estimated to range from several hundred up to about a thousand indi- viduals (Ott et al., 2002). Simulations ^Contribution 012 from the Grupo de Estudos de Mamiferos Aquaticos do Rio Grande do Sul (GEMARS), Rua Felipe Neri, 382/203, Porto Alegre 90440-150, Brazil. 582 Fishery Bulletin 102(4) Figure 1 Map of the study area showing the locations along the southern coast of Brazil where franciscanas were caught as bycatch between 1992 and 1998. studies on the effects of incidental captures on francis- canas in Rio Grande do Sul were carried out by using available data on vital rates, stock size, and bycatch estimates (e.g., Secchi, 1999; Kinas, 2002). All these studies showed that there is a decline in franciscana abundance in this region. Although the reproductive biology of the female fran- ciscanas have been studied in detail in Uruguay (Ka- suya and Brownell, 1979; Harrison et al., 1981), Rio Grande do Sul (Danilewicz et al., 2000; Danilewicz, 2003), and Rio de Janeiro (Ramos, 1997), there are few data about male reproduction. Kasuya and Brownell (1979) presented information on male reproduction for Uruguay, although their small sample size precluded them from estimating age and size at attainment of sexual maturity. In the Rio Grande do Sul coast, franciscanas are known to reproduce seasonally; births occur from Oc- tober to early February (about 75"7r from October to De- cember). Because the gestation period was estimated to last about 11.2 months, mating and conception may take place between November and early March I Danilewicz, 2003). Seasonal changes in testicular size and activity have been used to infer or corroborate mating seasons in some cetacean species (e.g., Neimanis et al., 2000 I. Nevertheless, it is not known if male franciscanas also undergo seasonal changes in the testicular activity. In this study, we describe the reproductive biology of male franciscanas from Rio Grande do Sul and present evidences for the species' mating system. Materials and methods Sampling procedures Data and samples collected from 121 specimens inci- dentally caught (889c) or beached (12%) along the Rio Grande do Sul coast between 1992 and 1998 were used for the analysis on reproduction of male franciscanas. The sampling of the incidentally caught animals was carried out through the monitoring of the commercial fishery fleet from Rio Grande (32°08'S; 52°05'W) and Tramandai/Imbe (29°58'S, 50°07'W). Stranded dolphins were sampled from systematic beach surveys conducted in an area with an extension of 270 km of sandy beaches, between Torres (29°19'S, 49°43'W) and Lagoa do Peixe (31°15'S, 50°54'W). Not all information could be collected from each car- cass; therefore sample sizes varied among parameters. Standard length (SL, n = 118) was measured by following the guidelines established by the American Society of Mammalogy (1961). The animals were weighed (rc = 97) and teeth were extracted and preserved dried or in a 1:1 mix of glycerin and alcohol (70%). Testes and epi- didymis were removed and fixed in 10'> formalin. Age determination Age was estimated by counting the growth layer groups (GLGs) in thin, longitudinal sections of teeth (ra=47). The teeth were decalcified in nitric acid or in RDO Danilewicz et al.: Reproductive biology of male Pontopona blainvillei from Rio Grande do Sul, southern Brazi 583 (a commercial mixture of acids) and sectioned on a freez- ing microtome. The 15-20 fim sections were stained with Mayer's hematoxylin and mounted on microscope slides with Canadian balsam or in glycerin. Poor and off-center sections were discarded in favor of new preparations. Three readers counted independently the number of growth layer groups in both the dentine and cementum. When reader estimates differed, the sections were reex- amined together and a best estimate was agreed upon. In this study, we considered one GLG to represent one year of age, which is the accepted model for the francis- cana (Kasuya and Brownell, 1979; Pinedo, 1991; Pinedo and Hohn; 2000). Reproduction In the laboratory, the testes were separated from the epididymis, weighed to the nearest 0.01 g (n = 107), and measured in three dimensions (length and two diam- eters perpendicular to each other in the middle of the testis) to the nearest 0.1 mm (;? = 104). The mean of these two diameters was called mean testis diameter. The weight of one of the gonads could not be recorded on some occasions (« = 8) and we assumed that both testes had the same weight. Then, relative testis weight was determined as the ratio of the combined testis weight to the animal weight. A 1-cm3 subsample of each testis from the central portion of the organ was removed and examined by using standard histological preparations. The tissue was embedded in paraffin, sectioned in 4-10 fim thick slides through a manual microtome, and stained with hematoxylin and eosin (H&E). Male sexual maturity status was determined by examining the testicular sections at a magnification of lOOx. In this study, we followed the classification criteria suggested by Hohn et al. (1985): 1 Immature — seminiferous tubules containing main- ly spermatogonias. Abundant interstitial tissue present between the seminiferous tubules and lu- men totally closed. 2 Pubertal — seminiferous tubules containing sper- matogonias and spermatocytes. Less interstitial tissue present between the seminiferous tubules than in immature animals. The lumen is partially opened. 3 Mature — seminiferous tubules containing sper- matogonias, spermatocytes, spermatids and, in many cases, spermatozoa. Interstitial tissue al- most nonexistent between the seminiferous tu- bules. The lumen is totally opened. The diameters of ten random circular seminiferous tubules were measured for each specimen (« = 93) with a scale present in the lens of the microscope in order to calculate the seminiferous tubule mean diameter. A maturity index (MI) was calculated as the ratio of the combined testes weight by the combined testes length aw/iL). An analysis of the variation along the year of the val- ues of relative and combined testes weight, and seminif- erous tubule mean diameter, was employed to assess re- productive seasonality. Values of these parameters were compared between months when mating and conception occur ("reproductive months": November-March) and months when they not occur ( "nonreproductive months": April-October). In order to increase the sample size of mature animals collected in reproductive months, data on testes weight from mature male franciscanas from Uruguay were included in the analysis (data supplied by Kasuya1). The mean age at attainment of sexual maturity (ASM) was estimated through the DeMaster (1978) method and the logistic regression. The DeMaster (1978) equation computes the mean as ASM = £a(/Q -f^), where fa = the fraction of sexually mature animals in the sample with age a; j = the age of the youngest sexually mature animal in the sample; and k = the age of the oldest sexually immature animal in the sample. The variance of the DeMaster method estimate is cal- culated as k variASM)=^[ifaa-fa)/Na-D], «=j where N = the total number of animals aged a. The logistic regression approach fits a sigmoid curve representing the probability that a franciscana of age a is sexually mature to the distribution of sexually mature and immature animals by age as Y = l/(l+e°+/,v) or In (1/1-1) = a + bx, where x = the age of the dolphin; b = the slope of the regression; and a = the intercept. To obtain the age when 50% of the animals are sexu- ally mature (Y=0.5), the last equation is simplified as ASM = -alb. Mean length and weight at sexual maturity was also estimated by the DeMaster (1978) method, by substitut- ing age for length and weight, respectively. The meth- od was slightly modified, as suggested by Ferrero and Walker (1993), and was calculated as 1 Kasuya, T. 1970-73. Unpubl. data. Teikyo University of Science and Technology. Uenohara, Yamanashi Prefecture, 409-0193, Japan. 584 Fishery Bulletin 102(4) Cmax LSM= X Uft-ft-i), Cmin where Cmax = the length or weight class of the largest or heaviest sexually immature animal; Cmin = the length or weight class of the smallest or lightest sexually mature animal; L = the lower value of the length or weight class t; and ft = fraction of mature animals in the length or weight class t. The specimens were pooled into length and weight inter- vals of 4 cm and 4 kg, respectively. The estimated variance of this method is also modi- fied and is calculated as var(MS) = M>2 £ [(/; (1 -/",)/ A^, -l]. (x=33.6 mm), respectively. The weight and length of the right testes ranged from 0.17 to 9.98 g (.v=2.62 g) and from 17.9 to 60.0 mm (.v=34.5 mm), respectively. The relationship of testes weight and testes length resulted in significant regression (P<0.0001) and correlation E ra 25 - 73 c 0) 5 0 2 4 6 8 10 12 14 16 18 20 22 Combined testes weight (g) Figure 2 Relationship between combined-testes weight and mean seminiferous tubule diameter in immature (open boxesi, pubertal (filled boxes), and mature (triangles) male franciscanas (Pontoporia blainvillei) from Rio Grande do Sul (re=59). Seminiferous tubule diameter A nonlinear regression demonstrated positive allometry (6>0.333) of the seminiferous tubule diameter to the combined testicular weight (6 = 0.39; 95% CI=0.35-0.44) (Fig. 2), and a strong correlation between these two vari- ables (F=343.6; r2 = 0.86; P<0.0001; y=59.4.x° 39). The relationship between the seminiferous tubule di- ameter and testes length is shown in Figure 3 and the relationship between the seminiferous tubule diameter and standard length is shown in Figure 4. In immature males, there was almost no increase in the seminif- erous tubule diameter with the increase of standard length (0.26 jim/cm) and total weight (0.5 ^m/kg). In mature males, however, seminiferous tu- bule diameter was significantly correlat- ed with standard length (6 = 1.06; F=4.4; r2 = 0.18; P=0.048; y = 1.4775.v-43.572) and there was no correlation with total weight (6 = 0.23; P=1.28; r2=0.07; P=0.27; y = 1.6132.\-+108.54). The differences of the seminiferous tu- bule mean diameters were statistically significant between immature, pubertal, and mature male franciscanas (ANOVA, Fs=255.4; df=87; P<0.001). Combined-testes weight and length, and sexual maturity There was almost no increment in mass of the combined-testes weight in imma- ture dolphins. An increment of only about 2.0 g in the combined-testes mass was observed in animals of 70.0 to 125.0 cm in length. For dolphins about 120.0-130.0 cm in length, the combined-testes mass sud- denly increased (Fig. 5), indicating the Danilewicz et al.: Reproductive biology of male Pontopona blainvillei from Rio Grande do Sul, southern Brazil 585 attainment of sexual maturity. The rate of testes-mass gain was 0.05 g/cm for imma- ture and 0.28 g/cm for mature dolphins. All animals with combined-testes weight higher than 5.0 g were sexually mature, and this finding may indicate that this parameter can be used as a reliable indi- cator of sexual maturity in male fran- ciscanas from Rio Grande do Sul (Table 1). However, the large variation in testes weight after the attainment of sexual maturity precludes a correlation between testes mass and standard length. Although testes length increases pro- gressively as standard length increases (Fig. 6), there is no abrupt increase in testes length at the moment of attain- ment of sexual maturity, as observed in the testes mass. A nonlinear regression (exponential) best fits this relationship (y =4. 5444 e0.0163.vi. As opposed to testes mass, there is a considerable overlap in the values of testes length of immature, pubertal, and mature franciscanas (Table 1), which makes testes length a less reli- able predictor of sexual maturity than testes mass. Age, length, and weight at sexual maturity Forty-seven specimens in the sample pro- vided information on age and reproduc- tive status (35 immature or pubertal, and 12 mature). The oldest immature animal was 5 years old and the youngest mature was 2 years old (Table 1). Average age at attainment of sexual maturity was esti- mated to be 3.6 years by the DeMaster method (SD = 0.47; 95% CI =2.7-4.5) and 3.0 years by the logistic equation Y = 1/(1+ eo.74-2 23i) The age structure of the sample studied is presented in Figure 7. Sexual maturity in relation to standard length was estimated for 110 males. The smallest mature and the largest immature males were 120.5 and 137.5 cm long, respectively. The av- erage length at sexual maturity was 128.2 cm (SD=1.49; 959r CI=125. 3-131.1 cm). Sexual maturity in relation to total weight was estimated for 90 males. The lightest mature and the heaviest immature males were 20.3 and 29.7 kg, respectively. The average weight at sexual matu- rity was 26.4 kg (SD = 0.88; 95% 01=24.7-28.1 kg). Index of testicular maturity The differences of the mean index of testicular maturity between immature (0.03), pubertal (0.04), and mature (0.11) dolphins were statistically significant (ANOVA, Fs=210.0 df=101, P<0.001). There was almost no overlap in the values of this index between mature specimens 225 -I ~ 200 of 17S E c fS 150' E c/> i O 125' f E too. c ° 50 - A A A^&— - & — ^ A A A A ■ A 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 hnsrns hnlers (ww) Figure 3 Relationship between testes length and mean diameter of seminifer- ous tubules in immature (open boxes), pubertal (filled boxes), and mature (trianglesi male franciscanas (Pontoporia blainvillei i in Rio Grande do Sul (ra=54). Data from pubertal animals are not included in the curves. 5 g 205 ■ A 185 - 165 - 145 - A 3 A^ AAA A^ A1 A A A A A A A A 125 ■ *f» 105 - 85 ■ 65 ■ 45 ■ D D rjPrP D 1 is? crP^1 25 - 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 itanoaro lentth (nm) Figure 4 Relationship between standard length and mean diameter of seminif- erous tubules in immature (open boxes), pubertal (filled boxesl. and mature (trianglesi male franciscanas (Pontoporia blainvillei) in Rio Grande do Sul l;i=91>. and immature and pubertal specimens (Table 1). All males with an index value higher than 0.07 were sexu- ally mature. These results indicate that the index of testicular maturity is a very good indicator of sexual maturity for franciscanas. Reproductive seasonality The null hypothesis that the combined and relative testis weight would be higher in the months when the females are reproductively active is rejected. No increase in the testes weight was observed during the months when most mating occurs (Fig. 8). There were no statistically significant differences in the combined-testes weight (ANOVA, Fs=2.28; df=34; P=0.48; n=35) and relative 586 Fishery Bulletin 102(4) £ o O V a nnj 60 70 80 90 100 110 120 130 140 150 160 Standard length V;ma Figure 5 Relationship between standard length and combined-testes weight in immature (open boxes), pubertal (filled boxes), and mature (triangles) male franciscanas ^Pontoporia blainvillei) in Rio Grande do Sul (tt=79). Data from pubertal animals are not included in the curves. testes weight (ANOVA, Fs=2.42; df=29; P=0.76; /? = 30) between reproductive and nonreproductive months. The analyses of the variation in the diameter of the seminiferous tubules throughout the year also did not indicate that the testes undergo seasonal changes. However, it is important to view this re- sult with caution because the sample size of mature males (and therefore the infor- mation on tubules diameter collected in reproductive months) was small (n=3). However, the presence of spermatids or spermatozoa (or both) in the seminiferous tubules may be also regarded as a direct evidence of testicular activity. Three ma- ture males (119<-) in the sample presented seminiferous tubules with spermatids or spermatozoa (or both) and were collected in nonreproductive months (May, June, and August). Although the epididymis of Table 1 Summarized information on age length mass, and testicul ar characteristics for ma e franciscanas iPontoporia blainvillei) in the Rio Grande do Sul at different sexual m aturity stages. Characteristics and maturity state ;? Mean Standard deviation Range Age (years) Immature 31 1.29 1.01 0-5 Pubertal 4 2.0 0.82 1-3 Mature 12 3.8 1.14 2-6 Standard length (cm) Immature 62 111.2 13.62 70.0-137.5 Pubertal 7 118.5 9.75 107.8-132.5 Mature 37 133.7 7.71 120.5-155.0 Total mass (kg) Immature 53 19.0 5.6 4.95-29.7 Pubertal 6 21.4 4.62 17.1-28.0 Mature 30 29.9 5.22 20.25-41.5 Mean diameter of seminiferous t ubules (/./m) Immature 54 69.6 12.2 50.0-105.0 Pubertal 6 95.0 19.2 74.5-121.2 Mature 33 154.1 21.7 113.0-197.0 Combined testes mass (g) Immature 63 1.59 0.84 0.33-4.78 Pubertal 7 2.73 1.28 1.30-4.8 Mature 37 10.24 3.94 4.27-20.08 Testes length (mm) Immature 62 27.2 4.9 15.7-35.5 Pubertal 7 32.6 6.2 25.0-41.0 Mature 35 45.4 5.6 31.6-59.7 Index of testicular maturity Immature 61 0.03 0.01 0.01-0.06 Pubertal 7 0.04 0.01 0.02-0.06 Mature 36 0.11 0.03 0.05-0.18 Danilewicz et al.: Reproductive biology of male Pontoporia blainvillei from Rio Grande do Sul, southern Brazil 587 a subsample of 10 mature males were examined histologically, we did not find any sign of spermatozoa. Discussion The high bilateral uniformity in tes- ticular weight and length presented by the franciscana is a characteristic shared with many other cetacean spe- cies. Studies on the striped dolphin, Stenella coeruleoalba (Miyazaki, 1977), the common dolphin, Delphinus delphis (Collet and Saint Girons, 1984), the sperm whale, Physeter macrocephalus (Mitchell and Kozicki, 1984), and the dusky dolphin, Lagenorhynchus obscu- rus (van Waerebeek and Read, 1994), among others, demonstrate the same pattern of testis symmetry. Given the similar dimensions of both testes in franciscanas, it is possible to extrapo- late the combined-testes weight by weighing only one testis without intro- ducing bias in the analysis. It is recom- mended, however, that the weight of the testes should be presented without the epididymis weight, as it was pre- sented in the most extensive compara- tive study on the subject (Kenagy and Trombulak, 1986). There is a negative allometry of the seminiferous tubule diameter in rela- tion to testis length, standard length, and total weight. This pattern is ac- centuated in immature males, in which the tubule diameters remain almost unchanged with the increase of the other variables. The lack of values for tubule diameters in the testes weight interval (2.5-6.0 g) and testes length interval (34-42 mm) just before the attainment of sexual maturity (Figs. 2 and 3) indicates that the increase in tubule size in relation to sexual matu- rity must occur very quickly, probably when the tubules are between 85 and 125 urn in diameter. Attainment of sexual maturity Length and weight at attainment of sexual maturity of male franciscanas in Rio Grande do Sul are very similar to those values estimated in previous estimates for Uruguay (Table 2). In con- trast to the present study, Kasuya and Brownell (1979) calculated mean length at sexual maturity for Uruguay as the 65 - 60 ■ CO E 55. E < 50- '2 « * c a <,, -0,0163* y= 4,5444e r! = 0.72 A . A ^ 4 A &/* A < O) 35 ■ c/1 8) 30- n 25 20 ° DEh 3- — ° □ 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 atcnecre Ignhth Ydma Figure 6 Relationship between standard length and testes length in immature (open boxes), pubertal (filled boxes), and mature (triangles) male franciscanas (Pontoporia blainvillei) in Rio Grande do Sul i;; = 99l. 60 -I 50- o c CD g "»■ CD < CD 30- O CD EC 1 10- . 1 1 I ■ . _ 0 year 1 year 2 years 3 years 4 years 5 years 6 years Figure 7 Age structure of male franciscanas (Pontoporia blainvillei ) collected in Rio Grande do Sul (n=48). Figure 8 Relationship between month and relative testes weight in mature male franciscanas (Pontoporia blainvillei) (7j=31) (l = January, 12 = December; filled boxes=the nonreproductive months, open boxes=reproductive months). Bars indicate 25^ and 75% percentiles. 588 Fishery Bulletin 102(4) Table 2 Comparison between average age, weight, and length at sexual maturity between male and female franciscanas from Rio Grande do Sul and Uruguay. The means of the animals from Rio Grande do Sul were estimated by using the DeMaster method (modified! and those from Uruguay- were estimated by using a linear regression to determine the moment when 50(7( of the animals are mature. Rio Grande do Sul Uruguay' Males Females2 Males Females Age Weight (kg) Length (cm) 3.6 3.7 26.6 32.6 127.4 138.9 2-4 2.7 25.0-29.0 33.0-34.0 131.4 140.3 ' Data from Uruguay were compiled from Kasuya and Brownell 11979' 2 Data from Damlewicz (2003). length where 50r* of the dolphins were mature through a linear regression. Applying this same approach, a LSM of 125.4 cm was estimated for Rio Grande do Sul — a value still very similar to the Uruguay estimate. Male franciscanas attain sexual maturity at less length and weight than do females in Rio Grande do Sul (Danile- wicz, 2003), as observed previously in Uruguay (Kasuya and Brownell, 1979) and Rio de Janeiro (Ramos et al., 2000). This is the first estimate of mean age at sexual ma- turity presented for male franciscanas. Kasuya and Brownell (1979) could not calculate ASM for Uruguay because of their small sample size («=25). Nevertheless, Kasuya and Brownell suggested that sexual maturity is attained when males are between 2 and 4 years of age. Franciscanas from Rio de Janeiro were considered mature when they were older than 2 years of age and larger than 115.0 cm in length (Ramos et al., 2000). However, histological analysis of the testes was not performed and Ramos et al. employed indirect methods to determine sexual maturity. Nevertheless, despite the uncertainties produced by the use of different criteria to determine sexual maturity, it was evident that there was substantial difference in the size at maturity be- tween males from Rio de Janeiro and those from Rio Grande do Sul and Uruguay. This difference is probably the result of the well-known distinct growth patterns of the franciscanas from these two regions (Pinedo, 1991) and does not necessarily reflect an early attainment of sexual maturity in males from the Rio de Janeiro population. The trade-off between growth and reproduction is the best-documented phenotypic trade-off in nature (Stearns, 1992) and has been studied in a wide range of taxa. Because animals from Rio de Janeiro invest less in growth than do animals from Rio Grande do Sul, it is still an open question whether the francisca- nas from the Rio de Janeiro have higher reproductive rates or start reproducing earlier than those from Rio Grande do Sul. The oldest male and female franciscana ever aged were 16 and 21 years-old, respectively (Kasuya and Brownell, 1979; Pinedo, 1994). These ages contrasts with the age distribution found in the present study, where the oldest specimen analyzed was 6 years old. Similar to what is observed in catches for several other small cetacean species (e.g.. Hector's dolphins — Slooten and Lad, 1991; harbor porpoise — Read and Hohn. 1995), a general feature of incidental catches for these spe- cies is the high entanglement rate of immature ani- mals. In all fishing communities studied in Argentina, Uruguay, and Brazil, a large proportion (>50%) of the specimens caught were less than three years old (e.g., Kasuya and Brownell, 1979; Corcuera et al.. 1994; Ott, 1998; Di Beneditto and Ramos, 2001). Although the precise reason for biased catch rates towards imma- ture individuals is not well understood, it could be a combination of factors, including the imbalanced age- structure of local populations (where there are fewer older individuals because of an extensive history as bycatch) and a behavior-related higher vulnerability to bycatch for immature individuals (i.e., juveniles can be more inquisitive and have less ocean experience so that they rove into the area increasing the chances of being entangled). The typically low proportion of old animals in bycatches may explain the characteristics of the data used in this study. Index of testicular maturity An index of testicular maturity may be very useful in studies where it is necessary to know the sexual maturity of a large sample of animals without the need of histological analysis, which is time consuming and requires expertise. Although Hohn et al. (1985) recom- mended the investigation of the applicability of this indirect index of sexual maturity for male cetaceans, the research on this subject has shown no progress. To date, the index of sexual maturity has been calculated only for the common dolphin, Delphinus delphis (Collet and Saint Girons, 1984), and from the pantropical spot- ted dolphin, Stenella attenuate! (Hohn et al., 1985). For both species, this index distinguished satisfactorily the mature from the immature and pubertal dolphins. Given the results presented, we also recommend the use of the index of testicular maturity as an alternative, nonhisto- logical method, to determine the sexual maturity of male franciscanas. Males with index values lower than 0.05 can be safely classified as immature, and males with index values above 0.08 can be classified as mature. It is recommended that for animals with intermediate values their testes be analyzed histologically so that their reproductive status may be determined definitively. Besides making intra- and inter-populational com- parisons possible, the index of testicular maturity also permits interspecific comparisons because size differ- ences between species are eliminated. The mean index of testicular maturity of mature franciscanas (0.12) is Danilewicz et al.: Reproductive biology of male Pontoporia blamvillei from Rio Grande do Sul, southern Brazil 589 considerably lower than mature pantropical spotted dolphins (1.9) (Hohn et al., 1985). This difference is a consequence of the relatively small increase of the testes weight of male franciscanas when sexual matu- rity is attained. Although male spotted dolphin show a marked increase of about 25-fold in testes weight at this moment, franciscanas show an increment in testes weight of about ninefold only. Reproductive seasonality The reproductive activities in male mammals are usually restricted to the periods when the females are in estrus (Lincoln, 1992). Reproductive seasonality in males has been reported for several cetacean species and popula- tions through the identification of temporal variations in the testes weight and histological characteristics. In species where the reproductive period is restricted for a few months, as with the dusky dolphin (Lagenorhyn- chus obscurus) and the harbor porpoise (Phocoena pho- coena), the testes weight presents marked fluctuations accompanying the reproductive period (Read, 1990; van Waerebeek and Read, 1994; Neimanis et al., 2000). Even in species with a diffuse reproductive period (i.e., with more than one peak for births per year) as in the case of dolphins of the genus Stenella in the tropical Pacific, it was possible to detect seasonal variation in the male reproductive rhythm (Perrin et al., 1976, Hohn et al., 1985). Because of the known seasonality for births for fran- ciscana (Kasuya and Brownell, 1979, Harrison et al., 1981, Danilewicz, 2003), it would be expected that the males would accompany the female rhythm, decreas- ing or even ceasing testicular activity in autumn and winter months. Kasuya and Brownell (1979) examined the seasonal change in testes weight in the months of January, June, and December. From our knowledge of the species' reproduction period, testes weight would be expected to be higher in December and January. However, the authors could not confirm this predic- tion and attributed the lack of seasonality to the small sample size of mature animals. Nevertheless, the lack of seasonality, even when the testes weight of the ma- ture males from Rio Grande do Sul are included, may indicate what is occurring in the population, and not be a bias introduced by a small sample size. In species that possess small testes, as in the case of the franciscana, the variation in the testicular activity may be better reflected by changes in the diameter of the seminiferous tubules and the rate of spermatogen- esis rather than by changes in the testes weight. Nev- ertheless, the preliminary results about these charac- teristics (mature males with spermatids or spermatozoa [or both] in the seminiferous tubules in nonreproductive months and little monthly variation in the diameter of the seminiferous tubules) also do not support the hypothesis of a male reproductive seasonality. The com- bination of results presented here indicates that testicu- lar activity is not completely interrupted in all males within the population, and that at least some of them may remain capable of fertilizing females during the year. This conclusion is supported by the observation of pregnancies outside the normal gestation season and that the births resulting from these pregnancies were estimated to take place in September and in late March (Danilewicz, 2003). The hormone and sperm production by the testes dur- ing periods when the females are not able to reproduce may represent an unnecessary energetic expense by the male (Dewsbury, 1982) and may be an explanation for the period of reproductive inactivity for males of several mammal species. In species with large relative testes weight, the maintenance of high levels of sperm production in the testes is a considerable energetic cost for the individual. However, as discussed earlier, this is definitely not the case for the franciscana. For this reason, we suggest that the small energy investment in producing sperm all over the year, due to the small testicular mass, may be an evolutionary advantage for male franciscanas in case of the appearance of off-sea- son reproductive females. Franciscana reproductive strategy Although important advances in the knowledge of fran- ciscana behavior in the wild have been made (e.g., Bor- dino et al., 1999; Bordino, 2002), there is no information on the species' reproductive behavior and its mating strategy remains unknown. Relative testis weight, sexual size dimorphism, and secondary sexual charac- teristics may provide indirect clues regarding mating strategy in franciscana and are discussed below. Relative testis weight In mammals, there is a func- tional relationship between relative testis weight and the species' mating system (Kenagy and Trombulak, 1986). Testes are relatively small in species presenting monogamy or extreme poliginy (several females + few males), i.e., where a male copulates with all females of a group or harem. Comparative studies have demonstrated that males tend to be larger than females and show secondary sexual characteristics in species present- ing extreme poliginy. On the other hand, the relative testis weight is high and the sexual size dimorphism is reduced or nonexistent in species where several males copulate with only one estrus female (polyandry). In this case, the evolution for a large testis is attributed to the sperm competition in a system where different males attempt to fertilize the same female and where a higher copulatory frequency and higher levels of sperm production are required (Harcourt et al., 1981; Kenagy and Trombulak, 1986). Using the data on 133 mammal species, Kenagy and Trombulak (1986) presented a function describing the relationship between body weight and combined-testes weight without epididymis. Applying their equation for the adult male franciscanas, we discovered that mature franciscanas have testes 3 to 12 times lighter than expected (mean = 6 times) for a mammal of its body weight. Indeed, among the 133 species analyzed, 590 Fishery Bulletin 102(4) the relative testes weight of the franciscana is heavier than that of gorilla {Gorilla gorilla), humpback whales (Megaptera novaeangliae), and fin whales iBalaenoptera physalus), indicating that sperm competition does not occur in franciscanas. Sexual size dimorphism Males are larger than females in most mammal species. Nevertheless, the reversed sexual size dimorphism (RSSD) (i.e., females are larger than males) is more common than previously thought and has been documented for 12 out of the 20 orders of mammals (Ralls, 1976, 1977). Among the odontocetes, four (Ziphidae, Pontoporiidae, Phocoenidae, and Delph- inidae) out of the eight families present RSSD. Although sexual selection may be the main reason why males are the larger sex in most mammal species, it has been systematically refused as an explanation in the cases where females are the larger sex (Ralls, 1976, 1977; Andersson, 1994). In species with RSSD, females do not mate with many males, they are not dominant, and are not more aggressive than males of the same species. Moreover, they do not show secondary sexual characteristics associated with intrasexual selection (e.g., horns in Artiodactyla and large canine teeth in Primates). Therefore, the occurrence of RSSD in mam- mals may be explained more satisfactorily by natural selection (Andersson, 1994). Slooten (1991) proposed an interesting hypothesis for the occurrence of RSSD in cetaceans, suggesting that a minimum size may be necessary for a newborn cetacean to survive. In odontocetes, the smallest mean sizes at birth are about 70-80 cm. Because the size of the newborn is directly related to the size of the moth- er, in species of small dimensions the females would suffer a selective pressure to be a larger size, so that they could produce offspring with the minimum viable size. This hypothesis is reinforced by the fact that most of the odontocete species with RSSD (e.g., Pontoporia blainvillei, Cephalorhynchus hectori, Cephalorhynchus commerssoni, Phocoena phocoena, Phocoena sinus) are the smallest species within the group. Moreover, spe- cies presenting RSSD also have larger relative size at birth than the other species within the taxonomic group (Ralls, 1976). The degree and direction of SSD (sexual size dimor- phism) in mammals is the result of the difference of the sum of all selective pressures affecting the female's size and the sum of all selective pressures affecting the male's size (Ralls, 1976). Thus, it is very probable that more than one factor may act selectively on animals of both sexes in Pontoporia, molding the degree and direction of SSD. We propose that the requirement of a neonate minimum viable size (70-80 cm in length) is one of the main selective pressures acting on female franciscanas. It is important to emphasize that other factors may also be influencing SSD in franciscana, and in some species it was evident that different selec- tive pressures could affect body size in opposing direc- tions in males and females and in different age classes (Grant, 1986; Andersson, 1994). Among the factors that may be simultaneously acting on franciscana body size are intrinsic genetic and physiological limitations, and the requirement of maintaining an optimum size for the species' ecological niche. Secondary sexual characteristics The presence and intensity of secondary sexual characteristics in males is a more precise indication of the degree of intrasexual selection than is body size (Andersson, 1994). In odonto- cete males, these characteristics are present in the form of "weapons," such as the tusk of the narwhal (Monodon monoceros) and the teeth in species of the genus Gram- pus, Physeter, Berardius, Hyperoodon, and Mesoplodon used in male-male combats (MacLeod. 1998). In spe- cies of these genus, the teeth were reduced in number, enlarged in size, and their form was modified (specially in males of Ziphiidae). The teeth of these species also lost their function in feeding because of a diet comprising almost exclusively cephalopods and were used uniquely in intrasexual combats. There is no evidence that the same evolutionary process occurred in male francisca- nas because their teeth are very small and numerous (around 200), their diet is primarily fish, and the number of combat scars is apparently low. These characteristics support the hypothesis that male-male combat must be very rare or even nonexistent in franciscanas. The sexual features presented in this study (extremely low testis weight, reversed sexual size dimorphism, ab- sence of secondary sexual characteristics in males, and a low number of scars in males) indicate the absence of sperm competition in the franciscana, and these features differ drastically from those characteristics of odonto- cete species where males combat each other for copula- tion. This finding may indicate that franciscanas form temporary reproductive pairs during the reproductive period, where a male pairs and copulate with only one female. Recently, Valsecchi and Zanelatto (2003) pro- vided molecular evidence suggesting that franciscanas may travel in kin groups that include mothers with their calves and the father of the youngest offspring. The au- thors also suggested that male franciscanas may prolong their bond with their reproductive partner, providing some form of paternal care. For a better understanding of franciscana social structure and mating system, the following suggestions are proposed: 1) an increase in the efforts of behavioral studies of free-ranging francisca- nas; 2) quantification of the intraspecific teeth scars in franciscanas of different sexes and reproductive status in order to confirm the absence of intrassexual aggres- sions among males; 3) investigation of the relationship of relative testis weight, SSD. and reproductive strate- gies in cetaceans, by phylogenetic methods (see Harvey and Pagel. 1991) to understand the evolution of these characters in this group. Acknowledgments This study could not be made without the cooperation and friendship of the fishermen from Tramandai/Imbe Danilewicz et al.: Reproductive biology of male Pontopona blainvillei from Rio Grande do Sul, southern Brazil 591 and Rio Grande. Many people collaborated in the collec- tion and necropsy of the dolphins, and the authors wish to thank Paulo Ott, Ignacio Moreno, Marcio Martins, Glauco Caon, Larissa Oliveira, Manuela Bassoi, Alexan- dre Zerbini, Luciana Moller, Luciano Dalla Rosa, Lilia Fidelix, and numerous volunteers for helping in this task. Gonad histology was partially done in Laboratorio de Histologia e Embriologia Comparada of Universi- dade Federal do Rio Grande do Sul and we thank Sonia Garcia and Nivea Lothhammer for their instructions and encouragement on this subject. Paulo Ott, Enrique Crespo and Silvana Dans participated in the age deter- mination procedures. Part of the age determination was also done in the Rio Grande and the first author thanks Cristina Pinedo and Fernando Rosas for their instruc- tion. The authors also thanks Norma Luiza Wiirdig, Iraja Damiani Pinto (CECLIMAR-UFRGS), and Lauro Barcellos (Director of the Museu Oceanografico) for their constant logistical support and for encouraging marine mammal studies in southern Brazil. Marcio Martins, Ignacio Moreno, Luiz Malabarba, and Monica Muelbert reviewed an early draft of this paper. We wish to thank Renata Ramos and three anonymous reviewers for their suggestions regarding the manuscript. Financial support was given by Cetacean Society International, Fundacao O Boticario de Protecao a Natureza, The MacCarthur Foundation, World Wildlife Fund, CNPq, UNEP, Yaqu Pacha Organization, and Whale and Dolphin Conser- vation Society. This paper is part of the first author's M.S. thesis, and The Coordenagao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) has granted him a graduate fellowship. The Conselho Nacional de Desenvolvimento Cientifico e Tecnologico of the Brazilian Government (CNPq) has granted graduate fellowships to E.R. Secchi (Grant no. 200889/98-2). Literature cited American Society of Mammalogy. 1961. Standardized methods for measuring and record- ing data on the smaller cetaceans. J. Mamm. 42: 471-476. Andersson, M. 1994. Sexual selection. Monographs in behavior and ecol- ogy, 599 p. Princeton Univ. Press. Princeton, NJ. Bordino, P. 2002. Movement patterns of franciscana dolphin {Pon- toporia blainvillei) in Bahia Anegada, Buenos Aires, Argentina. LAJAM (special issue) 1:71-76. Bordino, P., G. Thompson, and M. Iniguez. 1999. Ecology and behaviour of the franciscana {Pon- toporia blainvillei) in Bahia Anegada, Argentina. J. Cetacean Res. Manag. 1 (2):213-222. Collet, A., and H. 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Allen Press, Lawrence, KS. Ralls. K. 1976. Mammals in which females are larger than males. Q. Rev. Biol. 51:245-276. 1977. Sexual dimorphism in mammals: avian models and unanswered questions. Am. Nat. 111:917-937. Ramos, R. 1997. Determinacao de idade e biologia reprodutiva de Pontoporia blainvillei e da forma marinha de Sotalia fluviatilis no litoral norte do Rio de Janeiro. M.S. thesis, 95 p. Universidade Estadual do Norte Fluminense, Rio de Janeiro, Brazil. Ramos, R., A. P. Di Beneditto, and N. R. W. Lima. 2000. Growth parameters of Pontoporia blainvillei and Sotalia fluviatilis (Cetacea) in northern Rio de Janeiro, Brazil. Aquat. Mamm. 26 (11:65-75. Read, A. 1990. Reproductive seasonality in harbour porpoises, Phocoena phocoena, from the Bay of Fundy. Can. J. Zool. 68:284-288. Read, A., and A. Hohn. 1995. Life in the fast lane: the life history of the har- bour porpoises from the Gulf of Maine. Mar. Mamm. Sci. 11:423-440. Secchi, E. R. 1999. Taxa de crescimento potencial intrinseco de um estoque de franciscanas, Pontoporia blainvillei (Ger- vais & D'Orbigny, 1846) (Cetacea, Pontoporiidae) sob o impacto da pesca costeira de emalhe. M.S. thesis, 152 p. Fundacao Universidade Federal do Rio Grande, Rio Grande, do Sul, Brazil. [In Portuguese.] Secchi, E. R., A. N. Zerbini, M. Bassoi, L. Dalla Rosa, L. M. Moller, and C. C. Rocha-Campos. 1997. Mortality of franciscanas, Pontoporia blainvillei, in coastal gillnetting in southern Brazil. Rep. Int. Whal. Comm. 47:653-658. Secchi, E. R., Ott, P. H., and Danilewicz, D. 2002. Report of the fourth workshop for the coordi- nated research and conservation of the franciscana dolphin [Pontoporia blainvillei) in the western South Atlantic. LAJAM (special issue) 1:11-20. 2003. Effects of fishing bycatch and the conservation status of the franciscana dolphin, Pontoporia blainvillei. In Marine mammals: fisheries, tourism and manage- ment issues (N. Gales, M. Hindell, and R. Kirkwood, eds.), p. 174-191. CSIRO Publishing, Collingwood, Victoria, Australia. Slooten. E. 1991. Age, growth, and reproduction in Hector's dolphins. Can. J. Zool. 69:1689-1700. Slooten, E„ and F. Lad. 1991. Population biology and conservation of Hector's dolphin. Can. J. Zool. 69:1701-1707. Stearns, S. C. 1992. The evolution of life histories, 249 p. Oxford. Univ. Press, New York, NY. Valsecchi, E.. and R. C. Zanelatto. 2003. Molecular analysis of the social and population structure of the franciscana (Pontoporia blainvillei): conservation implications. J. Cetacean Res. Manag. 5il):69-75. Van Waerebeek, K.. and A. Read. 1994. Reproduction of dusky dolphins, Lagenorhyn- chus obscurus, from coastal Peru. J. Mamm. 75 i 11:1054-1062. 593 Abstract— Red snapper (Lutjanus campechanus) in the United States waters of the Gulf of Mexico ( GOM ) has been considered a single unit stock since management of the spe- cies began in 1991. The validity of this assumption is essential to manage- ment decisions because measures of growth can differ for nonmixing popu- lations. We examined growth rates, size-at-age, and length and weight information of red snapper collected from the recreational harvests of Ala- bama (n=2010), Louisiana (re=1905), and Texas (re=1277) from 1999 to 2001. Ages were obtained from 5035 otolith sections and ranged from one to 45 years. Fork length, total weight, and age-frequency distributions dif- fered significantly among all states; Texas, however, had a much higher proportion of smaller, younger fish. All red snapper showed rapid growth until about age 10 years, after which growth slowed considerably. Von Ber- talanffy growth models of both mean fork length and mean total weight- at-age predicted significantly smaller fish at age from Texas, whereas no differences were found between Ala- bama and Louisiana models. Texas red snapper were also shown to differ significantly from both Alabama and Louisiana red snapper in regressions of mean weight at age. Demographic variation in growth rates may indicate the existence of separate management units of red snapper in the GOM. Our data indicate that the red snapper inhabiting the waters off Texas are reaching smaller maximum sizes at a faster rate and have a consistently smaller total weight at age than those collected from Louisiana and Alabama waters. Whether these differences are environmentally induced or are the result of genetic divergence remains to be determined, but they should be considered for future management regulations. Red snapper (Lutjanus campechanus) demographic structure in the northern Gulf of Mexico based on spatial patterns in growth rates and morphometries Andrew J. Fischer Coastal Fisheries Institute School of the Coast and Environment Louisiana State University Baton Rouge, Louisiana 70803-7503 E-mail address: afische g Isu edu M. Scott Baker Jr. Coastal Fisheries Institute School of the Coast and Environment Louisiana State University Baton Rouge, Louisiana 70803-7503 Charles A. Wilson Coastal Fisheries Institute and Department of Oceanography and Coastal Sciences School of the Coast and Environment Louisiana State University Baton Rouge, Louisiana 70803-7503 Manuscript submitted 6 May 2003 to the Scientific Editor's Office. Manuscript approved for publication 19 April 2004 by the Scientific Editor. Fish. Bull. 102:593-603 12004). Red snapper {Lutjanus campechanus) in the United States waters of the Gulf of Mexico (GOM) are heavily exploited by both recreational and commercial fishermen (Wilson and Nieland, 2001; Shirripa and Legault1). Harvest, how- ever, has not proceeded without det- rimental affects on the population. Commercial landings have declined substantially from 6048 metric tons (t) in 1964 to 1207 t in 1990; recreational landings exhibited similar declines from 1937 t in 1981 to 481 t in 1990 (NMFS'2). In 1991, harvest restrictions including reef fish permits, seasonal fishing, fish quotas, creel limits, and minimum size limits were placed upon the red snapper fishermen by the Gulf of Mexico Fishery Management Council (GMFMC3) to increase the spawning potential ratio to 20%, which is indic- ative of recovery. These regulations have also been adopted for state waters in Alabama, Louisiana, and Texas. Despite the management actions, GOM red snapper remain overfished (Good- year4; Schirripa and Legault1). 1 Shirripa, M. J., and C. M. Legault. 1999. Status of the red snapper in the U. S. waters of the Gulf of Mexico; updated through 1998, 44 p. + appendices. Con- tribution rep. SFD-99/00-75 from Sus- tainable Fisheries Division. Miami Laboratory, Southeast Fisheries Science Center, National Marine Fishery Ser- vice, 75 Virginia Beach Drive, Miami, FL 33149-1099. [Not available from NTIS], 2 NMFS (National Marine Fisheries Service). 2003. Fisheries Statistics and Economics Division. Website: www. nmfs.noaa.gov. 3 GMFMC (Gulf of Mexico Fishery Man- agement Council). 1991. Amendment 3 to the reef fishery management plan for the reef fish resources of the Gulf of Mexico, 38 p. Gulf of Mexico Fish- ery Management Council, 3018 N. U.S. Hwy 301 Suite 1000, Tampa, FL. 33619- 2272. [Not available from NTIS], 4 Goodyear, C. P. 1995. Red snapper in U.S. waters of the Gulf of Mexico. Stock assesment report MIA-95/96-05, 171 p. Miami Laboratory, Southeast Fisheries Science Center, National Marine Fish- eries Service, 75 Virginia Beach Dr., Miami, FL, 33149-1099. [Not available from NTIS]. 594 Fishery Bulletin 102(4) 96c 94c 92 ; 90 88 : 86 : 84 82 ; 80 32° ? /> ^M, — , 30° Fourchon, Daulphin Island, AL , 28° 7* LA 26° S Port Aransas TX -**-^ 24° N 400 0 400 800 Kilometers A 22° 32 30 28 26 24 22 96 94° 92° 90 88° 86° 84 82 = 80 Figure 1 Map of the northern Gulf of Mexico showing the three red snapper iLutjanus campecha- nus) sampling locations. An underlying assumption crucial to a fishery man- agement plan is that the fish species being managed is a unit stock (Gulland, 1965). A stock is defined as the part of a fish population that is under consideration as an actual or potential resource (Ricker, 1975). Since management began in 1991, red snapper in the north- ern GOM have been considered a unit stock. Genetic studies to date have shown that there is little evidence to dispute this assumption (Camper et al., 1993; Gold et al., 1997, Heist and Gold, 2000). On the other hand, tag-recapture studies indicate that red snapper have the capacity to move great distances, making it pos- sible for separate stocks to develop (Patterson et al., 2001). The validity of an assumption of a single stock of red snapper is essential to management decisions because measures of growth, natural mortality, reproductive capacity, and recruitment can differ among nonmix- ing populations. Should separate red snapper stocks exist, management plans would have to be enacted for each defined stock in order to follow federal guide- lines. Even if a single large red snapper stock exists, management should be sensitive to both the diversity of habitats and user groups within the species area of occurrence. Because red snapper are arguably the most important recreational and commercial offshore fishery from Florida to southern Texas, every effort should be undertaken to develop the most effective and productive management plan. The objective of this study was to evaluate the stock structure of GOM red snapper based on growth rates and size-at-age information. We hypothesized that red snapper sampled from across the northern GOM would be indistinguishable in their growth rates and size at age — a uniformity indicative of a single unit stock. Methods and materials Red snapper were collected from the recreational har- vests of 1999, 2000, and 2001 from the northern GOM at Dauphin Island, Alabama, at Port Fourchon, Louisi- ana, and at Port Aransas, Texas (Fig. 1). A maximum of 75 fish were randomly selected and sampled from the daily catch of each charter boat or head boat while the captains and deck hands cleaned fish. These fish were not selected by size. Larger individuals (>6.8 kg) were opportunistically sampled from spear fishing and hook-and-line fishing tournaments in Alabama and Louisiana. In addition, a number of smaller fish (<406 mm. <457 mm during summer 1999) were randomly sampled during red snapper tagging cruises in Alabama. Morphometric measurements were recorded I fork length [FL] in mm, total weight [TW] in kg, and eviscerated body weight |BW| in kg), sex was determined by macro- scopic examination of gonads, and both sagittal otoliths were removed, rinsed, and stored in coin envelopes until processed. Fish weights were not recorded for 1999 Texas samples. A transverse thin section (containing the core) was taken from the left sagittal otolith of each individual. Sections were made with the Hillquist model 800 thin- sectioning machine equipped with a diamond embedded wafering blade and precision grinder (Cowan et al., 1995). When the left otolith was unavailable, the right otolith was sectioned. Examinations of otolith sections were made with a dissecting microscope with transmit- ted light and polarized light filter at 20x to 64x mag- nification Opaque annulus counts were made along the ventral side of the sulcus acousticus from the core to the proximal edge (Wilson and Nieland. 2001). Annulus counts were performed by two independent readers (AJF Fischer et al.: Demographic structure of Lutjanus campechanus in the northern Gulf of Mexico 595 and MSB) without knowledge of either date of capture or morphometric data. The appearance of the otolith section edge condition was coded as opaque or translu- cent after Beckman et al. (1989). Annuli were counted a second time when initial counts disagreed. In instances where a consensus between the two readers could not be reached, annulus counts of the more experienced reader (AJF) were used. Between-reader differences in annulus counts were evaluated with the coefficient of variation (CV), index of precision (D) (Chang, 1982), and average percent error (APE) (Beamish and Fournier, 1981). The periodicity of opaque zone formation was verified for each sampling location with edge analysis after Wilson and Nieland (2001). Ages of red snapper were estimated from opaque annulus counts and capture date with the equation described by Wilson and Nieland (2001): Day age= -182 + (opaque increment count ((»;-!) x 30) + d, 365) + where m = the ordinal number (1-12) of month of cap- ture; and d = the ordinal number (1-31) of the day of the month of capture. The 182 days subtracted from each age estimate are to account for the uniform hatching date assigned for all specimens (Render, 1995; Wilson and Nieland, 2001). Age in years was assigned by dividing day age by 365. Fork length-TW relationships were fitted with lin- ear regression to the model FL = a TWb from log1(l- transformed data for Alabama, Louisiana, and Texas specimens. Analysis of covariance (ANCOVA) was used to compare slopes and intercepts among sampling lo- cations (SAS, 1985). Variability in age. FL, and TW frequency distributions of red snapper were compared among states with the Komolgorov-Smirnov two-sample test (Tate and Clelland, 1957). Growth of red snapper was modeled for FL and TW with the von Bertalanffy growth equations. Because of differences in sample population size among states, weighted mean FL and mean TW at age were fitted for each state with nonlinear regression in the forms: FL, = LJ1 -e'-*"11) TW, = Wjl-el-'''"'!)''. where FL, = FL at age t\ TW, = TW at age /; L„ = the FL asymptote; W„ = the TW asymptote; k = the growth coefficient; t = age in years; and b = exponent derived from our length-weight regressions (SAS, version 5, 1985, SAS Inst, Cary, NO. Because of a lack of smaller individuals in all sample populations, no y-intercepts for t0 were specified and models were forced through 0. Larger individuals and Table 1 Numbers of red snapper (Lutjanus camped lanus) sampled from recreational sources by stai e and year. State Males Females Jnknown sex Total Alabama 1999 434 396 5 835 2000 355 415 7 111 2001 189 209 0 Total 398 2010 Louisiana 1999 367 339 31 737 2000 399 397 8 804 2001 160 179 25 Total 364 1905 Texas 1999 268 293 14 575 2000 278 284 22 584 2001 52 56 10 Total 118 1277 juveniles selectively sampled by size were excluded from the models to more accurately reflect a random sample. Likelihood ratio tests (Cerrato, 1990) were used to test for differences among states in models and in growth parameter estimates. Differential growth was evalu- ated for red snapper in the first 10 years of life when somatic growth is most rapid (Szedlmayer and Shipp, 1994; Patterson et al., 2001; Wilson and Nieland, 2001). Linear regressions of mean FL and mean TW at age for fishes aged 1 to 10 years were compared among states with analysis of covariance (ANCOVA) and tested for homogeneity of slopes. Results During the three-year study period, 5192 red snapper were sampled from the recreational harvest of the north- ern GOM (Table 1): 642 individuals from fishing tourna- ments, 71 undersize fish from tagging cruises, and 4479 random samples from recreational catches. The samples included 2502 males, 2568 females, and 122 individu- als of undetermined sex. The resultant male-to-female ratios were 0.96:1 for Alabama, 1:0.99 for Louisiana, 0.94:1 for Texas, and 0.97:1 for all states combined. A chi-square test indicated no significant difference in the number of males to females (j2=0.78, P=0.38). Fork lengths ranged from 237 to 916 mm (Fig. 2A). Speci- mens from Alabama ranged from 237 to 916 mm FL, Louisiana specimens ranged from 282 to 913 mm FL, and Texas specimens ranged from 266 to 846 mm FL. The FL frequency distributions of the random samples were different among all states (AL and LA, maximum difference (MD)=5.26; AL and TX, MD = 51.86; LA and TX, MD = 51.77)(Fig. 2A). 596 Fishery Bulletin 102(4) 30 -> 25 20 15 10 n ,nnM DAL ■ LA DTX tlk^^idirftr^.,. 125 250 300 350 400 450 500 550 600 650 700 750 800 850 900 Fork length (m) 50 45 40 35 30 25 20 15 10 5 0 B :; IV ryfk .ryryry r* n .rim m ri r. . . .fl 5 6 7 Total weight (kg) 10 >10 Figure 2 Distributions of (A) fork length in mm (/! = 5177) and (B) total weight in kg (n = 4531l for red snapper (Lutjanus campechanus) sampled from the 1999-2001 recreational harvests of Alabama. Louisiana, and Texas. Total weights of all fish sampled ranged from 0.11 to 17.35 kg (Fig. 2B). Specimens from Alabama ranged from 0.22 to 15.42 kg TW, Louisiana specimens were 0.42 to 17.35 kg TW, and Texas specimens ranged from 0.33 to 9.42 kg TW. Total weight-frequency distributions (in 0.5 kg increments) differed significantly between all states (AL and LA, MD = 5.37; AL and TX, MD = 53.68; and LA TX, MD = 52.28)(Fig. 2B). Significant differ- ences in red snapper FL-TW regression models were detected among states (ANCOVA test of homogeneity of slopes, F5 4522=23.36; P<0.001; r2=0.98; ANCOVA test for equal intercepts, F5 4522=22.77, P<0.001, r2=0.98); therefore, separate models were fitted for each state. The resultant equations were AL TW= 1.51 x 10-"' iFL:,,i:i) (F1;1965=102740; P<0.0001; r2=0.98); TX TW = 2.88 x 10-5 (PL2-92) LA TW = 1.02 x 10-r' iFL ') ^1; = 13345; P<0.0001; r2=0.95). (P 1;1856 =77981; P<0.0001; r2=0.98); Ages were obtained from 5035 transverse otolith sec- tions. Thirty fish had otolith sections deemed unread- able by both readers. The age estimates determined by the two readers were evaluated for reader agreement, precision, and average percent error for first and sec- ond readings of otolith sections by sample year. Table 2 gives APE, CV, D. percentage agreement (O), and per- centages of differences in age estimates (±1, 2, and 3 years). The readers agreed on age estimates for 4053 otoliths (80.5%) after the initial reading. Re-examina- tion of the 982 otolith sections for which annulus counts differed produced agreement for 5007 individuals. We compared the timing of opaque annulus formation among red snapper sample sites by plotting the monthly occurrence of maximum and minimum proportions of opaque otolith edges. Sample limitations of red snapper in Texas, however, prevented meaningful comparisons of Fischer et al.: Demographic structure of Lut/anus campechanus in the northern Gulf of Mexico 597 opaque annulus formation for this state. However, mini- mum proportions of opaque edges during the months of April through October may indicate that red snapper from Texas form an opaque annulus during the winter months. Proportions of opaque edges for Alabama and Louisiana were essentially the same: maximum propor- tions of opaque edges during the months of February and March followed by a decrease to minimum propor- tions during the months of May through November (Fig. 3). These findings are consistent with previous age and growth studies on red snapper in the northern COM (Patterson et al, 2001; Wilson and Nieland, 2001), indicating that the formation of one opaque annulus in the winter months is followed by the formation of one translucent annulus in summer. Annulus-based age estimates of red snapper from the northern GOM have also been validated to 55 years with otolith radiocarbon chronologies based on accelerator mass spectrometry 14C measurements (Baker and Wilson, 2001). Red snapper ages ranged from 1 to 45 years and the majority (90%) of individuals were between 2 and 6 years (Fig. 4). Alabama fish ranged from 1 to 35 years (re=1985), Louisiana fish ranged from 2 to 37 years (n=1864). and Texas fish ranged from 1 to 45 years (rc=1186). Modal ages were 4 years for Alabama and 3 years for Louisiana and Texas red snapper. We found significant differences among age-frequency distribu- tions from all states (AL and LA, MD = 9; AL and TX, MD = 33.84; and LA and TX, MD=24.84). Texas had a much higher proportion of younger individuals; 63% of sampled fish were aged at 3 years or less compared to only 30% of Alabama and 39% of Louisiana fish aged at 3 years or less. Red snapper growth was modeled from weighted mean FL at age and mean TW at age by using the von Berta- lanffy growth equation (Fig. 5, A and B). Resultant von Bertalanffy growth equations were Table 2 Differences between two readers in average percent error (APE). ?oefficient of variation (CV), index of precision (D). and in percentages of agreement ( O ) for counts of opaque annuli in red snapper (Lutjanus campechanus) otoliths after fi rst and second readings for each sample year. «=number of otoliths sa mpled. Year 1st reading 2nd reading 1999 in =2100) APE 0.483 0.499 CV 0.014 0.0008 D 0.010 0.0006 O 89.48% 99.43% ±1 8.62% 0.48% ±2 1.19% 0.095% ±3 0.71'. 2000 (n =2069) APE 0.487 0.499 CV 0.034 0.0006 D 0.024 0.0004 O 73.79f; 99.47% ±1 22.49', 0.53% ±2 1.78% ±3 1.93% 2001 in = 866) APE 0.459 0.498 CV 0.032 0.0005 D 0.023 0.0003 O 74.73* 99.42^ ±1 22.06% 0.58% ±2 2.27% ±3 0.94% ALFL. = 839(1 -e1-0381"1) (F, 15= 2824.9; P<0.0001; r2=0.95); LAFL„ = 847.8(1 - e'-° •25"") (F1; 13=5024.4; P<0.0001; r2=0.76); TXFL„ = 778.2(1 - e<-o.49tt)>) (F1;19=1452.1; P<0.001; r2=0.85); AL TW 17.05(1 -e (-0.15inii3.03 (F1;15=457.9; P<0.0001; r2=0.89); LA TW_._ = 12.61(1 - ec-o.32(»))3.03 (F114=122.02; P<0.0001; ;-2 = 0.18); TX TWrr, = 8.89(1 - e'-0-21"")2 84 (F1;12= 613.01; P<0.0001; r2=0.96). Models of mean red snapper FL at age for Alabama and Louisiana were markedly similar with likelihood ratio tests indicating no significant differences between red snapper from the two states (Table 3). However, the Texas model differed from both Alabama and Louisiana models. The Texas model displayed significant differ- ences from the other models in both Lm and in k. A comparison of the models of mean TW at age indicated no significant differences between Alabama and Loui- siana red snapper (Table 3). Differential growth in TW was found when comparing Alabama and Louisiana with the Texas model; significant differences were manifested in both WM and in k. The model failed to converge for estimating a common value of k for both Louisiana and Texas. We recognized that the larger red snappers from Louisiana might bias the data; therefore we compared growth for fish from 2 to 10 years of age — a time pe- riod when red snapper have demonstrated rapid linear growth (Szedlmayer and Shipp, 1994; Patterson et al., 2001; Wilson and Nieland, 2001). Linear regressions of mean FL at age for all individuals 2 to 10 years (Fig. 6A) were compared among states. We found no significant differences among states (ANCOVA test of homogeneity of slopes, F2;28=2.7; P=0.08; ANCOVA test for equal intercepts, F2.28=0.52; P=0.6). 598 Fishery Bulletin 102(4) Mean TW at age was also examined among states for red snapper 2 to 10 years in age as above (Fig. 6B). No significant differences were found between Alabama and Louisiana (ANCOVA test of homogeneity of slopes, F117= 0.1; P=0.75; ANCOVA test for equal intercepts, F1;'17= 0.26; P=0.66 for intercepts). However, a significant difference between slopes was detected when compar- ing Alabama and Texas red snapper (ANCOVA test of homogeneity of slopes, F1;16=19.68; P<0.0007; ANCOVA test for equal intercepts, F1;16=2.74; P<0.12). The same was found when comparing slopes for Louisiana and Texas red snapper I ANCOVA test of homogeneity of Figure 3 Marginal increment analysis of red snapper {Lutjanus campecha- nus) otoliths for specimens from Alabama (n = 1985l, Louisiana (n = 1864), and Texas . 45 -, 40 ■ J DAL 35 - ■ LA £. 30 - DTX Frequency o en | - 15 - n 10 - 1 5 - JJ 1 flflr^m-rm.^ . _ r». o - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 >15 Age (yr) Figure 4 Age distri butions for red snapper (Lutjanus campechanus) sam- pled from the 1999-2001 recreational harvests from Alabama, Louisiana , and Texas. slopes, Fl 16=9.62; P<0.008) but not when comparing intercepts' (Fh 16 = 0.64; P<0.44). Discussion Demographic variations in growth rates and in size- frequency distributions may indicate the existence of isolated management units of red snapper in the north- ern GOM. The recreational harvests of Alabama and Louisiana red snapper were dominated by individuals ranging from 375 to 425 mm FL, whereas the majority of Texas fish (69%) were 375 mm FL or less. It was within this size range (375-400 mm FL) that the significant differences in red snapper among states were detected. The FL distribu- tion of red snapper sampled in Texas also dif- fered from those for Alabama and Louisiana; there were very few large fish represented in the Texas sample population, partly because fishing tournaments (where larger individuals are targeted) were not sampled in Texas. Signifi- cant differences in TW frequencies among states were also detected at approximately 1 kg (the approximate weight of a red snapper 375-400 mm FL); 86% of Texas fish weighed 1 kg or less, compared to only 27% of Alabama fish and 28% of Louisiana fish in this size range. One factor possibly contributing to the modal size class difference was the type of fishing vessel used to catch the fish. The majority of Texas specimens I~95fr<) were sampled from headboats; whereas Louisiana and Alabama fish were obtained almost exclusively from char- terboats. This is not to say that charterboats were purposely excluded from the Texas sur- vey. On the contrary, red snapper were sampled from any and all available recreational fishing parties at the three individual sampling loca- tions. Differences in modal size and number of red snapper caught per person onboard charter- boats versus headboats may be inconsequential considering that both trip types used similar gear and targeted similar or the same fishing locations. It should be noted however that in the Texas study area, charterboats routinely frequented a wider array of fishing spots (rigs, hardbottom. wrecks, etc.) than did headboats, which typically return to the same few rigs and large structures over and over again iTolan 5), Our von Bertalanffy growth models on FL at age showed that red snapper from all three states exhibit a pattern of rapid, linear growth to approximately 10 years, after which maximum theoretical (asymptotic) FL is soon Tolan, J. 2003. Personal commun. Texas Parks and Wildlife Department, Resource Protection, 6300 Ocean Dr., Corpus Christi, TX 78412. Fischer et al.: Demographic structure of Lut/anus campechanus in the northern Gulf of Mexico 599 Table 3 Chi-square if), degrees of freedom (df ), and P-values for likelihood ratio tests for comparing FL and TW von Bertalanffy growth models and parameters among sample locations (states). AL= Alabama; LA= Louisiana: and TX=Texas. n/a=not available. r df P r df p AL-LA FL model 2.54 1,28 0.11 TW model 2.15 1,29 0.14 FL model 5.14 1,34 0.023 TW model 38.8 1,27 4.7x10" AL-TX LA-TX k FL model 13.67 1,34 0.0002 21.53 1,34 3.48xl0"6 5.8 1,32 0.015 k TW model 21.3 1,27 3.9x10" 37.8 1,27 7.97xl0-10 16.77 1.26 4.2xl0"5 10.16 1,32 0.001 15.1 1,26 0.001 k 9.8 1,32 0.002 n/a n/a n/a reached and growth in length becomes negligi- ble. This pattern of rapid growth was similar to that reported in previous studies (Szedlmayer and Shipp, 1994; Manooch and Potts, 1997; Patterson, 1999; Wilson and Nieland, 2001). However, our models predicted smaller L r and higher values of It. Because of the minimum size limits on the recreational fishery, very few fish under age 2 years (>300 mm FL) were in- cluded in our sample populations. We forced our models through t0 = 0 to more accurately pre- dict juvenile growth, which in turn increased our estimates of k. In addition, we had a much larger sample population that included more older, larger fish than most of the previously cited studies. These larger fish pulled the curve down, driving the lesser estimations of LM. The lack of significant differences in growth param- eters between the Alabama and Louisiana mod- els supports the findings of previous research, which indicates that Alabama and Louisiana red snapper grow at similar rates and reach comparable sizes (Patterson et al., 2001). How- ever, values of LM for Texas red snapper were significantly smaller than parameters predicted for Alabama and Louisiana red snapper. In- terestingly, Texas had a value of k that was significantly larger then that for Alabama and Louisiana and this would indicate that Texas fish obtain a smaller maximum theoretical FL but reach it at a faster rate then fish from Ala- bama and Louisiana. Von Bertalanffy growth models of mean weight at age produced similar results, in- dicating that Texas red snapper obtain sig- nificantly smaller maximum theoretical TW than fish from Alabama and Louisiana. Fish sampled from tournaments were excluded from all growth models to more accurately reflect 30 35 40 45 B 15 ■ 12 - 0 5 10 15 20 25 30 35 40 45 Age (yr) Figure 5 Observed (A) mean fork length (mml at age and (B) mean total weight (kg) at age for red snapper iLutjanus campechanus) from Alabama, Louisiana, and Texas. Plotted lines are weighted von Bertalanffy growth functions fitted to the data. 600 Fishery Bulletin 102(4) growth of a random population. Tournament anglers target large fish, possibly the fastest growing individu- als at a given age, and their catches may bias growth estimates (Ottera, 1992; Vaughan and Burton, 1993; Goodyear, 1995). Without these tournament fish, how- ever, the Alabama red snapper TW model did not reach an asymptote. Therefore the growth parameters for that model were poorly estimated. Notwithstanding, Alabama and Louisiana models did not differ signifi- cantly. Estimates of Wm and k predicted for Louisiana red snapper were slightly larger than previously re- ported for fish from the Louisiana commercial and recreational catches (Render, 1995). Although the Texas model predicted a value of Wc. that was significantly less than those for both Alabama and Louisiana red snapper, Texas had a growth coefficient (k) that was larger then that for Alabama. It appears that, as in the length models, Texas fish reach a smaller theoretical maximum weight but at a faster rate than Alabama fish. Louisiana fish attained maximum weight at a faster rate than Alabama or Texas red snapper. Our growth models indicate that although Texas red snap- 900 800 E E 700 600 *— c CD 500 400 O u. 300 200 100 0 12 10 A j A X '. ^T^' r \_ • AL 1 LA XTX — i 10 B 01 23456789 10 Age (yr) Figure 6 Scattergram with linear regression lines for relationships (A) between age (yr) and mean fork length (mm) and (B) age (yr) and mean total weight (kg) for red snapper (Lutjanus campechanus) aged 1 to 10 years from the 1999-2001 rec- reational harvests of Alabama. Louisiana, and Texas. Krror bars represent standard deviations from the mean. per grow in mass at a faster rate than Alabama fish. Texas red snapper are consistently smaller at age and reach smaller maximum sizes than those from Alabama and Louisiana and that there is a veritable difference in size at age and growth rates among regions. Similar demographic variations in growth rates among popula- tions have been previously noted for other marine fish species of the South Atlantic and GOM, such as gray snapper (Johnson et al., 1994; Burton 2001), and king mackerel (DeVries et al, 1990; DeVries and Grimes, 1997). Linear regressions of mean FL and mean TW at age for red snapper aged one to 10 years indicated that only TW was significantly different among sample regions. Texas red snapper were shown to differ significantly from both Alabama and Louisiana red snapper in re- gressions of mean weight at age. Although comparisons of FL at age for all regions were not significantly differ- ent, Texas fish were significantly smaller in mass (TW) at age than fish from Alabama and Louisiana. This difference was observed in all age classes. Our research efforts indicate that there is mounting evidence for discrete differences in size at age and in overall growth rates between red snapper sampled from the north central GOM (Louisiana and Alabama) and the southwest GOM (Texas). Texas red snapper are clearly reaching smaller maximum sizes and are consistently smaller (TWi at age than those collected from Louisiana and Alabama waters. Although the reasons behind these differences remain uncertain, logic indicates that factors such food availability, habitat prefer- ence, and actual population size may cause these differences between regions. The more productive, nutrient-rich waters of the Mississippi River and north-central GOM off Louisiana and Alabama may be more conducive to faster growth than the less fertile waters off Texas. Approximately 70-80% of GOM fishery landings come from the waters surrounding the Mississippi River delta (Grimes, 2001). The west- ern GOM (including the sampling area of Port Aransas, TX) is devoid of a contributing river sys- tem anything remotely similar to the Mississippi River. Draining 43% of the continental United States, the Mississippi River is the largest river system in North America and provides an enor- mous amount of nutrient-laden fresh water to the shallow continental shelf of the northern GOM. Although the mechanics by which the Mississippi River enhances fishery production remain uncer- tain. Grimes (2001) postulated that the discharge from the Mississippi primarily influences recruit- ment m the plume field. Increased growth rates associated with the Mississippi River plume com- pared with other regions of the GOM have been noted for a number of species, such as gulf menha- den (Warlen, 1988), king mackerel (DeVries et al., 1990), striped anchovy (Day, 1993), and yellowfin tuna (Lang et al., 1994). Fischer et al.: Demographic structure of Lut/anus campechanus in the northern Gulf of Mexico 601 In addition to increased food availability off of the north-central GOM, the amount and condition of pre- ferred habitat may have some effect on the observed differences in growth rates for Texas and those for Louisiana and Alabama. Approximately 95"% of all Louisiana fishes sampled in this study were harvested from waters surrounding nearshore (<50 km) oil and gas platforms. Similarly, about 95% of all Alabama fishes sampled were caught over artificial reef sites. The fact that there was no detectable difference in size at age and overall growth rates between Louisiana and Alabama red snapper therefore is not surprising, given the similarity in the habitats sampled and the proxim- ity of both locations to the Mississippi River discharge plume. Texas was the only area in which samples were routinely obtained from natural hard bottoms (40%), as well as from oil and gas platforms and artificial reefs (60%). Given that more than half of the Texas specimens were captured in the waters immediately surrounding artificial structures (i.e., oil and gas plat- forms), we can assume that habitat type is not be the sole source for the observed differences in growth rates among regions. Despite the current acceptance of a unit stock hypoth- esis for GOM red snapper, the species is not, and to our knowledge never has been, uniformly distributed across the northern GOM. The fishery for red snapper began in northwest Florida approximately 20 years before the Civil War (Collins, 1887) and during that time period was centered between Mobile, AL, and Fort Walton, FL (Camber, 1955). One hundred years of landings data indicate that the fishery, and possibly the population, has undergone a major shift from the natural outcrop- pings of the West Florida Shelf to oil and gas platforms of the north-central portion of the GOM (Shirripa and Legault1). Fishery-dependent data indicate that cur- rently there is a center of abundance of red snapper off southwest Louisiana and a second, smaller center off Alabama (Patterson et al., 2001; Goodyear4; Shirripa and Legault1). Patterson et al. (2001) stated that Loui- siana and Alabama accounted for 32.6% and 11.4%, respectively, of the combined recreational and commer- cial GOM landings from 1981 to 1998. This is especially surprising for Alabama, considering that its coastline accounts for only 3% of the GOM coastline from the Texas-Mexico border to the southern tip if Florida (Pat- terson et al., 2001). Red snapper have never been reported to be plentiful in Texas waters, despite the availability of suitable hab- itat in the form of natural hard bottom and the cur- rent high concentration of oil and gas platforms. In a historical report on red snapper fishing in the GOM, Camber ( 1995 ) reported that although a few red snap- per were taken from the "Galveston Lumps" or the "Western" fishing grounds off Texas, the fishery never fully developed in this region during the latter part of the nineteenth century. Commercial landings for red snapper from the GOM indicated that Texas accounted for approximately only 18% of the total catch during the time period 1981-95 (Goodyear6). In a recent fish- ery-dependent survey of recreational headboat discards and landings in Texas coastal waters, red snapper less than the minimum legal size (15 inches) made up 64% of the catch (Dorf. 2000). In the latter study, Galveston, Port Aransas, and Port Isabel were surveyed to canvas a large portion of the Texas coast. Discard-to-landing ratios were as high as 211:1 in the waters off Galveston and were possibly indicative of the paucity of legal-size red snapper in Texas waters. Of the three sampling locations. Port Aransas had the lowest discard-to-land- ing ratio (5.2:1) and the largest mean fish length and weight (387 mm, 0.9 kg) — length and weight data that are consistent with a 3-yr-old fish from our Texas (Port Aransas) specimens. The majority of Texas fish (63%) were aged at 3 years or less. Age distribution, along with FL and TW distributions, may indicate that red snapper are being harvested from Texas waters just as they reach legal size. Given the vast differences in historical landings data between the northern and southwest GOM, the highly disproportionate discard- to-landing ratio reported for headboats in Texas wa- ters (Dorf, 2000), and the large number of young fish sampled in Texas, it is not inconceivable to speculate that there are fewer red snapper available for harvest in Texas waters. Demographic variation in growth rates may indicate the existence of separate management units of red snap- per in the GOM. Our data indicate that the red snapper inhabiting the waters off Texas are reaching smaller maximum sizes at a faster rate, but are consistently smaller (TW) at age than those collected from Louisi- ana and Alabama waters. Whether these differences are environmentally induced or result from genetic diver- gence remains to be determined. The more productive, nutrient-rich waters of the Mississippi River and north- central GOM off Louisiana and Alabama may be more conducive to faster growth than the less fertile waters off Texas. Fishing pressure and its effects on population size may also be leading to the observed differences in growth rates. Fishery-dependent landing data and dis- proportionate discard-to-landing ratios in Texas waters loosely support the concept that fewer red snapper are available for harvest in the southwest GOM. Regardless of the cause, the existence of demonstrable demographic differences argues for the delineation of multiple red snapper management units in the GOM. Acknowledgments Funding for this research was provided by the U.S. Department of Commerce Marine Fisheries Initiative (MARFIN) program (grant number NA87FF0424). We 6 Goodyear, C. P. 1996. An update of red snapper harvest in U.S. waters of the Gulf of Mexico. Report MIA-95/96- 60, 21 p. Miami Laboratory, Southeast Fisheries Center, National Marine Fisheries Service. 75 Virginia Beach Dr. Miami, FL., 33149-1099. [Not available from NTIS]. 602 Fishery Bulletin 102(4) thank Forrest Davis, John Gold, Jessica McCawley, Linda Richardson, Jim Tolan, Melissa Woods, Candace Aiken, and many others for help with sampling red snap- per. We also thank Josh Maier and Brett Blackman for otolith sectioning. We thank Steve Tomeny and his boat captains and crew (Port Fourchon, LA), as well as all boat captains and crews in Dauphin Island, Alabama, and Port Aransas, Texas, for graciously allowing us to sample fish from their charter fishing vessels. We also thank Yvonne Allen for providing the map in Figure 1. Finally we would like to thank Dave Nieland for time spent fielding questions concerning statistical analysis and for a constructive review of this manuscript. Literature cited Baker, M. S. Jr., and C. A. Wilson. 2001. Use of bomb radiocarbon to validate otolith sec- tion ages of red snapper Lutjanus campechanus from the northern Gulf of Mexico. Limnol. Oceanogr. 46:1819-1824. Beamish, R. J., and D. A. Fournier. 1981. A method for comparing the precision of a set of age determinations. Can. J. Fish. Aquat. Sci. 38:982-983. Beckman, D. W., C. A. Wilson, and A. L. Stanley. 1989. Age and growth of red drum, Sciaenops ocel- latus, from offshore waters of the northern Gulf of Mexico. Fish. Bull. 87:17-28. Burton, M. L. 2001. Age. growth, and mortality of gray snapper, Lut- janus griseus, from the east coast of Florida. Fish. Bull. 99:254-265. Camber, C. I. 1955. A survey of the red snapper fishery of the Gulf of Mexico, with special reference to the Campeche banks. Fla. Board Conserv. Tech. Ser. No. 12:1-64. Camper, J. D., R. C. Barber, L. R. Richardson, and J. R. Gold. 1993. Mitochondrial DNA variation among red snapper (Lutjanus campechanus) from the Gulf of Mexico. Mol. Mar. Biol. Biotech. 2:154-161. Cerrato, R. M. 1990. Interpretable statistical tests for growth com- parisons using parameters in the von Bertalanffy equation. Can. J. Fish. Aquat. Sci. 47:1416-1426. Chang, W. B. 1982. A statistical method for evaluating the repro- ducibility of age determination. Can. J. Aquat. Sci. 39:1208-1210. Collins, J. W. 1887. Report on the discovery and investigation of fishing grounds made by the Fish Commision Steamer Albatross during a cruise along the Atlantic Coast and in the Gulf of Mexico. Rep. U.S. Comm. Fish. 13:217-311. Cowan, J. H. Jr., R. L. Shipp, H. K. Bailey IV, and I). W. Hnywick. 1995. Procedure for rapid processing of large otoliths. Trans. Am. Fish. Soc. 124:280-282. Day, G K 1993. Distribution, abundance, growth and mortality of striped anchovy, Anchoa hepsetus, about the discharge plume of the Mississippi River. M.S. thesis, 45 p. Univ. West Florida, Pensacola. FL. DeVries, D. A., and C. B. Grimes. 1997. Spatial and temporal variation in age and growth of king mackerel, Scomeromorus cavalla. 1977-1992. Fish. Bull. 95:694-708. Devries, D. A., C. B. Grimes. K. C. Lang, and D. B. White. 1990. Age and growth of king and Spanish mackerel larvae and juveniles from the Gulf of Mexico and U.S. south Atlantic. Environ. Biol. Fishes 29:135-143. Dorf, B. A. 2003. Red snapper discards in Texas coastal waters: a fishery-dependent onboard survey of recreational head- boat discards and landings. In Fisheries, reefs, and offshore development (D. R. Stanley and A. Scarborough- Bull, eds.i, p. 155-166. Am. Fish. Soc. Symposium 36. Am. Fish. Soc, Bethesda. MD. Gold J. R., F. Sun, and L. R. Richardson. 1997. Population structure of red snapper from the Gulf of Mexico as inferred from analysis of mitochondrial DNA. Trans. Am. Fish. Soc. 126:386-396. Goodyear, C. P. 1995. Mean size at age: an evaluation of sampling strat- egies using simulated red grouper data. Trans. Am. Fish. Soc. 124:746-755. Grimes, C. B. 2001. Fishery production and the Mississippi River discharge. Fisheries 26:7-26. Gulland, J. A. 1965. Fish stock assessment: a manual of basic methods, 223 p. John Wiley & Sons, New York, NY. Heist, E. J, and J. R. Gold. 2000. DNA microsatellite loci, and genetic structure of red snapper in the Gulf of Mexico. Trans. Am. Fish. Soc. 129:469-475. Johnson, A. G, L. A. Collins, and C. P. Kelm. 1994. Age-size structure of gray snapper from the South- eastern United States: a comparison of two methods of back-calculating size at age from otolith data. Proc. Annu. Conf. Southeast Assoc. Fish and Wildl. Agencies 48:592-600. Lang, K. L.. C. B. Grimes, and R. F. Shaw. 1994. Variations in the age and growth of yellowfin tuna larvae. Thunnus albacares, collected about the Mississippi River plume. Environ. Biol. Fishes 39:259-270. Manooch, C. S., Ill, and J. C. Potts 1997. Age and growth of red snapper. Lutjanus campecha- nus, Lutjanidae, collected along the southeastern United States from North Carolina through the east coast of Florida. J. Elisha Mitchell Sci. Soc. 113:111-122. Ottera. H. 1992. Bias in calculating growth rates in cod (Gadus morhua L.) due to size selective growth and mortality. J. Fish Biol. 40:465-467. Patterson, W. F. III. 1999. Aspects of the population ecology of red snapper Lutjanus campechanus in an artificial reef area off Alabama. Ph.D. diss.. 164 p. Univ. South Alabama, Mobile. AL. Patterson. W. F III. James H. Cowan Jr.. Charles A. Wilson, and Robert L. Shipp. 2001. Age and growth of red snapper, Lutjanus campecha- nus, from an artificial reef area off Alabama in the northern Gulf of Mexico. Fish. Bull. 99:617-627. Render. .Ill 1995. The life history (age, growth, and reproduction) of red snapper [Lutjanus campechanus) and its affinity Fischer et al.: Demographic structure of Lut/anus campechanus in the northern Gulf of Mexico 603 for oil and gas platforms. Ph.D. diss., 76 p. Louisiana State Univ., Baton Rouge, LA. Ricker, W. E. 1975. Computation and interpretation of biological statis- tics offish populations, 382 p. Bull. Fish. Res. Board Can., Ottawa. Szedlmayer, S. T., and R. L. Shipp. 1994. Movement and growth of red snapper, Lutja- nus campechanus. from an artificial reef area in the northeastern Gulf of Mexico. Bull. Mar. Sci. 55(2-3): 887-896. Tate. M. W.. and R. C. Clelland. 1957. Non-parametric and shortcut statistics in the social, biological, and medical sciences, p. 93-94. Interstate Printers and Publishers, Inc., Danville, IL. Vaughan, D. S., and M. L. Burton. 1993. Estimation of von Bertalanffy growth parameters in the presence of size-selective mortality: a simulated example with red grouper. Trans. Am. Fish. Soc. 123:1-8. Warlen, S. M. 1988. Age and growth of larval gulf menhaden, Brevoortia patronis, in the northern Gulf of Mexico. Fish. Bull. 86:77-90. Wilson, C. A., and D. L. Nieland. 2001. Age and Growth of red snapper, Lutjanus campechanus, from the northern Gulf of Mexico off Louisiana. Fish. Bull. 99:653-664. 604 Abstract— The population struc- ture of walleye pollock \Theragra chalcogramma) in the northeastern Pacific Ocean remains unknown. We examined elemental signatures in the otoliths of larval and juvenile pollock from locations in the Bering Sea and Gulf of Alaska to determine if there were significant geographic variations in otolith composition that may be used as natural tags of population affinities. Otoliths were assayed by using both electron probe microanalysis (EPMA) and laser ablation inductively coupled plasma mass spectrometry iICP-MS). Ele- ments measured at the nucleus of otoliths by EPMA and laser abla- tion ICP-MS differed significantly among locations. However, geographic groupings identified by a multivariate statistical approach from EPMA and ICP-MS were dissimilar, indicating that the elements assayed by each technique were controlled by sepa- rate depositional processes within the endolymph. Elemental profiles across the pollock otoliths were generally- consistent at distances up to 100 fim from the nucleus. At distances beyond 100 /im, profiles varied significantly but were remarkably consistent among individuals collected at each location. These data may indicate that larvae from various spawning locations are encountering water masses with differing physicochemical properties through their larval lives, and at approximately the same time. Although our results are promising, we require a better understanding of the mechanisms controlling otolith chemistry before it will be possible to reconstruct dispersal pathways of larval pollock based on probe-based analyses of otolith geochemistry. Elemental signatures in otoliths of pollock may allow for the delineation of fine-scale population structure in pollock that has yet to be consistently- revealed by using population genetic approaches. Elemental signatures in otoliths of larval walleye pollock (Theragra chalcogramma) from the northeast Pacific Ocean* Jennifer L. FitzGerald Simon R. Thorrold Biology Department, MS 35 Woods Hole Oceanographic Institution Woods Hole, Massachusetts 02543 E-mail address (for J L. FitzGerald): ifitzgerald awhoi edu Kevin M. Bailey Annette L. Brown NOAA Alaska Fisheries Science Center 7600 Sand Point Way NE Seattle, Washington 91185 Kenneth P. Severin Department of Geology and Geophysics University of Alaska Fairbanks P.O. Box 755780 Fairbanks, Alaska 99775-5780 Manuscript submitted 4 August 2003 to the Sceintific Editor's < X'fice. Manuscript approved for publication 28 May 2004 by the Scientific Editor. Fish. Bull. 102:604-616(20nl The "stock" concept is a central tenet of modern fisheries science because it represents the fundamental manage- ment unit of marine fisheries (Begg and Waldman, 1999). This emphasis, in turn, places a premium on accu- rate identification of groups of fish whose population statistics are largely independent of other groups. However, stock identification has often proved problematic in marine fishes. For instance, the stock structure of wall- eye pollock {Theragra chalcogramma* across the North Pacific Ocean has been a topic of investigation for many years. Early studies were based on phenotypic characteristics of pol- lock, such as meristics and morpho- metries (Serobaba. 1977; Hinckley, 1987; Temnykh, 1994). Other studies have focused on genotypic markers, such as DNA and allozyme analyses (Grant and Utter, 1980; Mulligan et al., 1992; Shields and Gust, 1995). These approaches resulted in only the broadest characterization of pollock stock structure but have been able to distinguish populations from the eastern and western Pacific (Bailey et al., 1999). Quasi-isolated subpopula- tions may be at least demographicallv isolated on smaller spatial scales. For instance, within the Gulf of Alaska, spawning pollock aggregate at specific locations in Shelikof Strait, Prince William Sound, and in the Shumagin Islands region (Bailey et al., 1999). However, the extent of larval dis- persal from the spawning sites and the degree of spawning site fidelity of adult pollock to these locations remains unknown. The difficulties associated with de- termining stock structure in fishes are essentially the same ones that currently limit our ability to deter- mine population connectivity in ma- rine systems (Thorrold et al.. 2002). Tag-recapture studies using tags have limited applicability in the case of pollock. Adults are located deep in the water column and are sensitive to barotrauma during the process of being caught, brought to the surface, and tagged. Traditional population genetic approaches may be similarly Contribution 11219 from the Woods Hole Oceanographic Institution. Woods Hole, MA 02543. Fitzgerald et al.: Elemental signatures in otoliths of larval Theragra chakogramma 605 ineffective because of the low level of exchange required to maintain genetic homogeneity, at least over ecological time scales, and the low level of genetic drift associated with large populations (Waples, 1998; Hellberg et al., 2002). However, preliminary studies have indicated that otolith geochemistry may prove to be a useful natural tag of population structure in walleye pollock (Severin et al., 1995). Otoliths are accretionary crystalline struc- tures located within the inner ear of teleost fish. They are formed through concentric additions of alternating protein and aragonite layers around a central nucleus. The use of otoliths as natural geochemical tags is con- tingent on the metabolically inert nature of the otolith and the fact that once deposited, otolith material is neither resorbed nor metabolically reworked (Campa- na and Neilson, 1985; Campana, 1999). The chemical composition of otoliths also reflects to some degree the physicochemical characteristics of the ambient water (Bath et al., 2000). If the water where pollock reside has distinct oceanographic characteristics, then many of the elements incorporated into the otoliths should differ among locations. Migrations between water masses at some age will, therefore, be recorded in the chemical composition of the otolith at the appropriate daily incre- ment. Natural geochemical signatures in otoliths may therefore be useful markers of environmental history throughout the life of the individual and in turn, fish stock composition (e.g., Campana et al., 1995). The use of geochemical signatures in otoliths as natu- ral tags requires accurate and precise assays of otolith composition. Electron probe micro-analysis (EPMA) has been commonly used for probe-based analyses of otolith chemistry (Gunn et al., 1992). However, detection lim- its of approximately 100 ,ug/g limit the technique to a relatively small number of minor elements in otoliths, including Na, CI, K, and Sr (Campana et al., 1997). Most of the elements measured by EPMA are probably controlled by physiological rather than environmen- tal factors, which may limit their usefulness in stock identification studies (Campana, 1999). Nonetheless, a number of researchers using EPMA have reported geo- graphic differences in otolith chemistry (e.g., Thresher et al., 1994). More recently, attention has focused on inductively coupled plasma mass spectrometry (ICP-MS) to assay elements that are typically below the detection limits of EPMA. Laser ablation ICP-MS uses focused Nd:YAG or excimer lasers to ablate specific locations on the otolith. The vaporized material is then swept up by a carrier gas into a plasma torch and analyzed by mass spectrometry. Limits of detection of the technique are typically on the order of 0.1-l^g/g, allowing for quan- tification of several elements that cannot be assayed by using EPMA including Mg, Mn, Ba, and Pb (Thorrold et al., 1997; Thorrold and Shuttleworth, 2000). These observations led Campana et al. (1997) to conclude that EPMA and laser ablation ICP-MS were complementary and that there is little overlap in the elements that are accurately measured by the two techniques. Yet few studies of otolith geochemistry have attempted to use both approaches on the same samples. The objectives of this study are to determine if larval walleye pollock from different geographic localities can be distinguished based on elemental signatures in their otoliths. By analyzing sagittal otoliths with both EPMA and laser ablation ICP-MS, we hoped to identify greater differences among locations than would have been pos- sible by using either technique in isolation. If success- ful, the study may provide a powerful tool for determin- ing stock structure and tracing migration pathways of walleye pollock in the north Pacific. These data could then be used by managers of one of the world's largest single species fisheries to direct the sustainable harvest of this considerable natural resource. Materials and methods All fish used in the study were collected in the spring and summer of 1999 from Alaska Fisheries Science Center research cruises in the Bering Sea and Gulf of Alaska (Fig. 1, Table 1). Fish of birth year 1999 were collected within three months of spawning time to minimize the likelihood of larval transport from other regions. In the case of the Yakutat samples, fresh juvenile pollock were removed from Pacific cod guts. Samples were collected only when the pollock were readily identifiable and heads were intact. Otoliths showed no visible sign of degradation from digestive processes. Juvenile pollock were frozen whole and transported to the laboratory for otolith removal. Otoliths were removed from the fish and mounted on petrographic slides in LR White resin (acrylic, hard- grade). Larval otoliths were ground on one side to expose the nucleus by using 500-grit paper and were polished with 0.25-um grit diamond paste. Juvenile otoliths were ground and polished in the sagittal plane on both sides to maximize clarity of the nucleus during microanalysis. Electron probe microanalysis After having been polished, the otoliths were cleaned with Formula 409® and coated with a 30-nm layer of carbon. They were subsequently analyzed with a Cameca SX-50 electron microprobe equipped with four wavelength dispersive spectrometers. A 15keV, 10 nA, 4-/jm diameter beam was used for all analyses. Counting times, standards, detection limits, and analytical errors are summarized in Table 2. Although Mg was analyzed in all otoliths, in most cases it was below detection limits and was therefore not used in the statistical analysis. Laser ablation ICP-MS After having been ground and polished, otolith sections were decontaminated before elemental analysis by using laser ablation ICP-MS. Sections were rinsed in ultra- pure water, scrubbed with a nylon brush in a solution of ultrapure H,,0, triple rinsed with ultrapure 1%HN03, 606 Fishery Bulletin 102(4) •;"- North Bering Sea 1$' ■"\T7 60N Bristol 15V"^C' bay • j *?p* Prince William Sound -55N SE Bering Sea *>•.. Shelikof Strait ^ A Yakutat •**>&* 160W I Figure 1 Locations of sampling sites for larval and juvenile walleye pollock (Theragra chalcogramma the Gulf of Alaska and Bering Sea. ) in sonified for 5 minutes in ultrapure H90, and finally triple rinsed again with Milli-Q water. The section was dried under a positive flow hood for 24 hours and stored in a polyethylene bag. Elemental analyses were conducted with a Finnigan MAT Element2 magnetic sector field ICP-MS and Mer- chantek EO LUV266X laser ablation system (Thorrold and Shuttleworth, 2000). Instrument set-up was simi- lar to that outlined by Giinther and Heinrich (1999). An Ar gas stream was used to carry ablated material from the laser cell to the ICP-MS. The carrier gas was then mixed with the Ar sample gas and a wet aerosol (1% HN03) in the concentric region of the quartz dual inlet spray chamber. The wet aerosol was supplied by a self-aspirating PFA micro-flow (20 /./L/min) nebulizer attached to a CETAC ASX100 autosampler. Diameter of the 266-nm laser beam was nominally 5 j.im, repeti- tion rate was 5 Hz, and the scanning rate was set at 5 /im/sec. A typical run for an individual otolith consisted of a blank sample (l%HNO:! only), a standard sample, five laser samples, and then another blank and standard. The number of laser samples in a run ranged from 5 to 15, depending upon the size of the otolith. All laser runs began with a 70 fim x 70 /.im raster, centered on the otolith nucleus. The laser software was then used to trace out concentric lines, 720 /jm in length and ap- proximately 40 /jm apart, which followed the contour of individual growth increments from the raster to the otolith edge. This approach produced reasonably high spatial resolution (30-50 u,m) for life history scans across otoliths while allowing sufficient acquisition time to maintain measurement precision. We examined Mn/Ca, Sr/Ca, and Ba/Ca ratios in the pollock otoliths by monitoring 48Ca, 55Mn, 86Sr, and 138Ba. Quantification followed the approach outlined by Rosenthal et al. (1999) for precise element/Ca ra- tios using sector field ICP-MS (Thorrold et al., 2001). Quality control was maintained by assaying a dissolved aragonite standard (Yoshinaga et al., 2000) every five samples. The standard was introduced at the appropri- ate time by moving the autosampler probe from the solution containing the 1% HNO;j to the standard solu- tion, while maintaining the carrier gas flow through the ablation cell. Elemental mass bias was calculated by reference to known values of the standard, and a correction factor was then interpolated and applied to the laser samples bracketed between adjacent standard measurements. Average u; = 40) within-run precisions (RSD) of the standard measurements were all less than 1% (Mn/Ca: 0.16%, Sr/Ca: 0.16%, and Ba/Ca: 0.33%). Long-term (5-month) estimates of the standard mea- surements (n=40), again uncorrected for changes in mass bias over time, were less precise (Mn/Ca: 5.6%, Sr/Ca: 3.7%, and Ba/Ca: 5.6%). However, laser samples were corrected for changes in mass bias by using the laboratory standard. Precision of the technique was ap- proximately 1% for all the ratios that we measured. Statistical analyses All elemental data were initially examined for nor- mality and homogeneity of variance by using residual analysis (Winer, 1971) and were found to conform to the assumptions of ANOVA without the need for data transformation. We therefore assumed that require- Fitzgerald et al.: Elemental signatures in otoliths of larval Theragra cha/cogramma 607 Table 1 Location, collection date, standard length range (mm), and sample sizes (n) of larval and juvenile walleye pollock iTheragra chalcogramma) capt ured from the southeast Bering Sea (SE Bering) North Bering Sea I N Ber ing), Bristol Bay. Shelikof Strait. Prince William Sound (PWSi, and Yakutat, and analyzed by laser ablation ICP-MS (ICP-MS) and electron probe microanalysis (EPMA). Area Date SL range (mm) n (total) in ICP-MS ) n (EPMA) SE Bering 23 May-27 July 1999 5.3-42.2 117 8 30 N Bering 18-23 July 1999 15.6-30.7 45 9 25 Bristol Bay 22-24 July 1999 85.1-135.7 75 11 28 Shelikof 27-28 May 1999 3.6-7.9 46 25 PWS 7 July-19 August 1999 35.2-66.0 11 4 6 Yakutat 15 July,1999 — 50 6 24 Table 2 Counting times for each element itime seconds], standards, limits of detection [LOD. 7rweight, 99<7f confidence limits] and ana- lytical errors 1 Error. '/c weight. 1 stand ird deviation]) for electron probe microanalysis (EPMA). Detection limits and analytical errors were calculated by following the procedures of Scott et al. (19951. N/A = not applicable. Element Time Standard LOD Error Na 60 Halite ( CM Taylor) 0.029 0.023 Mg 60 OsumilitelUSNM 143967) 0.019 0.022 P 60 Apatite (Wilberforce) 0.036 0.027 S 60 Gypsum (CM Taylor) 0.023 0.017 CI 46 Halite 1 CM Taylor) 0.027 0.015 K 46 OsumilitelUSNM 143967) 0.019 0.012 Ca 20 CalciteiNMNH 136321) N/A 0.245 Sr 120 Strontianite (Smithsonian R-10065) 0.036 0.019 merits for the MANOVA were also met by the data. Among-location differences in the elemental composition of larval pollock in specific regions of the otoliths were compared by using one-factor multivariate analysis of variance (MANOVA) and one-factor analysis of variance (ANOVA). We treated location as a fixed factor in both MANOVA and ANOVA tests. Because of difficulties col- lecting pollock larvae, we were unable to achieve equal replication of sites within locations. We therefore pooled samples from collections within a location by randomly selecting fish from each location for subsequent analysis. However, the lack of replication at the within-location level necessarily restricted our ability to draw general conclusions concerning spatial variability in otolith composition beyond the samples analyzed in the pres- ent study. All a posteriori comparisons among locations were performed by using Tukey's honestly significant difference (HSD) test (experimentwise error rate = 0.05). Multivariate differences in elemental signatures from the MANOVA were visualized by using canonical dis- criminant analyses (CDA). All analyses were conducting by using the SAS statistical program (SAS, version 6, 1990, SAS Inst. Inc., Cary, NO. Comparisons of elemental profiles across otoliths were made with repeated measures ANOVA. We tested the following null hypotheses: 1) there was no variation in trace element profiles across individual otoliths (i.e., from the nucleus to the edge), 2) there were no differ- ences in mean element concentrations among locations, determined by averaging data across individual otolith profiles, and 3) there were no differences in the pat- tern of element profiles across otoliths among locations. Otolith profiles with missing values were removed, and therefore we were able to use MANOVA for the repeated measures analysis. The multivariate approach to re- peated measures is generally more conservative than univariate repeated measures analysis. However, the multivariate test does not assume sphericity of orthogo- nal components, requiring only that the data conform to multivariate normality with a common covariance ma- trix for individual larvae at each location (Littell et al., 1991). The approach still requires that adjacent points on the trajectories be equidistant. Therefore samples from EPMA were assigned to a distance category at intervals of 15 ftm (0 //m, 15 fim, 30 /.im , 45 /jm, 60 f/m, 75 /im, and 90 j/m) across the otolith, to a distance of 90 ^m from the nucleus. Samples were averaged when more than one measurement was available within a distance category. Laser ablation ICP-MS samples were assigned to a distance category at intervals of approxi- 608 Fishery Bulletin 102(4) Table 3 EPMA results of one-factoi ANOVA l degrees of freedom |df]; sums of squares [SS] mean square [MS]) at two positions (0- 20 ,um and 20-45 pm from the nuc leus) in otoliths of larval walleye pollock (Theragra c hal ■ogramma i collected from six locations: three locations in the Berin I Sea southeast B ;ring Sea [SB]; North Bering Sea |NB] Br stol Bay [BB] I and three in the Gulf of Alaska (Prince William Sound [PW]; Shelikof Strait [SH]- and Yakutat |YK1 ***= sign ficant at a = 0.05; ns = nonsignificant. A posteriori multiple comparisons among locat ons were conducted by using Tukey's hone 3tly significant difference ( HSD I. Loca- tions are ordered I left to right I from lowest to highest concentrations, and lines link locations that are not significantly different (experimentwise error rate = = 0.05) Element Source df SS . MS F P *>■* *° *o * * _•' " O . " A 4 .»-* II ' t o ° *4 » • •4-20246 Canonical variate 1 Figure 2 Plot of first two canonical variates con- trasting multivariate elemental sig- natures in otoliths of walleye pollock (Theragra chalcogramma) determined by using electron probe microanaly- sis, at 0-20 (im from the nucleus (A) and 20-40 Jim from the nucleus (B). Larvae were collected from the North Bering Sea (▲), southeast Bering Sea (O), Bristol Bay IB), Shelikof Strait !♦), Prince Wiiliam Sound lO>. and Yakutat (ft). ■ »\ J8 o - ° . ° 0OA * * i *tAcf H - ■ A. ° . ^ A V -3-113 Canonical variate 1 Figure 3 Plot of first two canonical variates contrasting multivariate elemental signatures in otoliths of walleye pol- lock (Theragra chalcogramma) deter- mined by electron probe microanalysis at 0-20 /im from the nucleus iAi and 20-40 jim from the nucleus IB). Larvae were collected from the North Bering Sea (A), southeast Bering Sea (O). and Bristol Bay ■ We used CDA to visualize multivariate differences among locations in reduced dimensional space. Three groups were readily discernible in a plot of the first two canonical variates (Fig. 2). Samples from the North Ber- ing Sea, the southeast Bering Sea, and Shelikof Strait formed one group separated from Yakutat, Bristol Bay, and Prince William Sound samples along the first ca- nonical variate. The second canonical variate separated Yakutat samples from Bristol Bay and Prince William Sound individuals. Elemental signatures at 20-45 jum from the otolith nucleus were distributed similarly in canonical space to samples from the otolith nucleus (Fig. 2). Three groupings were apparent in the canoni- cal plot, and Bering Sea larvae were separated from Bristol Bay and Prince William samples on canonical variate one, and Yakutat samples were separated from all other locations on canonical variate two. We then conducted a similar analysis with only samples from the southeast and North Bering Sea and Bristol Bay. Elemental signatures of larvae from the Bering Sea separated from Bristol Bay on canonical variate one. The southeast Bering Sea samples separated from the North Bering Sea along canonical variate two, although not as clearly as with the elemental signatures from the Bering Sea and Bristol Bay (Fig. 3). Elemental profiles across otoliths varied significantly, as determined by repeated measures ANOVA, among the five locations for Na, P, S, and Sr (Table 4). Both S and Sr concentrations declined from high values at the nucleus to significantly lower values towards the edge of the otolith (Fig. 4). Repeated measures ANOVA also provided a test of the differences among locations when data were averaged over the otolith profiles. Sig- nificant differences among locations were detected for 610 Fishery Bulletin 102(4) a. 5.5 5 45 4 3.5 3 25 2 1.5 1 05 0 1.5 I 5 $ I I J E. u i i llfl f i E_ to 2.5 E 15 0 15 30 45 60 75 90 0 15 30 45 60 75 90 Distance from nucleus (mm) Figure 4 Profiles of elemental concentrations, determined by electron probe microanalysis, from the nucleus out to a distance of approximately 90 Jim in the otoliths of larval walleye pollock iTheragra chal- cogramma) collected from the North Bering Sea (A), southeast Bering Sea (O), Bristol Bay (■), Prince William Sound (O), and Yakutat ("I. Individual points are mean ( + SE) values grouped at 15-f/m intervals. five elements (Sr, K, S, P, and Na). Finally, the interac- tion term (positionxlocation) in the repeated measures ANOVA tested the hypothesis that the shape of the elemental profiles differed among locations. There was a significant interaction between profile and location for K. Laser ablation ICP-MS We quantified Mn/Ca, Sr/Ca, and Ba/Ca ratios in the otoliths of larval walleye pollock using laser ablation ICP-MS. Both Mn and Ba were found at trace levels in otoliths, with average values of approximately 3 jimo}/ mol and 6 umol/mol, respectively. Strontium was present in the otoliths at an average concentration of approxi- mately 2.2 mmol/mol. A MANOVA detected significant differences among locations from a raster centered on the nucleus (MANOVA; Pillai's trace = 0.85; Flz99=3.26; P<0.0005), and from the average values of lines 40-80 pm from the nucleus (MANOVA; Pillai's trace = 0.99; F1299=4.1;P<0.0001). Univariate ANOVA and a posteriori multiple compari- sons by using Tukey's HSD revealed that Mn/Ca, Sr/Ca, 5 - B 5 - 0 o 0 - -"*dy ■ ■ ■ ■ 5 - 5 - ■ -4-2024 deamahcei eenhe(Jg Figure 5 Plot of the first two canonical variates contrasting multivari- ate elemental signatures in oto- liths of walleye pollock iTheragra chalcogramma) determined with laser ablation ICP-MS, at 0-40 fan from the nucleus (A) and 40-80 fim from the nucleus (Bi. Larvae and juveniles were collected from the North Bering Sea (At, south- east Bering Sea (O). Bristol Bay ■ Prince William Sound (♦), and Yakutat ( I. and Ba/Ca ratios varied significantly among locations at the otolith nucleus and at positions 40-80 um from the nucleus (Table 5). Samples from the North Bering Sea had consistently lower Sr/Ca and Ba/Ca ratios than those from the southeast Bering Sea at both positions. However, we noted only subtle differences among the Gulf of Alaska and Bristol Bay samples. We found a total of three groupings in canonical plots of multivariate elemental signatures from the otoliths of larval walleye pollock (Fig. 5). Samples from the North Bering Sea and Bristol Bay were separated along ca- nonical variate one. A third grouping, including larvae from the southeast Bering Sea, Prince William Sound, and Yakutat, clustered together in the center of the canonical plot. Samples from the nucleus and 40-80 fim outside the nucleus showed very similar geographic patterns. Repeated measures ANOVA detected significant dif- ferences in both Mn/Ca and Ba/Ca profiles from the nu- Fitzgerald et al.: Elemental signatures in otoliths of larval Theragra chalcogramma 611 Table 4 EPMA results from repeated-measures ANOVA of elements profiles across otoliths from walleye pollock Theragrc chalcogramma) larvae collected at five locations in the Ber ing Sea and the Gulf of Alaska. With in-subject effects (pr ofile and profilexlocationl tested bv using MANOVA (Pillai's trace I, and between sub ects effect (location I te sted by using ANOVA I degrees of freedom [df]: sums of s quares [SS]; mean squares [MS]) ** = significant at a = 0.05; ns = nonsignificant. Element Source df Pillai's trace or SS MS F P. Individual points are mean (±SE) values grouped at 40-jAm intervals. cleus out to a distance of approximately 200 jjm in the walleye pollock otoliths (Fig. 6, Table 6). The univariate test of location, averaged over the individual otolith profiles, was significant for both Sr/Ca and Ba/Ca. We also found significant interactions between profile and location for Mn/Ca, Sr/Ca, and Ba/Ca ratios (Table 6). Manganese values increased from the nucleus to the otolith edge at all locations, indicating that the signifi- cant interaction was generated by the observation that the profile from the North Bering Sea was considerably flatter than profiles from Bristol Bay and southeast Bering Sea. Strontium trajectories were more dynamic; profiles from some locations increased from the nucleus to the edge (Bristol Bay and Prince Williams Sound), 4.5 3.5- ' I li'f'Mi1 * ft ft ft ft ft ft ft ft ft z CO O CO O O * ft HHf . . I ft! *;**** tp 0 6 10 14 18 22 26 30 34 38 42 46 50 54 58 Distance from nucleus (mm x 10'2) Figure 7 Profiles of elemental ratios, determined by using laser ablation ICP-MS. from the nucleus out to a distance of approximately 600 fim in the otoliths of juvenile walleye pollock (Thuragra ehaleogramma) collected from Bristol Bay ■ and Yakutat i I. Individual points are mean (± SE) values grouped at 40-;/m intervals. profiles from other locations decreased (North Bering Sea and Yukatat), and a single location (southeast Ber- ing Sea) showed no obvious trend. Finally, profile varia- tions in Ba/Ca ratios among locations were dominated by a sharp increase in Ba/Ca ratios across the otoliths in the southeast Bering Sea samples. Profiles were ef- fectively horizontal for the other four locations. Otoliths in walleye pollock collected from Bristol Bay and Yakutat were significantly larger than those from the other four locations. We were, therefore, able to con- duct extended profiles in these otoliths out to a distance of approximately 600 fim (Fig. 7). After starting at similar values at the nucleus, Mn/Ca and Sr/Ca profiles from the two locations quickly diverged and appeared to vary largely independently over the remaining time periods. The Ba/Ca profiles also appeared to be vary- ing independently between the two locations, although the relative magnitude of differences between the two locations was smaller than for either Mn/Ca or Sr/Ca profiles. Fitzgerald et al.: Elemental signatures in otoliths of larval Theragra chalcogramma 613 Table 6 Laser ablat on ICP-MS results from repeated-measures ANOVA of elementa profiles across otoliths from walleye pollock l Ther- agra chalcogramma l larvae collected at fi ire locations in the Bering Sea and the Gulf of Alaska. Within-subject effects I profile and profile x location) were tested by using MANOVA iPillai's trace), and between subject s effect location) tested by using ANOVA (degrees of freedom [df]; sums of squares [SS]; mean squares [MS]). *** = significant at a = 0.05; ns = nonsignificant Element Source df Pillai's trace or SS MS F PBsp 0 „„„,„/), we considered the weighted average of the two estimators above: Bsp-Optimai = (("'' estimator 1 + (1-wO estimator 2), with weight w: 0<«'<1. We must choose the weight w of estimator 1 in order to minimize the variance ( Var(Bsp 0 „,„„„/)) of the esti- mator B.Sp.0p,„„o/. Var(Ssp.oP,„„n/» = Var(B»e+(l-w)(e+e')) = Varie +{l-w)e I = Var(e) + ( l-u;)2Var(e') + 2( 1-w i covariance(e.e') = a2+il-w)'2al2+2(l-wtc><71p where p = correlation between e and e'. For Var (Bsp Optimal t° be a minimum, the w derivative must be zero, yielding 628 Fishery Bulletin 102(4) 0.4 0.6 DEPM weighting Figure 4 The relationship between error variance between the two estimators for spawn- the optimally weighted estimate of Bsp (i.e., B SPOPTIUAL ). Error variance is shown for a range of error correlations from r = -0.9 to -0.2. 0 = ( 1-w ) 0; i.e., a2 + ct,2 + 2p ct,ct< a- + e; i.e., 2p < (-rjj- + e)laxa. This requires p < -0.5 when crl = a, and e is small. This relation has an important role in our decision of what is the best estimator. In Figure 4 the error variance for the estimator Bsp optimal 's shown for various DEPM weightings and a theoretical range of error correlations (i.e., between e and e) from r = -0.9 to -0.2. Our aim was to choose a DEPM weighting that provides minimal error variance along the most stable regions of the suite of error cor- relation curves, i.e., where the error correlation curves are flattest. The error correlation curves from -0.4 to -0.7 were the most stable and across these the DEPM weightings from 0.3 to 0.7 had the smallest error vari- ance. Therefore we choose 0.5 as our preferred DEPM weighting, which lies centrally within a stable part of the range of theoretical error correlations. Results The decline in spawning area in each region (Fig. 2) corresponded to declines in Bsp DEm ( Table 1), which in turn were reflected by the Bsp .optimal estimates (Fig. 5). We recognize that imbalance in the intensity of samples between years poses a problem for the interpolation of data between sampling stations but we contend that the collapse in distribution observed is of sufficient contrast to be a reliable reflection of the estimated 709f decrease in Sardinops biomass that resulted from the 1998-99 epidemic (Gaughan et al., 2000; Ward et al., 2001). Note that we have used Albany (Fig. 2A) as the primary sup- port for this contention because of the larger data set. The same pattern was observed at all regions, although it was not so marked for the west coast Sardinops (Fig. 2D) because estimated Bsp (this term hereafter is used generically) had already declined substantially between 1996 and 1998. Despite sometimes large intervals between consecutive surveys, there were two broad patterns in the trends for Sardinops Bsp during the 1990s (Fig. 5). Within each region on the south coast (Albany, Bremer Bay, and Esperance), Bs P DEPM remained relatively high in the early to mid 1990s before decreasing substantially by 1999. In contrast to the results from south coast DEPM surveys, the west coast estimated BSP_DEPM fluctuated widely (Table 1). This fluctuation resulted in a rela- tively poor fit of the optimal model and correspondingly wide CIs. Since 1996, when substantially more samples were routinely collected during each survey on the west coast, there has also been a decrease in Bsp consistent with that observed on the south coast. Inconsistency in the determination of variability es- timates around some B^PI)EPM estimates precludes any definitive statements about the relative precision of the Gaughan et al.: Distribution of Sardinops sagax off southwestern Australia 629 60000 ■ Albany 50000 • 30000 ■ 20000 ■ ■ . I , ■ 10000 ■ 0 ■ ■ 1994 1996 1998 2000 80000 - 3remer Bay 60000 - 40000 - ■ \ .p 20000 - " / 0 - "\ £ Year Figure 5 Sardinops sagax BSP_DEPM estimates Icircles with error bars) and Bsp qptimai estimates (squares with accompanying 95rr confidence interval boundaries! at four regions of southwestern Australia. In each case the confidence intervals that extend below the .v-axis have not been shown. Bspoptimal estimates. Notwithstanding this, the CIs for the optimal estimates always encompassed the DEPM point estimates. Because the CIs were so broad in rela- tion to the point estimates, only the point estimates for BSP_DEPM and BSP 0PTIMAL are further compared. The estimated Bsp .optimal indicates that for Albany the spawning biomasses were underestimated in 1992 and 1999 and overestimated in 1997 and that the differ- ence between estimates in each case was greater than 25% (Fig. 5, Table 4). Although the DEPM estimated that the Albany Bsp remained steady between 1995 (17,544 t) and 1997" (18,597 t), the PPS almost halved from 0.33 to 0.19 for these same surveys (Tables 1 and 2). For Bremer Bay the estimates for Bsp DEPM and the Bsp-optimal were within 20% (Fig. 5, Table 4). In Esper- ance the B SP OPTIMAL estimate indicates that the DEPM underestimated Bsp by 199r in 1994, but overestimated Bsp by 37% in 1999. The DEPM estimates of Sardinops Bsp on the west coast had the poorest fit against PPS. Thus, the optimal estimates of BSP differed by >30% in four of the five DEPM-based Bsp estimates. In particular, the 1994 and 1999 DEPM estimates were too low. and those for 1996 and 1998 were too high. Discussion Egg presence-absence analysis, i.e., proportion of positive stations (PPS), was used to objectively assess changes in the spawning area of Sardinops along the south and lower west coasts of WA between 1991 and 1999. The 630 Fishery Bulletin 102(4) 60000 - Espe ranee 50000 - 40000 ■ 30000 ■ . ■. • 20000 ■ 10000 ■ 0 ■ ■ 1992 1994 1996 1998 2000 aoooo - . West Coas 60000 - 40000 - ■ V ■ 20000 - \ y\ ■ 0 - / \ i 1994 1996 Year Figure 5 (continued) collapse in distribution was evident in 1999 for three of the four regions examined and has been attributed to a combination of fishing mortality, several years of poor recruitment, and two mass mortality events (Murray and Gaughan, 2003). The spawning stock in Albany and Bremer Bay decreased to a point where the annual total allowable catch (TAC) in these fisheries was reduced to zero. The concurrent decreases in Bsp _DEPM and PPS at the south coast regions in 1999, estimated shortly after the progression of an epidemic mass mortality (Gaughan et al., 2000), indicates a positive relationship between Bsp-depm and PPS. This widespread response provides support for the concept of using the PPS-BSP DEPM rela- tionship to objectively detect, albeit retrospectively, particularly suspect estimates of Bsp_DEPM. The marked decline in Bsp in 1999 to a very low level at Albany provided sufficient contrast in the time series of data to allow detection of an overestimation of spawning biomass in 1997. Although the difference may not appear to be overly large, the critical factor in this particular case is that the Bsp of 18,597 metric tons (t) was seen to be healthy, whereas an estimate of 13,660 t would have clearly indicated to the Management Advi- sory Committee a downward trend in Sar-dmops Bsp. In turn, such a result would have supported the contention that the stock was in decline, which was expected be- cause of several years of poor recruitment, as evidenced by catch-at-age data (Gaughan et al., 2002). Further- more, in 1998, during the 6 months prior to the mass mortality, the purse-seine fleet in Albany experienced significant difficulties in meeting catch expectations, which also indicated that the stock was at a low level. Although we cannot assess precision of the revised es- timates of Bsp, it is likely that the Bsp-OPTIMAI ^01" 1997 still overestimates the actual stock size. The evidence for a decline in Bsl, at Bremer Bay from 1994 to 1999, as suggested by the decline in PPS, was supported by trends in catch curves for that period, which showed very low levels of recruitment (Gaughan et al., 2002). The recruitment trends ensured that the annual TACs for Bremer Bay after the mid 1990s did not increase but instead were gradually reduced. The Gaughan et al.: Distribution of Sardmops sagax off southwestern Australia 631 Table 4 Comparison of estimates of spawning biomass for Sardi nops sagax from four regions in southwest era Aust ralia. Estimates obtained by using the daily egg produc ion method (DEPM) were re-estimated by using a model that considered the proportion of positive stations iPPS. see text and Table 2) during each of the DEPM surveys. In turn, a weighted or optimal, estimate was derived from the p •evious two estimates. The difference and ratio between the optimal and the DEPM esti mates are provided for comparison. Optimal estimates that fall outside the 95ri confidence intervals for the DEPM estimates and optimal:DEPM ratios that differ from 1 by greater than 0.25 are shown in bold type. DEPM estimate PPS estimate Optimal estimate DEPM-PPS estimate OptimahDEPM Albany 1991 19,300 20,190 20,209 -890 1.05 1992 16,994 25,456 21,811 -8462 1.28 1993 23,432 22,823 23,653 609 1.01 1994 31.330 26,334 29.438 4996 0.94 1995 17,544 14.484 16,347 -1197 0.93 1997 18,597 8339 13.660 10,258 0.73 1999 89 6584 3488 -6495 39.19 Bremer Bay 1992 19.280 21,346 22,689 -2066 1.18 1993 44,010 25,195 37.407 -6603 0.85 1994 28,458 24.495 29,204 3963 1.03 1999 4156 4199 4645 -43 1.12 Esperance 1993 32,252 23,542 28,220 -4032 0.88 1994 20,080 26,838 23,827 -6758 1.19 1995 31.900 28,721 30,705 3179 0.96 1999 17.396 4238 10.875 13,158 0.63 West coast 1993 41,250 48,318 43.048 -7068 1.04 1994 8714 27,350 17,049 -18,636 1.96 1996 60,228 20,968 39,845 39,260 0.66 1998 18,985 9117 13,723 9868 0.72 1999 5725 10.940 7714 -5665 1.35 very poor fit for Esperance may reflect the low sample size or may be indicative of a certain level of decoupling of BSP and PPS not evident in the other south coast regions. The 1996 estimate for the west coast was hampered by poor estimation of adult parameters resulting from a low number of adult samples obtained; the Bsp DEPM estimate for that year appeared to be much too high and, intuitively, was not used as the basis for making management decisions at that time. The precautionary decision to use the lower bound rather than the "best" estimate from the 1996 west coast DEPM survey was therefore justified. In contrast, the estimate of B^pDEP!il of 8714 t in 1994 for the west coast Sardmops stock appears to have been too low. The lack of an obvious collapse in distribution off the west coast was partly due to the marked changes in the intensity and dis- tribution of sampling after 1996. Another contributing factor may have been a change in the distribution of the spawning adults because of the anomalously warm water in the Indian Ocean in the late 1990s (Webster et al., 1999) during the last major La Nina. The PPS of only 0.10 in 1998, before the epidemic mortality, may therefore have been the result of behaviorally mediated changes in the distribution of Scuxlmops in response to the warmer than average water temperatures (Gaughan et al., 2000). We recognize that other factors may also have influenced the distribution of Sardinops off the west coast but our relatively short time series of data precluded development of more definitive, alternative hypotheses at this time. The potential for unusual en- vironmental conditions to influence spawning behavior applies equally to the south coast Sardinops; interpreta- tion of PPS data therefore also requires consideration of environmental conditions in each case. As our time series of biomass estimates is extended through further DEPM surveys, hypotheses regarding the influence of the environment will be further developed. Prelimi- nary hypotheses have already been presented to the Management Advisory Committee and thus form part of current management deliberations. The results from this retrospective analysis will im- mediately be used to reassess the Bsp estimates ob- tained for Sardinops in WA during the 1990s before 632 Fishery Bulletin 102(4) refitting them to Hall's (2000) integrated models for the three adult assemblages on the south coast of WA. The integrated model is tuned with BSPDEPM estimates. Therefore, replacing BSPDEPM estimates with BSP_0PTI_ MAL estimates will result in a model that better simu- lates the size of the Sardinops stocks off southern WA. Although the changes may appear trivial, it is impor- tant that re-estimating the most deviant estimates of Bspdepm can De undertaken in a manner that satisfies demands by stakeholders, including industry, for open- ness and clarity in the provision of scientific advice. As further DEPM surveys are conducted to assess the status of the Sardinops stocks in the five to six years following the 1998-99 mortality event, more reliable re- lationships between PPS and Bsp DEPM will be developed. To assist this process, the relative merits of the data for individual DEPM surveys can also be re-examined, particularly those data that this study has indicated to have resulted in poor estimates of BSPDEPM. An ongoing iterative approach that employs retrospective analyses will be undertaken in an attempt to continuously re- duce the variance of the PPS-BSP _DEPM relationship. This approach will permit further refinement of Hall's (2000) integrated model, a process already in prog- ress (Stephenson et al.5) and will therefore contribute to increased confidence in the scientific advice that is provided for management of the Sardinops fisheries in WA. Eventually, PPS alone may be sufficient to provide an indication of spawning biomass with an acceptable level of precision. Besides contributing to the integrated model, the BSP optimal point estimates obtained over nearly a decade in each of four management regions now provide a clearer indication of potential maximum biomass levels against which industry members can plan their businesses. Be- cause of the highly variable recruitment of many small pelagic fish, purse-seine businesses that target fish such as Sardinops should not invest at levels that require an economic return based on maximum biomass sizes. For the purse-seine fishing zones in southern WA the maxi- mum spawning biomass from which purse-seine indus- try members can expect their TAGS to be determined are as follows: west coast 40,000 t, Albany 29,000 t, Bremer Bay 37,000 t, and Esperance 30,000 t. Although these values provide an upper limit to business plan- ning, maximum biomasses should not represent invest- ment targets. These values provide an indication of the maximum size for the industry but, because of the "natural and social disarray" that can result "from har- vesting marine fish species at the crest of their produc- tion" (Smith 2000), the industry should be structured at a level that focuses on longer-term average biomass and that includes industry's ability to survive during periods of low stock size. Maximum and average Bsp for Sardinops at each of the four management regions Stephenson, P., N. Hall, and D. Gaughan. 2004. Unpubl. data. Department of Fisheries. Western Australian Ma- rine Laboratories, North Beach, Western Australia 6920, Australia. in southwestern Australia will be further investigated during ongoing development of the integrated model and as more information becomes available. Conclusion Even large numbers of plankton samples can result in imprecise estimates of egg production for use in DEPM calculations (e.g., Mangel and Smith, 1990). Relative trends in spawning area that can be obtained from the same survey by using egg presence-absence analysis provide a secondary means of assessing trends in the status of stocks. This egg presence-absence analysis will be particularly useful for stocks already assessed by using DEPM surveys and more so for those that do not have large amounts of ancillary information, such as long time-series of catch-at-age data, or meaningful effort data. Detection of either upwards or downwards bias in estimates of Bsp will be considered in the integrated model and also communicated to industry members to increase their understanding of the stock in each region. Although this review of biomass trends of Sardinops during the 1990s cannot change how Sardinops were managed during that period, an increased understand- ing of both the stock sizes and the science behind the biomass assessments will facilitate ongoing manage- ment processes. Acknowledgments The authors sincerely thank all Department of Fish- eries staff involved with the collection and analysis of historical ichthyoplankton-survey data, in particu- lar Stuart Blight, Gary Buckenara, Cameron Dawes- Smith, Rick Fletcher, Kieren Gosden, Matt Robinson, Rob Tregonning, Ken White, Bruce Webber, and other personnel on PV Baudin and PV McLaughlan. We also thank Kevern Cochrane (FAO) for a detailed review of an earlier version of this manuscript. We are grateful to the Fisheries Research and Development Corporation (Canberra) that provided funding through Project 92/25 for the earlier DEPM surveys. Literature cited Alheit. J. 1993. Use of the daily egg production method for estimat- ing biomass of clupeoid fishes: a review and evaluation. Bull. Mar. Sci. 53:750-767. Fletcher, W. J. 1991. A test of the relationship between otolith weight and age fin- the pi Ich a rd Sa rilin o/j.s ■ nctipili-hnrdua Can J. Fish. Aquat. Sci. 48:35-38. 1995. Application of the otolith weight-age relationship for the pilchard, Sardinops sagax neopilchardus. Can. J. Fish. Aquat. Sci. 52:657-664. 1999. Vertical distribution of pilchard tSardinops sagax) Gaughan et al.: Distribution of Sardinops sagax off southwestern Australia 633 eggs and larvae off southern Australia. Mar. Freshw. Res. 50:117-122. Fletcher, W. J„ N. C. H. Lo, E. A. Hayes, R. J. Tregonning, and S. J. Blight. 1996. Use of the daily egg production method to estimate the stock size of Western Australian sardines (Sardinops sagax). Mar. Freshw. Res. 47:819-825. Fletcher W. J., and N. R. Sumner. 1999. Spatial distribution of sardine i Sardinops sagax) eggs and larvae: an application of geostatistics and resampling to survey data. Can. J. Fish. Aquat. Sci. 56:907-914 Fletcher. W. J., and R. J. Tregonning. 1992. The distribution and timing of spawning by the Australian pilchard (Sardinops sagax neopilchardus) off Albany, Western Australia. Aust. J. Mar. Freshw. Res. 46:1437-1449. Fletcher, W. J., R. J. Tregonning, and G. J. Sant. 1994. Interseasonal variation in the transport of pilchard eggs and larvae off southern Western Australia. Mar. Ecol. Prog. Ser. 111:209-224. Gaughan, D. J„ W. J. Fletcher, and J. P. McKinlay. 2002. Functionally distinct adult assemblages within a single breeding stock of the sardine, Sardinops sagax: management units within a management unit. Fish Res. 59:217-231. Gaughan, D. J.. R. W. Mitchell, and S. J. Blight. 2000. Impact of mortality, possibly due to herpes virus, on pilchard Sardinops sagax stocks along the south coast of Western Australia in 1998-99. Mar. Freshw. Res. 51:601-612. Hall, N. G. 2000. Modelling for fishery management, utilising data for selected species in Western Australia. Ph.D. diss. ,199 p. Murdoch Univ., Perth, Australia. Hunter, J. R„ and N. C. H. Lo. 1997. The daily egg production method of biomass estima- tion: some problems and potential improvements. Oz- eanografika 2:41-69. Mangel, M„ and P. E. Smith. 1990. Presence-absence sampling for fisheries man- agement. Can. J. Fish. Aquat. Sci. 47:1875-1887. Murray, A. G., and D. J. Gaughan. 2003. Using an age-structured model to simulate the recovery of the Australian pilchard (Sardinops sagax) population following epidemic mass mortality. Fish. Res. 60:415-426. Parker, K. 1985. Biomass model for the egg production model. In An egg production method for estimating spawning biomass of pelagic fish: application to the northern anchovy. Engraulis mordax (R. M. Lasker, ed.i, p. 5-6. NOAA Tech. Rep. NMFS 36. Picquelle. S.. and G. Stauffer. 1985. Parameter estimation for an egg production method of anchovy biomass assessment. In An egg production method for estimating spawning biomass of pelagic fish: application to the northern anchovy, Engraulis mordax (R. M. Lasker, ed.i, p. 7-15. NOAA Tech. Rep. NMFS 36. Smith, P. E. 2000. Pelagic fish early life history: CalCOFI overview. In Fisheries oceanography: an integrated approach to fish- eries ecology and management (P. J. Harrison and T. R. Parsons, eds.l, p. 8-28. Blackwell Science. Oxford, UK. Smith. P. E„ H. Santander. and J. Alheit. 1989. Comparison of the mortality rates of pacific sar- dine, Sardinop sagax. and Peruvian anchovy, Engraulis ringens, eggs off Peru. Fish. Bull. 87:497-508. Uphoff, J. H. 1993. Determining striped bass spawning stock status from the presence or absence of eggs in ichthyoplankton survey data. N. Am. J. Fish. Manag. 13:645-657. Ward T. M., F. Hoedt, L. McLeay, W. F. Dimmlich, M. Kinloch, G. Jackson, R. McGarvey. P. J. Rogers, and K. Jones. 2001. Effects of the 1995 and 1998 mass mortality events on the spawning biomass of sardine, Sardinops sagax, in South Australian waters. ICES J. Mar. Sci. 58(4):865-875. Webster P. J., A. M. Moore, J. P. Loschnigg, and R. R. Leben. 1999. Coupled ocean-atmosphere dynamics in the Indian Ocean during 1997-98. Nature 401:356-360. Wolf, P.. and P.E.Smith. 1986. The relative magnitude of the 1985 Pacific sardine spawning biomass off southern California. CalCOFI Rep. 27:25-31. Zenitani. H., and S. Yamada. 2000. The relation between spawning area and biomass of Japanese pilchard, Sardinops melanostictus, along the Pacific coast of Japan. Fish. Bull. 98:842-848. Appendix 1— Derivation of model III from model II We start with model II "SP_DEPM ~ K, )""•-> + er For brevity, y is used to denote PPS. We first note that y'~=yy"'~1'- Now we obtain the Taylor series expansion of yU-D about. v=l giving Because y is a proportion, it satisfies 040 mm TL) moving in from adjacent rockpools and presumably to abun- dant species competing for access to vacant habitat. The main recolonizers were those species found in highest numbers in initial samples, such as Bathygobius cocosensis, Enneaptery- gius rufopileus, and Girella elevata. Defaunation did not affect the size composition of fishes, except during autumn and winter when juveniles (<30 mm TL) recruited to rockpools. It appears that Bass Point rockpool fish assemblages are largely con- trolled by postrecruitment density- dependent mechanisms that indicate that recolonization may be driven by deterministic mechanisms. Fish recolonization in temperate Australian rockpools: a quantitative experimental approach Shane P. Griffiths Environmental Science and Institute for Conservation Biology University of Wollongong Wollongong, New South Wales, Australia Present address: CSIRO Marine Research 233 Middle Street Cleveland, Queensland 4163 Australia Email address: shanegnffiIhsacsiro.au Ron J. West Environmental Science University of Wollongong Wollongong, New South Wales, Australia Andy R. Davis Institute for Conservation Biology University of Wollongong Wollongong, New South Wales, Australia Ken G. Russell School of Mathematics and Applied Statistics University of Wollongong Wollongong, New South Wales, Australia Manuscript submitted 28 April 2003 to thr Scientific Editor's Office. Manuscript approved for publication 5 May 2004 by the Scientific Editor. Fish. Bull. 102:634-647(2004). Rocky intertidal fishes are faced with many biotic (competition and food availability) and abiotic (temperature and salinity) factors that can influ- ence their distribution and abundance (Gibson. 1982). Despite occupying a dynamic environment, the fish assem- blages in intertidal rockpools have been widely shown to remain persis- tent through time (Grossman. 1982. 1986; Collette. 1986). These commu- nities can also rapidly return to their original state after major or even catastrophic perturbations (Moring, 1996). Such resilience is less common among assemblages of invertebrates (Connell, 1972; Astles, 1993) because recolonization of substrata is normally dependent upon successful larval settlement (Paine and Levin, 1981). In contrast, fish can rapidly colonize available habitat by larval recruit- ment from the plankton (Willis and Roberts, 1996. Beckley, 2000; Griffiths 2003a) but also by the relocation of subadults and adults from adjacent rockpools (Beckley, 1985a; Griffiths, 2003a). Under natural conditions rock- pools can be defaunated by events such as hurricanes (Moring, 1996) and, in some regions, by seasonal freezing of rockpool water (Thomson and Lehner, 1976; Moring, 1990). These events can create new microhabitats or open existing ones for fish to colonize, and therefore have the potential to change fish assemblage structure. Understanding recolonization pro- cesses of intertidal fish assemblages is integral for predicting the conse- quences of natural or anthropogenic impacts on the intertidal community. The role of disturbance and recoloni- zation processes in structuring inter- Griffiths et al.: Fish recolonization in temperate Australian rockpools 635 Tasman Sea •^Shellharbniir Bass Point 5 km Figure 1 Map illustrating the four sampling sites at Bass Point and the location of the study loca- tion in the Illawarra region. New South Wales, Australia. tidal rockpool fish assemblages has received considerable attention in many countries of the world (Bussing, 1972; Matson et al., 1986; Yoshiyama et al., 1986; Prochazka and Griffiths, 1992; Lardner et al., 1993; Prochazka, 1996; Faria and Almada, 1999; Silberschneider and Booth, 2001). Such studies have identified patterns in the rates of recovery, variation in species and size com- position of recolonizing fish assemblages (Polivka and Chotkowski, 1998; Beckley, 2000), and homing abilities of many intertidal fishes (Green 1971; Yoshiyama et al., 1992; Griffiths. 2003b). Rockpools can be regarded as "island' habitats (Under- wood and Skilleter, 1996) among an inhospitable rocky landscape. Therefore, there is probably a balance be- tween immigration (recruitment and relocation) and emigration (mortality) of fishes after a disturbance, sensu the equilibrium theory of island biogeography (MacArthur and Wilson, 1967). After a period of time, the number of species and individuals in a defaunated rockpool can be expected to reach an asymptote when a carrying capacity is reached. It is difficult to generalize about recolonization rates of rockpools by fishes from the current literature mainly owing to the diversity of methods used, their differing effectiveness in sampling fish, and the varying intensity of the sampling regime. For example, most studies have used only small sample sizes (<10 pools) and have sampled at a range of time intervals from days (Mistry et al., 1989; Matson et al., 1986; Polivka and Chotkowski, 1998) to years (Thomson and Lehner, 1976; Lardner et al., 1993; Mahon and Mahon, 1994). A second problem in measuring and comparing fish recolonization patterns between stud- ies is that many researchers have sampled fish using an anesthetic (Mahon and Mahon. 1994; Pfister, 1995, 1997) or ichthyocide (Beckley, 1985a, 1985b, 2000; Wil- lis and Roberts, 1996; Silberschneider and Booth, 2001), which may affect subsequent catches (Yoshiyama et al., 1986) and possibly result in fish assemblages never reaching preperturbation conditions (see Mok and Wen, 1985; Lockett, 1998). Nonetheless, recolonization of rockpools by fishes is generally a rapid process, beginning within days, or even hours, after defaunation (Collette, 1986). and com- plete recolonization to preperturbation levels can take a few weeks (Collette, 1986; Faria and Almada. 1999) to several months (Mok and Wen. 1985; Willis and Rob- erts, 1996; Polivka and Chotkowski, 1998). The aims of this study were to quantitatively deter- mine 1) the period required for intertidal rockpools to recover to preperturbation levels, 2) the fish species (permanent residents, opportunist, or transients) respon- sible for recolonizing rockpools, 3) whether recoloniza- tion patterns differ between the four sites at Bass Point and between the times of year when defaunation took place, and 4) whether fish comprise different life-history stages before and after a disturbance (sampling) — by examination of length-frequency distributions. Methods Study site and experimental design Spatial and temporal variation in fish recolonization pat- terns were investigated in three separate studies under- taken along the north- and south-facing rocky platforms at Bass Point (34°58'S, 150°93'E), New South Wales, Australia (Fig. 1). Bass Point is a large rocky headland that extends approximately 3 km into the Tasman Sea. Two short-term recolonization studies (each around 5 636 Fishery Bulletin 102(4) months in duration) were undertaken in spring-summer and autumn-winter (hereafter referred to as spring and autumn studies, respectively), and a long-term recolo- nization study spanned a 12-month period. Rockpools for each of the three studies were selected at four sites at Bass Point, NSW, which are named Maloney's Bay (MB), The Chair (TO, Gravel Loader (GL), and Beaky Bay (BB) (Fig. 1). Each of the four sites are separated by about 1 km. Rockpools were selected at each site (50-200 m apart) according to similar physical param- eters (i.e., volume, surface area, and substrate type) and particularly according to their vertical elevation on the rock platform. Because higher pools might have less chance of fish recolonization because they are less frequently inundated by seawater (Griffiths et al., 2003). every effort was made to select pools located in the mid- intertidal zone (1-1.5 m above MLLW [mean lower low water]) and, although pools were visually similar, they varied in volume, ranging from 762 to 2160 liters (or 0.76-2.16 m3). The bottom of the rockpools consisted of pebbles, cobbles, and small boulders. For the short-term studies, four rockpools were sam- pled and fish removed at each of the four sites. In the spring study (beginning 7 September 1999), they were then resampled 1 week, 1 month, and 3 months af- ter the preceding sampling date (referred to as the "1-week," "1-month," and "3-month" samples in this article). This study ended on 8 February 2000, after a period of 5 months. After this date a period of at least three months was given for pools to re-establish fish assemblages before beginning the autumn study on 15 May 2000. Rockpools were sampled in exactly the same manner as for the spring study, with sampling ending on 17 September 2000. For each study, 64 samples were taken giving a total of 128 samples for the short-term studies. It is important to note that although every ef- fort was made to resample pools after exactly the same time intervals, this was not possible because of daily time and height of tides and wave heights. For example, for the "1 week" samples, the number of days between samples was actually between 7 and 10 days. To determine whether frequent sampling in the short- term studies affected the structure of rockpool fish as- semblages, a long-term study was undertaken by using four different rockpools at the same four sites that were sampled in the short-term studies. Four rockpools at each site were considered adequate because Griffiths (2003a) was able to detect significant differences in the numbers of fish species and individuals in rockpools between sites and months using four rockpools per site in the same region that was surveyed in our study. Rockpools were initially sampled on 22 September 1999 and then resampled twice at intervals of six months (20 April 2000 and 11 September 2000). A total of 48 samples were taken for this study. Data collection Fish were collected by hand after completely emptying each rockpool with a VMC 12V battery-powered bilge pump of 9029 L/h capacity by using the methods of Griffiths (2000). A thorough search of each pool was conducted by overturning all rocks and boulders, search- ing all crevices and shaking algal fronds until all fish that could be seen were removed. Fishes were identified and total lengths (TL) were measured. Fork length (FL) was also measured for economically significant species. Fish were categorized as being juveniles (<30 mm), subadults (30-40 mm), or adults (>40 mm). Fish were then released alive into rockpools or the shallow subtidal 10-30 m away from the rockpool being sampled, which was considered to be the approximate distance that fish may be displaced by waves and surge during significant natural disturbances, such as storms. Each species was categorized by its residential status in rockpool habitats according to the definitions of Griffiths (2003c) in order to better understand the types of fish responsible for recolonization. These categories were "permanent resi- dents," "opportunists," and "transients." Statistical analyses A repeated-measures ANOVA (RM-ANOVA) was used (SPSS vers. 6.1; SPSS, Chicago, IL) to test for sig- nificant differences in the numbers of species and indi- viduals between sampling intervals (within-subjects factor) and sites (among-subjects factor). Short- and long-term experiments were analyzed with two sepa- rate RM-ANOVAs. For the short-term study a third factor of season (i.e., spring or autumn; among-sub- jects factor) was added. All factors were considered fixed. Assumption of sphericity of the variance-covari- ance matrix was tested by using Mauchly's criterion and, if violated, F tests were performed with Green- house-Geisser-adjusted degrees of freedom. Student- Newman-Keuls iSNK) tests were used for a posteriori comparisons among means (numbers of species and individuals) in RM-ANOVAs. Nonmetric multidimensional scaling (nMDS) was used to examine similarities in fish assemblage struc- ture between sampling intervals and sites. Data were fourth-root transformed, to reduce the influence of highly abundant taxa, and a similarity matrix was constructed by using the Bray-Curtis similarity coef- ficient (Clarke, 1993). Stress values are given for all ordination plots; these values describe the quality of the representation of multidimensional relationships of the data in a two-dimensional plane. Stress factors of less than 0.2 (<0.2 is considered to give a good rep- resentation of sample "relatedness" and to prevent the prospect of drawing false inferences) were obtained for each ordination (Clarke. 1993). Analysis of similarities (ANOSIM) was used to test whether fish assemblages in a priori groups differed sta- tistically (Clarke, 1993). Abundance data for each spe- cies were pooled for the four rockpools at each site and time. Each ANOSIM comparison involved generating 4999 random permutations of the data to calculate the probability that observed differences in the structure of the fish assemblages among a priori groups could arise Griffiths et al.: Fish recolonization in temperate Australian rockpools 637 by chance. Similarity percentages (SIMPER) were used to determine which species were responsible for differ- ences between selected groups. This analysis involved calculating the average contribution of each species in each pair of groups and comparing this contribution to the overall dissimilarity of fish assemblages between the groups. All multivariate analyses were carried out with PRIMER (Plymouth routines in multivariate eco- logical research) software (version 5.2.2, PRIMER-E Ltd., Roborough, Plymouth, UK). Results Composition of rockpool fish assemblages A total of 3658 fish representing 38 species and 19 families was caught in 176 samples from 32 rockpools at Bass Point between 7 September 1999 and 22 September 2000 (Table 1), corresponding to densities of 0.5 and 19 species/m3 (mean 4.4 [±2.9] /m3) and 0.5 and 80 fish/m3 (mean 15.6 | ±14.6] /m3), respectively. The most numeri- cally abundant taxa were permanent rockpool residents representing the families Gobiidae iBathygobius cocosen- sis), Tripterygiidae iEnneapterygius rufopileus), Clinidae iHeteroclinus whiteleggi and H. fasciatus), Blenniidae (Pa?-ablenrjius intermedins), and Gobiesocidae {Aspasmo- gaster costatus), although the temporary resident Girella elevata was the third most abundant species. The ten most numerically abundant species represented 92% of the catch (Table 1). Three species, G. elevata, Scorpis lineolatus, and Myxus elongatus, represented by 504 fish were considered to be of economic significance. All economically important fishes were caught as juveniles in the rockpools and 89% of the fish measured less than 100 mm FL. Numbers of species and individuals For the short-term studies, the mean number of species differed significantly between sampling intervals and sites (RM-ANOVA, Table 2). With respect to the site factor, there were significantly more species caught at BB than at the other three sites and the latter three sites did not differ from each other (SNK test). Only the "1-week" samples accounted for significantly fewer species than the initial samples (Fig. 2). However, the mean number of species caught in the "1-month" and "3-month" samples did not differ significantly from the initial samples at all sites (Fig. 2). The mean number of individuals differed significantly between sampling intervals and sites, although there was also a significant time x site interaction (RM-ANO- VA, Table 2). A close investigation of the significant interval xsite interaction, with primary interest in the interval factor, revealed that the number of individuals in the initial samples did not differ significantly from samples taken after three months at the exposed sites (MB and TC), but they did differ significantly at shel- tered locations (GL and BB) (Fig. 2). It appeared that the Number of species £ 50 -5 30 _ I 10 Number of individuals ;ni1^inW GL Site Figure 2 Mean i±SE) numbers of species and individuals (m~3) caught in rockpools at Bass Point, New South Wales between 7 September 1999 and 17 September 2000 during the short-term recolonization studies (combined for spring and autumn) between sampling intervals separated by 1 week, 1 month, and 3 months. Key to sites: Maloney's Bay (MB). The Chair (TC I, Gravel Loader (GL), Beaky Bay o ■' o A Slits, = n l J The Chair A A ■ ♦ * ♦ Stress = 0 1 l Gravel Loader ■ A o o*8 A A Sires, A = U.I6 Gravel Loader O ■ »CAO ° 4 o o Stress = i 15 Gravel Loader ■ * * o O ■ Stress = 0.03 Beaky Bay ° ' «"'»o Beaky Bay Beaky Bay ♦ A* Short-term study: ■ Initial o 1 week o 1 month A 3 months Long-term study: ■ Initial « 6 months A 12 months Figure 5 Nonmetrie multidimensional scaling (MDSl plots for comparison of fish assemblages from four sites at Bass Point in respect to initial samples and those taken after 1 week, 1 month, and 3 months during (A) spring and iBi autumn short- term studies, and after 6 months and 12 months during (C) the long-term study. Each coordinate represents a single mckpool sample. Stress values are shown. Griffiths et al.: Fish recolonization in temperate Australian rockpools 643 f$ Autumn Initial n=81 C Long-term Initial 1=115 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 Total length (mm) 0 10 20 30 40 50 60 70 80 Figure 6 Length-frequency distributions (in 2-mm intervals) for Bathygobius cocosensis caught in the initial samples and after 1 week, 1 month, and 3 months later in the short- term study and caught in the initial sampling and 6 months and 12 months later in the long-term study. Samples were pooled for all sites during the (Al spring and (Bl autumn short-term recolonization studies and for all sites during (C) the long- term recolonization study. Studies were conducted between 7 September 1999 and 22 September 2000. Samples sizes are shown. Results of ANOSIM testing during the spring, autumn. Table 6 for differences in fish assemblage structure among sampling intervals at four sites at Bass Point and long-term recolonization studies. Significant results are shown in bold. Site Spr ng Autumn Long term R P value R P value R P value Maloney's Bay The Chair Gravel Loader Beaky Bay 0.002 0.185 0.083 0.025 0.474 0.057 0.165 0.383 0.029 0.393 0.014 0.422 0.027 0.389 0.726 0.000 -0.101 0.063 0.190 0.044 0.752 0.284 0.051 0.348 For rockpool fish assemblages in southeastern Aus- tralia, a period of one week appears insufficient for recolonization of all species if fish are removed during sampling, whereas intervals of one to three months appear sufficient for rockpool fish assemblages at most Bass Point locations to recolonize to preperturbation levels. It is possible that recolonization times may be decreased if all fish are returned to rockpools immedi- ately after sampling. However, this should not provide a foundation for subsequent studies with other defauna- tion methods, such as anesthetics or ichthyocides. The possible residual effects of these other sampling meth- ods, such as the mortality of mobile and sessile inver- tebrates or the residues from chemical anesthetics and ichthyocides are possible factors that may complicate fish recolonization patterns (see Lockett, 1998) and certainly require additional investigation. Nonethe- less, in recolonization studies with chemical sampling methods similar recolonization times as those of the present study were found. For example, recolonization of rockpools defaunated by ichthyocides was shown to be complete within 1 month (Grossman, 1982; Prochazka, 1996) and 3 months (Beckley, 1985a; Willis and Rob- erts, 1996; Polivka and Chotkowski, 1998). Spatial variability in fish recolonization patterns was not definitive with regard to species composition 644 Fishery Bulletin 102(4) Spring Autumn /~> Long-term Initial n=73 i — i i F1 i — I 0 10 20 30 40 50 60 Total length (mm) Figure 7 Length-frequency distributions (in 2-mm intervals) for Enneapterygius rufopileus caught in the initial samples and after 1 week, 1 month, and 3 months later in the short-term study and caught in the initial sampling and 6 months and 12 months later in the long-term study. Samples were pooled for all sites during the (A) spring and (B) autumn short-term recolonization studies and for all sites during (C) the long-term recolonization study. Studies were conducted between 7 September 1999 and 22 September 2000. Samples sizes are shown. because samples were generally widely dispersed in nMDS ordination plots. The relatively low stress values (<0.2) indicate that high variability in fish assemblages at the level of individual rockpools is probably respon- sible for the patterns observed. However, some spatial variability in fish recolonization patterns was evident (Fig. 2) and appeared to be dependent to some extent on exposure of sites to predominant swell. There is evidence to suggest that wave exposure can affect the distribution of intertidal fishes (Gibson, 1972; Ibanez et al., 1989), although there is apparently no study that has investigated this effect in relation to fish recoloni- zation in rockpools. In the present study, recolonization appeared more rapid at wave-exposed sites (MB and TC) compared to more sheltered sites. This may have been the result of the close distance between rockpools at exposed sites (within meters of each other), whereas at both sheltered sites, rockpools were significantly farther apart. Consequently, defaunated rockpools at exposed sites may recolonize more quickly if the major recolonizers are derived from neighboring rockpools as has been documented elsewhere (Beckley, 1985a; Polivka and Chotkowski, 1998). Fish recolonization patterns were not influenced by the time of year that rockpools were defaunated in either short-term or long-term studies. The numbers of species and individuals consistently returned to pre" perturbation levels within a few weeks, but this return to previous levels may partially be a consequence of the relatively small number of species that are nor- mally found in rockpools at any given time. In such situations a significant differences could only occur if large-scale changes in abundances were recorded. The lack of temporal variation in recolonization rates was surprising because recolonization was expected to be more rapid during summer, when the larvae of residents and warm water transients are expected to be avail- able for settlement (Beckley, 1985a; Willis and Roberts, 1996). Recruitment was not the major mechanism driv- ing fish recolonization in the present study because the majority of recolonizers were subadults and adults that would have relocated from nearby rockpools. Although many of the fish captured in each pool were tagged, the vast majority offish caught in the same rockpool in subsequent sampling events were not tagged. Griffiths (2003b) showed that the common recolonizing species in the present study moved between a few rockpools within a limited home range. Therefore, postsettlement fishes from surrounding rockpools were probably moving into the study rockpools between each sampling event. The movement of postsettlement fishes from adjacent rockpools also appears to control the resilience of rock- Griffiths et al.: Fish recolonization in temperate Australian rockpools . 645 pool fish assemblages. Therefore, the composition of spe- cies in newly recolonized rockpools is probably depen- dent upon the relative abundances of species in nearby rockpools. Species having the highest local abundances, such as B. cocosensis and E. rufopileus, are therefore more likely to be the primary recolonizers because va- cant habitats have a higher probability of being located by these species during high-tide excursions throughout the intertidal zone (also see Polivka and Chotkowski, 1998). These species are also versatile and can exploit a range of microhabitats and, as a result, can occupy almost any rockpool within the intertidal zone (Griffiths et al., 2003). This is particularly true for B. cocosensis. In contrast, less abundant species such as H. whiteleggi often occupy more specific, and perhaps less abundant, microhabitats such as algal cover (see Marsh et al.. 1978; Bennett and Griffiths, 1984) that may require longer periods to locate than more abundant habitats, such as cobble-covered substratum. Processes regulating fish assemblages The structure of multispecies assemblages can be regarded as being regulated by either deterministic or stochastic processes (see Grossman, 1982). Assemblages regulated by deterministic processes generally occur in environments where conditions are constant or fluctuate consistently over time. The structure of these assem- blages is generally predictable. This can be maintained through a number of factors including partitioning of resources in finite supply (Schoener, 1974; Behrents, 1987) and interspecific competition, which prevents any single species being competitively dominant (Buss and Jackson, 1979). In contrast, assemblages regulated by stochastic pro- cesses generally exist in unpredictable environments. Here, the resources are available on a random or pe- riodic basis, which prevents superior competitors from dominating the assemblage (Sale, 1977, 1978). The suc- cess of particular species can be compared to winning a "lottery" for living space (Sale, 1977, 1978, 1982). Consequently, stochastically regulated assemblages are generally species rich (Sale, 1977). Rockpool fish assemblages are often persistent for lengthy periods, even after catastrophic natural dis- turbances, such as hurricanes (Moring, 1996), and con- tinual experimental eliminations (Grossman, 1982; Collette, 1986). For example, Collette (1986) found two species — Pholis gunnellus and Tautogolabrus adsper- sus — to be dominant over 19 years of study in two New England rockpools, whereas the rank of dominant spe- cies in the rockpools of Barbados showed no evidence of change over six years (Mahon and Mahon. 1994). Similar stability and persistence were evident in the present study, where B. cocosensis, E. rufopileus and G. elevata were consistently the highest ranked species in each collection for all three studies, regardless of the period between sampling. This finding may indi- cate that deterministic processes probably regulate the Bass Point fish assemblage. If this is the case, it may seem ironic because the intertidal zone is subjected to a high frequency of stochastic events. It would be easy to assume that such events could eliminate fishes from rockpools and thus leave microhabitats for other species to exploit. This kind of process has been documented for some sessile intertidal invertebrate assemblages that rely on the availability of vacant substrata for success- ful recruitment of larvae (see examples by Raffaelli and Hawkins. 1996). However, the locomotory capabili- ties and morphological and physiological adaptations of resident intertidal fishes allow them to cope with such disturbances by being able to cope temporarily with ad- verse conditions (Martin. 1995). As a result, the abun- dance of resident species may be little affected under normal disturbance regimes. Conclusions The results of this study have significantly increased an understanding of the patterns of recolonization of rock- pools by fishes and some of the processes that underpin these patterns. Such an understanding of recoloniza- tion processes may improve our ability to predict the consequences of significant natural and anthropogenic disturbances on not only the fish assemblages but also on other intertidal community assemblages that may be maintained by the presence offish (see Coull and Wells, 1983; Connell and Anderson, 1999). On a more technical note, the recolonization rates observed in the present study may provide insight for other researchers aiming to stud}' natural temporal variation of rockpool fish assemblages by minimizing the possibility of confounding effects of sampling. This may be particularly important for long-term monitoring programs, such as for marine protected areas (MPAs). that may require detection of changes in community structure over time. Finding sufficient numbers of simi- lar-size pools at a single location for monitoring can be difficult: therefore repeated visits to the same rockpools may often be required. For southeastern Australian rockpools. we feel that a period of one to three months is required before resampling the same rockpools with the methods employed in this study. Although fish were not returned to rockpools immediately after sampling in the present study, we feel that this practice may significantly increase recolonization rates. However, the results of the present study should not provide a foundation for studies using other defaunation methods, such as anesthetics or ichthvocides. because other fac- tors, such as chemical residues remaining in rockpools, may complicate fish recolonization patterns. Further investigation into these other factors will be necessary in the future. Acknowledgments We sincerely thank Jade Butler and Alan Griffiths for help with fieldwork. This paper is partly based upon 646 Fishery Bulletin 102(4) research included in a Ph.D. by S. P. Griffiths funded by an Australian Postgraduate Award. 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Ecol. 160:115-130. 648 Abstract — We have studied the repro- ductive biology of the goldlined sea- bream (Rhabdosargus sarba) in the lower Swan River Estuary in Western Australia, focusing particularly on elucidating the factors influencing the duration, timing, and frequency of spawning and on determining potential annual fecundity. Our results demonstrate that 1) Rhab- dosargus sarba has indeterminate fecundity, 2) oocyte hydration com- mences soon after dusk (ca. 18:30 hi and is complete by ca. 01:30-04:30 h and 3) fish with ovaries containing migratory nucleus oocytes, hyd rated oocytes, or postovulatory follicles were caught between July and November. However, in July and August, their prevalence was low, whereas that of fish with ovaries containing substan- tial numbers of atretic yolk granule oocytes was high. Thus, spawning activity did not start to peak until September (early spring), when salini- ties were rising markedly from their winter minima. The prevalence of spawning was positively correlated with tidal height and was greatest on days when the tide changed from flood to ebb at ca. 06:00 h. i.e., just after spawning had ceased. Because our estimate of the average daily prevalence of spawning by females during the spawning season (July to November) was 36.5%, individual females were estimated to spawn, on average, at intervals of about 2.7 days and thus about 45 times during that period. Therefore, because female R. sarba with total lengths of 180, 220, and 260 mm were estimated to have batch fecundities of about 4500, 7700, and 12,400 eggs, respectively, they had potential annual fecundities of about 204,300, 346,100 and 557,500 eggs, respectively. Because spawn- ing occurs just prior to strong ebb tides, the eggs of/?, sarba are likely to be transported out of the estuary into coastal waters where salini- ties remain at ca. 359«. Such down- stream transport would account for the fact that, although R. sarba exhib- its substantial spawning activity in the lower Swan River Estuary, few of its early juveniles are recruited into the nearshore shallow waters of this estuary. Manuscript submitted 9 June 2003 to the Scientific Editor's Office. Manuscript approved for publication 28 April 2004 by the Scientific Editor. Fish. Bull. 102:648-660(2004). Factors influencing the timing and frequency of spawning and fecundity of the goldlined seabream (Rhabdosargus sarba) (Sparidae) in the lower reaches of an estuary S. Alexander Hesp Ian C. Potter Centre for Fish and Fisheries Research School of Biological Sciences and Biotechnology Murdoch University South Street Murdoch, Western Australia 6150, Australia E-mail address (for I C Potter, contact author): i.pottera'murdoch edu.au Sonja R. M. Schubert Ernst-Moritz Arndt Universitaet, Hansestadt Greifswald F.-L.-Jahn StraBe 15a 17487 Greifswald, Germany The goldlined seabream {Rhabdosar- gus sarba) is an important recreational and commercial fish species in numer- ous regions throughout the Indo-west Pacific (van der Elst, 1988; El-Agamy, 1989; Kuiter, 1993). Although this species is a protandrous hermaphro- dite in certain regions, e.g., the waters of Hong Kong and South Africa (Yeung and Chan, 1987; Garratt, 1993). it is a rudimentary hermaphrodite in a range of environments in Western Australia (Hesp and Potter, 2003). Rudimentary hermaphrodites are those species in which the juveniles possess gonads consisting of both immature testicular and ovarian tissues that, in adults, develop permanently into either func- tional testes with rudimentary ovar- ian tissue or functional ovaries with rudimentary testicular tissue (Buxton and Garratt, 1990). In Western Aus- tralia, R. sarba attains similar maxi- mum lengths, i.e., 346-370 mm, in temperate marine coastal waters and the lower reaches of the Swan River Estuary on the lower west coast of Australia and in a large subtropical embayment ca. 800 km farther north (Hesp et al., 2004). However, the max- imum age recorded for this species in the estuary. 7 years, was far less than that for the other two environments: temperate marine coastal waters (11 years) and a large subtropical embay- ment (13 years) (Hesp et al., 2004). Although R. sarba is typically re- garded as a marine species that fre- quently uses estuaries as a nursery area (e.g., Wallace, 1975; Potter and Hyndes, 1999; Smith and Suthers, 2000), it spawns in the lower Swan River Estuary as well as in coastal waters outside this estuary (Hesp and Potter, 2003). However, this sparid attains maturity later in the estuary than in those nearby coastal marine waters. If this indication that the on- set of spawning for R. sarba in the Swan River Estuary is related to the attainment of higher salinities in the spring, it would parallel the situation recorded for the spotted seatrout (Cy- noscion nebulosus) in the estuaries of the Gulf of Mexico where this species completes its entire life cycle ( Brown- Peterson et al., 2002). Despite the importance and wide- spread occurrence of R. sarba. and the great value of fecundity data for stock assessments (Hunter et al., 1992: Nichol and Acuna, 2001), on- ly one attempt has apparently been made to estimate the annual fecun- dity of wild populations of this sparid (El-Agamy, 1989). Although El-Aga- my (1989) recognized that R. sarba is a "fractional" spawner and has a Hesp et al.: Timing and frequency of spawning and fecundity of Rhabdosargus sarba 649 protracted spawning season, he recorded the fecundity of this species as the number of larger eggs (diameter >180 fjm) estimated to be present in the ovaries of mature females just prior to the commencement of the spawning period. Thus, the very strong possibility that some eggs with diameters <180 /jm would have been destined to have become fully mature and released at some stage during the protracted spawning season, i.e., the species has indeterminate fecundity, was not taken into account. In species with indeterminate fecundity, the distri- bution of oocyte diameters essentially forms a contin- uum, reflecting the continuous maturation of oocytes throughout the spawning season and thus the progres- sion through to maturity of some of the small and pre- vitellogenic oocytes that were present at the beginning of the spawning period. Consequently, counts of the standing stock of larger oocytes found just prior to the onset of spawning will result in an underestimate of the potential annual fecundity of such species (Hunter et al., 1985. 1992; Lisovenko and Andrianov, 1991). Estimation of the annual fecundity of species with indeterminate fecundity thus requires a combination of data on batch fecundity and spawning frequency (Hunter et al.. 19851. Batch fecundity, i.e., the number of oocytes released during a single spawning event, can be estimated by counting the number of hydrated oocytes present in ovaries immediately prior to that spawning (Hunter et al., 1985). The frequency with which a fish spawns during the spawning period can be determined from the frequency of mature female fish possessing ovaries with either hydrated oocytes or postovulatory follicles (POFs) of a known age (Hunter and Macewicz, 1985). The spawning of many marine species of teleosts and invertebrates is correlated with lunar periodicity and the associated tidal cycles (e.g., Schwassmann, 1971; Taylor. 1984; Greeley et al., 1986; Hoque et al., 1999), with the spawning of such fish species typically peaking around the full or new moon (or both) (e.g., Johannes. 1978; Taylor and DiMichele. 1980; Greeley et al., 1986). Many fish and invertebrates with pelagic eggs spawn on high or ebb tides that enable eggs and the subsequent larval stages to be transported away from spawning areas, in which planktivorous predators are concentrated. This process thus reduces the likelihood of those early life cycle stages being subjected to predation (Taylor, 1984; Johnson et al., 1990; Morgan, 1990). The fact that there is very little recruitment of the early 0+ individuals of R. sarba into the lower Swan River Estuary, where ex- tensive spawning occurs, indicates that tides transport the eggs of this species from spawning areas in the estuary into coastal marine waters (Hesp and Potter, 2003; Hesp et al., 2004). This investigation, which involved a detailed study of the females of R. sarba in the lower Swan River Estuary, had the following aims: 1) to test the hypothesis that R. sarba has indeterminate fecundity; 2) to establish the period during the day when the oocytes of R. sarba become hydrated and when ovulation and spawning occur; 3) to establish whether R. sarba spawns mainly when salinities are high and thus approach those of the marine waters in which this species typically breeds and whether spawning is correlated with the strength and type (ebb vs flood) of tide in the lower reaches of the Swan River Estuary; 4) to estimate the average frequency of spawning for R. sarba during the spawning period; 5) and to determine the relationship between batch fecundity and fish length, and to use this rela- tionship, in combination with the average spawning frequency, to calculate the potential annual fecundity of R. sarba of different sizes. Materials and methods Tide, lunar phase, and salinity The maximum daily tidal heights at the mouth of the Swan River Estuary were calculated by using the tidal prediction data of the Coastal Data Centre at the Depart- ment of Planning and Infrastructure, Government of Western Australia (http://www.coastaldata. transport. wa.gov.au). The maximum tidal range at the mouth of the Swan River Estuary is small, i.e., <0.8 m, and tides can be diurnal or semidiurnal, depending on the time of year (Spencer, 1956). Salinity was measured on each sampling occasion by using a Yellow Springs Instru- ments salinity meter (YSI model number 30, Yellow Springs Instrument Co., Inc., Yellow Springs. OH). Sampling During 2001 and 2002, female Rhabdosargus sarba were collected by seine netting in nearshore shallow waters at distances of ca. 2.5 to 5 km from the mouth of the Swan River Estuary, and by rod and line fishing in water depths of 10-12 m at a distance of ca. 150 m from the shore (for details of sampling region and seine net, see Hesp and Potter, 2003). Sampling was under- taken at least once weekly between July and November, the period when R. sarba reach maturity in the lower Swan River Estuary (Hesp and Potter, 2003). It was restricted to the hours between dusk (ca. 18:00 h) and dawn (ca 06:00 h) because extensive seine netting and angling during the day in our earlier study failed to yield any R. sarba. The failure to capture R. sarba by these methods during daylight reflected the offshore movement of this species from the shallows prior to dawn and a far stronger targeting of bait by the large numbers of the banded toadfish {Torquigener pleurogramma) that feed in the offshore waters of the lower estuary during the day. Because the lower reaches of the Swan River Estuary act as a shipping harbor, alternative sampling methods, such as gill netting and spearing, could not be used to catch R. sarba during the day. The data for 2000 and 2001 were augmented by those derived from fish collected from the same location by using the same methods in 1998 and 1999 (Hesp and Potter, 2003). In total, the results of the present study are based on an examination of over 2000 R. sarba, of which 510 were 650 Fishery Bulletin 102(4) Table 1 Characteristics of each macroscopic stage in the development of the ovaries of Rhabdosargus sarba, and its corresponding histo- logical characteristics. Adapted from Laevastu (19651. Terminology for oocyte stages follows Wallace and Selman (1989). Stage Macroscopic characteristics Histological characteristics I Virgin Ovary is very small and strand-like. II Immature and resting Small and transparent. Yellowish-orange in color. Oocytes not visible through ovarian wall. Ill Developing Slightly larger than stage II. Reddish color. Oocytes visible through ovarian wall. Rhabdosargus sarba is a rudimentary hermaphrodite, sensu Hesp and Potter (2003). Thus, the gonads of small juveniles contain only connective tissue. Larger juveniles possess gonads (ovotestes) in which each ovarian lobe consists of an immature ovarian and testicular zone, separated by connective tissue. The ovotestes develop later into gonads containing almost entirely ovarian tissue (functional ovaries) or, in the case of males, gonads containing almost entirely testicular tissue (functional testes). Ovigerous lamellae highly organized. Chromatin nucleolar and perinucleolar oocytes dominate the complement of oocytes. Oogonia sometimes present. Chromatin nucleolar oocytes present in all subsequent ovarian stages. Chromatin nucleolar, perinucleolar and cortical alveolar oocytes present. Cortical alveolar and yolk granule oocytes abundant. IV Maturing Larger than stage III. Reddish-orange in color. Yolk granule oocytes visible through ovarian wall. V Mature Larger than stage IV occupying half to two Yolk granule oocytes predominant. thirds of body cavity. Extensive capillaries visible in ovarian wall. VI Spawning Hydrated oocytes visible through ovarian Migratory nucleus oocytes, hydrated oocytes, or postovula- wall. Note that fish with ovaries in "spawn- tory follicles present, ing condition" can only be detected macro- scopically when caught during the hydration period. VII Spent Smaller than V and VI and flaccid. Some yolk Some remnant yolk granule oocytes present, all or almost al granule oocytes visible through ovarian wall. of which are typically undergoing atresia. VIII Spent and Small and dark red. recovering Extensive scar tissue present. Ovarian lamellae becoming reorganized. No yolk granule oocytes present. females with stage-V (mature) or stage-VI (spawning) ovaries (see Table 1 for definitions of these stages). During the above sampling, R. sarba was collected for up to 2 hours at intervals commencing at 18:30, 21:30, 00:30, and 03:30 h on 1-2 September 2001 and for up to 2 hours at intervals commencing at 18:30 and 22:30 h on 13 September 2001. One of the ovarian lobes of up to five fish caught during each of these above time intervals was cut into several pieces, preserved in 10'' neutrally buffered formalin solution and used for determining the distributions of oocyte diameters at the above different times. The other lobe was used for histology to determine the oocyte stages present in that lobe, and thus, by extrapolation, also the stages of the oocytes in the lobe that had been preserved in formalin. The resultant comparisons were used, in conjunction with data from other times, to elucidate the pattern of oocyte development during hydration and the duration of hydration and timing of ovulation. Gonadal staging and histology of ovaries The sex, total length (to the nearest 1 mm), and total weight and gonad weight (to the nearest 0.01 g) of each fish were recorded. From its macroscopic appearance, each gonad was assigned to one of the following stages in maturation, based on the scheme of Laevastu (1965), i.e., I = virgin, II = immature and resting, III = developing, IV = maturing, V = mature, VI = spawning, VII = spent, VIII = spent and recovering. The corresponding histolog- ical characteristics of each macroscopic stage are shown in Table 1. When hydrated oocytes could be seen through the ovarian wall of a fish, a note was made as to whether they were distributed throughout the ovary or were in Hesp et al.: Timing and frequency of spawning and fecundity of Rhabdosargus sarba 651 the ovarian duct and thus whether or not ovulation had commenced at the time of capture of that fish. For all histological studies of the gonads, part of the mid region of one of the ovarian lobes was placed in Bouin's fixative for ca. 48 hours, dehydrated in a series of ethanols, embedded in paraffin wax, cut into 6-um sections, and stained with Mallory's trichrome. The ova- ries were fixed within 1-3 hours of capture of the fish. To test the hypothesis that R. sarba has indetermi- nate fecundity, the diameters of 100 oocytes in histo- logical sections of stage-VI ovaries of two fish caught during the spawning period were measured to the near- est 10 /jm by using an eyepiece graticule in a compound microscope and the stage of each of those oocytes was recorded. Measurements were restricted to oocytes in which a nucleus was visible in their center to ensure that the oocytes had been sectioned through their center and that the diameters were thus measured accurately. This approach could not be used to measure the oocyte diameters of hydrated oocytes in histological sections because the nucleus of these oocytes undergoes germi- nal vesicle breakdown. Histological sections of numerous ovaries were used to determine the timing of the formation and degen- eration of postovulatory follicles (POFs). An age was assigned to the POFs found in ovaries of fish caught at different times of the day, based on the timing of ovulation and the degree to which those POFs had degenerated (Hunter and Goldberg, 1980; Hunter and Macewicz, 1985). Histological sections were also used to determine the relative abundance of the different stages of atresia in ovaries at different times during the spawning period. The jars containing the ovarian lobes that had been preserved in formalin at the different time intervals on 1. 2, and 13 September 2001 (see earlier) were shaken until the oocytes of each ovary had become evenly sus- pended in the solution. The resultant solution from each ovary was then passed through a 125-/jm sieve to remove the smallest oocytes, and we were able thus to focus our study more specifically on the vitellogenic oocytes. Comparisons of the appearance of the larger oocytes under a dissecting microscope with those of the different oocyte stages in histological sections of the other ovarian lobe of the same fish were used to allocate the oocytes observed under the dissecting microscope to a specific stage in oocyte development. Each oocyte in a representative subsample of 100 oocytes from each formalin-preserved ovarian lobe was measured under a dissecting microscope with an eyepiece graticule. This approach enabled the diameters of hydrated oocytes to be measured accurately, which was not possible with histological sections (see earlier). Categorization of stages in atresia, fecundity estimates, and spawning frequency On the basis of their histological characteristics, atretic oocytes were allocated to either the a or /3 stages, by using the criteria of Hunter and Macewicz (1985). Mature ovaries were categorized according to the proportions of their a and /3 atretic oocytes (Hunter and Macewicz, 1985). Thus, atretic state 0 = ovaries with yolked oocytes but no a atretic oocytes; atretic state 1 = ovaries in which less than 509c of the yolked oocytes are in the a stage of atresia; atretic state 2 = ovaries in which less than 509c of the yolked oocytes are a atretic and atretic state 3 = ovaries which contain no yolked oocytes but do pos- sess |3 atretic oocytes. During the present study, atretic state 1 ovaries were further divided into three categories on the basis of the percentage of a atretic yolk granule oocytes in histological sections, namely early (<10%), mid (10-359? I and late (36-50%) atretic state 1, an approach similar to that adopted by Farley and Davis (1998). The batch fecundities of 31 R. sarba were estimated from the number of hydrated oocytes in one of the ovar- ian lobes of fish that had been preserved in 109c neu- trally buffered formalin. These fish were chosen because histological examination of their other ovarian lobe dem- onstrated that the ovaries were in atretic state 0 or early state 1, i.e., less than 10% of their yolk granule oocytes were atretic and newly formed POFs were not present (Hunter et al., 1992; Nichol and Acuna, 2001). The for- malin-preserved ovarian lobe was dried with blotting pa- per and ca. 180-200 mg of tissue was removed from each of its anterior, middle, and posterior regions and weighed to the nearest 1 mg. These pieces of tissue were placed on separate slides, covered with 309c glycerol and examined under a dissecting microscope. The oocytes were then teased apart and the number of hydrated oocytes record- ed. The number of hydrated oocytes in each of the three pieces of ovarian tissue of known weight were then used, in conjunction with the weight of both ovarian lobes, to estimate the total number of hydrated oocytes (=batch fecundity) that would have been present in the pair of ovarian lobes of each fish. The prevalence of spawning on any given night is expressed as the percentage of female fish with hydrated eggs (ovarian stage VD among all fe- male fish with stage-V I mature) and stage-VI (spawning) ovaries. These estimates were based on an examination of samples collected between 22:00 and 01:30 h, when it was possible to determine which female fish were going to spawn in the ensuing few hours (see Hunter et al., 1985, for further details of this method). Results Although mean monthly salinities in the lower Swan River Estuary in late spring to early winter were close to that of full strength sea water (359cc), they fell pre- cipitously to a minimum of 23%c (minimum individual value=14'?c) in August, and then rose sharply in early to mid-spring (Fig. 1). Staging of the ovaries and confirmation of indeterminate fecundity The characteristics of each macroscopic stage of the ovaries of R. sarba and the corresponding histologi- 652 Fishery Bulletin 102(4) 40 35 30 25 20 15 L 80 60 S 40 20 J 0 Month Figure 1 Mean monthly salinities I±1SE) at the bottom of the water column in the lower Swan River Estuary throughout the year and the preva- lences of atresia in mature ovaries of Rhabdosargus sarba between July and November, which are shown as histograms, together with the number of fish examined. Closed rectangles on the horizontal axis refer to summer and winter months, and the open rectangles to autumn and spring months. cal characteristics are presented in Table 1. Because stages V and VI could be distinguished macroscopi- cally only during the period of oocyte hydration, the macroscopic data for these two stages had to be com- bined for other times. The diameters of the oocytes in histological sections of an ovarian lobe from each of two mature female R. sarba caught during the spawning season — oocyte diameters that were typical of those from mature R. sarba during this period — formed an essentially continuous distribution (Fig. 2). This distri- bution reflected the presence of oocytes at all stages in development from chromatin nucleolar oocytes to yolk granule oocytes and demonstrated that R. sarba has indeterminate fecundity sensu Hunter et al. (1985). Thus, the potential annual fecundity is not fixed prior to the commencement of the spawning period and conse- quently the potential annual fecundity of R. sarba has to be estimated by using a combination of batch fecundity and spawning frequency. Period of hydration and spawning The diameters of oocytes in ovaries of fish collected at intervals on 1-2 September 2001 and 13 September 2001 and which had been retained on the 125-^im sieve, produced a modal class that, for each time interval, fell between 420 and 600 fim (Fig. 3). At ca. 18:30 h on 1 September 2001, the oocyte diameters formed a single mode, and the vast majority of oocytes were less than 720 ;im and produced a modal class at 420-539 um (Fig. 3). However, by ca. 21:30 h on the same evening. the maximum diameter of the oocytes had increased markedly and the distribution of the oocyte diameters was beginning to become bimodal. with modal classes at 480-539 and 780-839 ^m. By 00:30 h on 2 September, the oocyte diameter distributions had become markedly bimodal, and the modal diameter class of the largest oocytes at this time, and also at 03:30 h, lay between 840 and 959 ^(m (Fig. 3). The oocyte diameter frequen- cies on 13 September were essentially the same as those at similar times on 1 September; the distributions were unimodal at 18:30 h and bimodal at 22:30 h (Fig. 3). The oocyte diameters of each fish within a given time slot on 1, 2, and 13 September exhibited essentially the same distribution. Histological sections showed that, at 18:30 h on 1 September 2001, most of the mature ovaries contained migratory nucleus stage oocytes, i.e., oocytes in which the nucleus was migrating towards the edge of the cy- toplasm and a conspicuous lipid droplet was present in the cytoplasm (Fig. 4A). However, it was difficult at this time to distinguish migratory nucleus oocytes from yolk granule oocytes under a dissecting microscope (Fig. 4B). By 21:30 h, the yolk and lipid of the larger oocytes had begun to coalesce and the nucleus could sometimes be seen near the edge of the cytoplasm (Fig. 4Cl. Their relatively larger size, translucent appearance, and one's ability to detect their lipid droplet enabled these hydrat- ing oocytes to be far more readily distinguished from yolk granule oocytes under a dissecting microscope than was the case earlier in the evening (cf. Fig. 4, B and D). By 00:30 h. the largest oocytes had increased Hesp et al.: Timing and frequency of spawning and fecundity of Rhabdosargus sarba 653 further in size and all of their lipid and yolk material had coalesced (Fig. 4E). Under the dissecting micro- scope, these hydrated oocytes were of similar appear- ance to the corresponding oocytes at 21:30 h (Fig. 4F). Although mature fish with ovaries containing the above stages in oocyte hydration were frequently found in nearshore shallow waters, the numbers of such fish in these waters declined markedly after about 00:30 h and none of the few fish caught there after this time contained recently formed POFs. However, fish with ovaries containing newly formed POFs were caught in offshore deeper waters. Histological examination demonstrated that, when hydrated oocytes were present in the ovarian duct, the ovary contained recently formed POFs, which are formed by the thecal and granulosa layers of the oocytes that surround the zona radiata externa (Fig. 5A). Newly formed POFs (0-6 h old) possess a conspicuous lumen and their granulosa cells contain prominent darkly stained nuclei (Fig. 5B). These newly formed postovula- tory follicles were first observed in the ovaries of females caught at ca. 01:30 h and were present in the ovaries of several fish caught in the ensuing four hours. In con- trast, no newly formed POFs were found in the ovaries of R . sarba at dusk, i.e., ca. 18:30 h. At this time, the POFs comprised one of two morphological forms. The first and least degenerate form was less well organized than newly formed POFs and its nuclei were becoming pycnotic (Fig. 5C); the second form was smaller and highly degenerate and its nuclei had become far less visible or undetectable (Fig. 5D). The least degenerate of the two forms of POFs in ovaries of fish caught at ca. 22:00 and 01:00 h (Fig. 5, D and E) represents stages in degeneracy that are intermediate between those of the two different forms described above for the ovaries of fish caught at 18:30 h. These POFs were thus compact and, although some of their nuclei were still detectable, they were markedly pycnotic. Chromatin nucleolar oocytes Perinucleolar oocytes D Cortical alveolar oocytes Yolk granule oocytes 30 20 10 ■ Chromatin nucleolar oocytes E3 Perinucleolar oocytes fJJ Cortical alveolar oocytes B Yolk granule oocytes 0 100 200 300 400 500 Oocyte diameter (urn) Figure 2 Percent frequency distributions for the oocyte diameters of different oocyte stages in histological sections of stage- VI ovaries of two female Rhabdosargus sarba. Influence of salinity and tides on spawning Both a and /5 atretic oocytes were frequently observed in the ovaries of R. sarba. The chorion (zona radiata) of the early a atretic vitellogenic oocyte was distorted, fragmented, and had moved inwards (Fig 6A). By the /3 atretic stage, the yolk and lipid had been resorbed and a large proportion of the oocyte volume was occupied by vacuoles (Fig. 6B). Sixty-two percent and 72% of the stage-V and stage- VI ovaries sectioned in July and August, respectively, were at mid or late atretic state 1, i.e., 11-50% of their yolk granule oocytes were a atretic (Fig. 1). However, the prevalence of these mid-late state-1 ovaries declined precipitously to 28% in September, as salinities rose markedly, and remained at a similar level until the end of spawning in late November. Histological sections showed that, in July and August, only 39r/c of the 57 pairs of ovarian lobes of R. sarba that were macroscopically assigned as stage V and stage VI contained migratory nucleus oocytes, hydrated oocytes, or POFs, i.e., were at stage VI. However, in the following two months, 76% of the 88 pairs of ovarian lobes of R. sarba, that were macroscopically assigned as stage V or stage VI, were shown by histology to be at stage VI. During September, when spawning activity was great- est, the prevalence of spawning (PS) was positively correlated (P<0.05) with maximum daily tidal height (T). PS = 91.72T + 20.73 (/-2 = 0.46, number of sampling occasions=10) (Fig. 7A). Data for the same days as those used to provide the points shown in Fig. 7A demonstrated that the preva- lence of spawning (PS) is inversely correlated with the difference in hours between the time when spawning is believed to cease (ca. 06:00 h, see later) and the time of high tide. PS = -8.26(T) + 78.22 (r2=0.49, number of sampling occasions=10) (Fig. 7B). Thus, the prevalence of these "spawning" females was greatest on those days when the time that the tide was about to change from flood to ebb coincided with the time when R. sarba is considered to cease spawning. 654 Fishery Bulletin 102(4) 1 and 2 September 1 and 2 September 30 r 20 10 0 30 20 10 g- 30 lL 20 10 0 30 20 10 0 L 18:30 h n = 5 Ql i i i — i — i — i — i — t — i — i — i 21:30h n = 5 tn= 00:30 h rt = 5 hJ i i — i — 1_ _j — i — i — i 03:30 h n = 2 r *, 105 § 100 o CD ~ 9.5 o 1 9.0 9 ■ : \^^ • * c ^^-""» 8.5 • • • 8.0 • 7.5 S.2 5.3 54 55 5.6 In total length (mm) Figure 8 Relationship between batch fecundity ( = number of hydrated oocytes) and total length Imml for Rhabdosar- gus sarba. Spratelloicles robustus was particularly numerous in some of our seine-net catches, a movement of the eggs ofi?. sarba out of the estuary would also enhance their chances of avoiding predation by that species. A downstream transport of eggs would account for the relatively few young 0+ juveniles that are recruited into the nearshore shallow waters of the estuary ( Hesp et al., 2004). Indeed, substantial recruitment into these nearshore waters, presumably as a result of immigra- tion from coastal marine waters, does not occur until R. sarba is about one year old and about 140 mm in length (Hesp et al.. 2004). Because R. sarba settles at a length of ca. 12 mm (Hesp et al., 2004) and ca. 30 days of age (Neira1), this immigration back into the estuary does not occur until 11 months after settlement. In contrast to the situation in the Swan River Estuary, R. sarba elsewhere typically spawns in marine waters and their larvae often enter estuaries on flood tides (e.g.. Miskiewicz, 1986; Neira and Potter, 1992). a marine environment in which salinity remained con- stantly at ca. 35', 675 674 672 671 670 669 668 667 666 665 ' i 34WN- i i. 696 B9! 694 693 692 691 -k£ l8" 1034 r 776 717 716 715 714 713 ^ ^ 70S • " 706 705 704 703 70? l 1 738 735 '- 11 730 - 775 724 723 72? 7?1 741 4ft| 715%, i 1033 1 i f 777 755 754 753 75? 751 749 748 746 745 743 74? 740 760 759 758 775 774 773 772 771 770 769 768 767 766 - 765 764 763 V 75V, 32 i 895 820 819 818 817 816 815 314C ^13 812 811 810 809 IB 805 804 803 802 ' WOWN- 10 841 4 838 837 836 835 834 633 832 831 830 828 827 826 825 846 824 823 822 h 858 857 856 855 854 853 851 85o\ 848 847 845 844 843 ft 896 876 875 874 873 872 871 870 869 868 867 866 865 864 863 862 861 *V 894 893 892 891 890 889 888 887 888 885 884 883 882 881 880 879 878 ^ 897 916 n 1 I Figure 1 Fishing activity for the market squid in the Southern California Bight, from 26 April 1992 to 28 May 2000. (At Composite satellite image of squid fishing vessel lights (black marks). Permanent sources of lights (e.g., city lights, offshore oil platforms, naval installations) are removed. CalCOFI stations 83.42 ("A"; 34.18°N, 119.51°W) and 83.51 ("B"; 33.88°N, 120.13°W) are indicated. (B) Squid landings as reported by California Dept. Fish and Game fishing blocks. Gray: blocks that account for 6.8 million kg (2%) or more of the land- ings from blocks 651-896. Black: blocks that account for 20.5 million kg (6%) or more. Latitudinal blocks 1032-1035 are indicated. Santa Cruz Island is marked "999" to aid correspondence with A. 664 Fishery Bulletin 102(4) Squid fishing vessels could not always be distinguished as light boats or seiners and therefore were recorded as "squid fishing vessels." The numbers of squid fishing vessels showed large skew in their frequency distribution. These data were transformed by x' = log10(x+l). Similarly, proportion lunar phase was transformed by x' = arcsin(V.r), and detected light pixels were transformed by x' = log]0(.v+l) to correct for skew (Zar, 1984). These transformations produced normally distributed data acceptable for re- gression analysis. With these transformed variables, multiple stepwise regression (forward selection) was performed with the software S-Plus 2000 (MathSoft Inc., Cambridge, MA) to examine the effects of squid fishing vessels and the proportion lunar phase on de- tected light pixels. Squid fishing vessels and proportion lunar phase showed very little correlation (r=-0.09). Fishery characteristics, 1992-2000 For quantitative analysis of the fishery data, we aggre- gated the nightly satellite data (i.e., light pixels detected on the water) into calendar quarters, as suggested by the within-year distribution of squid landings in the bight (Butler et al., 1999). To standardize conditions of light detection, we excluded all data after 28 May 2000, because this was the starting date of mandatory shield- ing of the high intensity lights of the lightboats. This regulation was enforced by California's Department of Fish and Game to reduce light pollution by the light- boats. The shields did not totally obscure the lightboats from detection by the satellites (authors' pers. obs.) but made the emitted light less bright, and, hence, less detectable by the satellites. Thus, our data for fishing effort spanned calendar quarters from Jul-Sep 1992 to Jan-Mar 2000. We included a quarter for analysis if it contained 10 or more nights of acceptable images. By these criteria, we described effort for 24 of the 31 cal- endar quarters. The mean number of nights per quarter was 26 (range=10-72 nights). The quantity (kg) and location of landed market squid were recorded by California Department of Fish and Game (CDFG) throughout the 1992-2000 study pe- riod and were made available to the authors. During this study period, squid fishing in the bight occurred exclusively at night (Vojkovich, 1998). The squid were landed at port within several hours after being caught; therefore the landings for a given day corresponded to the previous night's effort. Squid fishermen reported the locations of their hauls by CDFG fishing blocks. We defined catch taken from the Southern California Bight as that from blocks 651-896 and 1032-1035 (Fig. 1). Blocks 651-896 are typically 10' latitude x 10' longitude and can be used to locate regions of high catch. Blocks 1032-1035 are large latitudinal bands, generally 30' wide, that encompass blocks 651-896. We used blocks 1032-1035 in calculating the total catch in the bight, but not in depicting the location of the catch. To construct the abundance index of landings per unit of effort (LPUE). we first estimated the number of squid fishing vessels for each night of satellite data, us- ing the regression results of the ground-truthing work (see "Results" section). We then summed the nightly estimated number of vessels for each calendar quarter. For those nights for which we had estimated numbers of vessels, we also summed the landed catch within each calendar quarter. To arrive at LPUE for the quarter, we divided the summed landings by the corresponding summed effort. Environmental data We used the multivariate ENSO index (MEI) to indicate overall environmental conditions over the course of the 1992-2000 study period. The MEI is a multivariate index that incorporates sea level pressure, surface zonal and meridional wind components, sea surface tempera- ture, surface air temperature, and cloudiness (Wolter and Timlin, 1998). The MEI index is calculated for the tropical Pacific (i.e., between 10°N and 10°S, from Asia to the Americas), and its monthly values appear on the website http://www.cdc.noaa.gov/~kew/MEI/table. html.2 Analysis of the location of fishing effort over the course of the traditional squid fishing season in the bight led to an investigation of oceanographic data for waters surrounding Santa Cruz Island in March. Spe- cifically, we examined sea temperature from two sourc- es. First, we obtained sea surface temperature for all satellite nights in March 1993-2000 from the Physical Oceanography Distributed Active Archive Center (PO. DAAC) at California Institute of Technology (Pasadena, CA). These data were reported for 18x18 km grids, which were approximately the size of the 10'xlO' fish- ing blocks. We selected the grid that covered block 686 to represent the northern shore of the island, and that which covered block 708 to represent the southern shore (Fig. IB). For each year in the 1993-2000 period, we calculated mean March temperature for both blocks. The second source of sea temperature was the da- tabase maintained by the California Cooperative Oce- anic Fisheries Investigations (CalCOFI). Since 1950. the CalCOFI program has conducted quarterly survey cruises along transects perpendicular to the southern California coast. This system of transects incorporates 66 geographically fixed stations. At each station, a conductivity-temperature-depth (CTD) instrument is deployed. Details on survey methods appear on the web- site http://www-mlrg.ucsd.edu/calcofi. html. :1 along with the publicly accessible database. For April 1993-2000, we obtained temperatures at sea surface and at 75 me- ters depth at two stations (Fig. 1A): 83.42 (northeast of Santa Cruz Island: 34.18°N, 119.51°W) and 83.51 (southwest of Santa Cruz Island; 33.88DN, 120.13 W). - NOAA-CIRES Climate Diagnostics Center website. I Ac- cessed 3 November 200.3.1 3 California Cooperative Oceanic Fisheries Investigations website. [Accessed 3 November 200.3.1 Maxwell et al.: Fishery dynamics of Loligo opalescens 665 One measurement was made at each station at sea surface and at 75 meters depth during April (n = 8 for both depths). Results Ground-truthing: aerial observations of boat activity Nonsquid vessels used weak lights (i.e., much less than 30,000 watts), which did not show in the satellite images. On average, 23 squid fishing vessels were observed each night by the aerial surveys (range = 0-64 vessels, n=26 nights). The 20:00-midnight observation period was the peak time for attraction of squid by the light boats. Although the squid vessels did change location during this time, they typically left their lights running to continue searching for squid. The number of squid vessels explained much of the varia- tion in detected light pixels; proportion lunar phase failed to enter the analysis as a signifi- cant variable (Table 1). Detected light pixels increased with the number of squid vessels (Fig. 2). The regression analysis yields the following simpli- fied equation: log10(p, +l) = 1.25xlog10(x, +1), (1) where xt = observed number of squid vessels; and pt = detected light pixels for night t. We used inverse prediction to estimate the number of squid vessels for each satellite night (Et\ in the 1992-2000 period (Zar, 1984). The estimated number of squid vessels was found by the equation 3.0- ♦ ♦ ♦ ^. ♦ * + ♦ ,' «> 2.0- ♦ ♦ *- ' ' ♦ "cB * *'<• ♦ 5. „' ♦ S ' ♦ .D) ' ♦,-.* • o ra 10- S *" o s "* <> *** 0.0 1.0 2.0 Log10 (observed squid vessels + 1 ) Figure 2 Plot of log10-transformed number of squid vessels and detected light pixels. Regression line taken from the statistics in Table 1. Table 1 Multiple stepwise (forward selection) regression of de- tected light pixels on squid fishing vessels (transformed: x'=log10(x+l) and proportion lunar phase (transformed: .r'=arcsin( Vr). r2=0.64; ANOVA: Fl 24 = 42.66, P<0.0001. Variable Coefficient ±SE Squid fishing vessels INTERCEPT Proportion lunar phase 1.25 ±0.19 0.07 ±0.24 not entered <0.0001 >0.75 not entered 4=io"«-(ft+imB-i=1J»/ft+i-i- (2) The ground sample distance of the satellite data is 2.7 km, which means that multiple squid vessels may potentially fit into one pixel of detected light. This could result in an underestimation of effort. The severity of this problem can be assessed by examining the coeffi- cient of the simple linear regression of log-transformed variables represented by Equation 1. One of four sce- narios is possible: 1) boats are not aggregated (coef- ficient^), 2) boats are aggregated regardless of the number of boats on the water (coefficients), 3) boats are aggregated only when many boats are on the water (coefficientl). The coefficient in Equation 1 is 1.25, which fails to significantly differ from 1.00 (f-test for regression coefficient: t = 1.305, j30 = 1, df = 24, P > 0.2, two-tailed; power < 0.5, retrospec- tively calculated; Zar, 1984). This result suggests that very little clumping of the boats occurred (scenario 1), or that the degree of clumping was independent of the number of boats on the water (scenario 2). Although the statistical power of this ^-test is not high (power<0.5), we conclude that the data provide more support for sce- narios 1 and 2 over scenarios 3 and 4. Either scenario, 1 or 2, allows for a comparison of the relative values of estimated effort and LPUE within a time series. Fishery characteristics, 1992-2000 A composite satellite image of all squid fishing activity in the Southern California Bight during the 1992-2000 study period revealed major concentrations of effort off the Channel Islands, especially Santa Rosa, Santa Cruz, Anacapa, and Santa Catalina (Fig. 1A). Squid fishing occurs close to the island shores and is bounded by the 100-m contour. During the study period, 379.2 billion kg of squid were landed in the bight: 341.2 billion from blocks 651-896 (Fig. IB), and the remainder from the large blocks 1032-1035. The main areas of fishing activ- ity, as indicated by satellite, are consistent with the blocks of high catch (Fig. IB). We note that blocks 682 666 Fishery Bulletin 102(4) and 720, although areas of high catch, do not appear on the satellite composite because the mainland shore was excluded from light detection. Further, much activity was evident around Santa Barbara Island (block 765). Although this block represented 4.0 million kg (18th out of the 127 blocks), it did not rank highly enough for inclusion in Fig. IB. Analysis of temporal trends in the fishery showed peaks in landed catch for the bight in the fall and winter quarters (Oct-Dec and Jan-Mar, respectively; Fig. 3A). There was a near absence of catch during c > J= o >-.o P L±J~g si <5 to 5 o> B .-c m o o > OS *- ZJ x cr " — to HI i- D <° a. °- _i Ol o S 30- rs.tt az 1992 1993 1994 1995 1996 1997 1998 1999 2000 Quarter Figure 3 Time series of market squid fishery data in Southern California Bight, by calendar quarter (Jul-Sep 1992 to Jan-Mar 2000). The Jan-Mar quarters are marked by dashed vertical lines. (A) Landings are in kg (blocks 651-896, 1032-1035). (B) Mean ±SE nightly fishing effort, in estimated number of squid vessels. (C) Landings per unit of effort (LPUEl: summed landings (kg) on satellite nights were divided by summed effort (estimated number of squid vessels) on the corresponding nights. most of 1997-98 (Fig. 3A), which corresponded to the strong El Nino event during this period (Fig. 4). Effort data revealed surges in the Oct-Dec quarters before the 1997-98 El Nino (Fig. 3B). The Oct-Dec quarter of 1998 signalled a resumption of fishing effort follow- ing El Nino, but effort levels for 1999 and early 2000 were lower than pre-El Nino levels. Interestingly, squid abundance, as measured by landings per unit of effort (LPUE), showed a rapid increase from the El Nino lows, and squid abundance for 1999-2000 reached the high- est values of the time series (Fig. 3C). Analysis of boat locations along the Channel Islands revealed a shift over the course of the fishing season. Compiling the satellite data to yield composite images in multiyear sets, we found that fishing activity in October consis- tently included the north shore of Santa Cruz Island (Fig. 5, A,C,E). In contrast, fishing ac- tivity in March showed considerable reduction along the north side of Santa Cruz Is., but activ- ity continued along the island's southern shore (Fig. 5, B,D,F). Composite images for December and January were also examined for all of the multiyear sets. December marked a transitional stage from the activity in October to reduction of fishing in March along the northern shores. In all multiyear sets, the December lights along northern Santa Cruz Island were more scat- tered and less dense than those in October. January images were very similar to those for March. Although data from March 1993-95 in- dicated little fishing activity, a composite image for January 1993-95 was very similar to that for March 1999-2000: light banks occurred off southern Santa Cruz, southeastern Santa Rosa, and around Anacapa, but were virtually absent from northern Santa Cruz and Santa Rosa. Water temperatures around Santa Cruz Is- land did not consistently differ between north- ern and southern waters. March sea surface temperatures, measured by satellite, were very similar for the island's northern and south- ern shores (Table 2). April sea surface tem- peratures, measured at CalCOFI stations, were slightly warmer to the northeast of the island (Table 2). Temperatures at 75 meters, however, were nearly identical for the two CalCOFI sta- tions (Table 2). Discussion The satellite images and landings data corrobo- rated spatial and temporal patterns of fishing activity for the market squid. For the period 1992-2000, both data sets indicated intense harvesting along the Channel Islands of Santa Rosa, Santa Cruz, Anacapa, and Santa Cata- lina. The satellite images captured additional information, such as fishing activity being Maxwell et al.: Fishery dynamics of Loligo opalescens 667 Table 2 Water temperature ( C I for the northern and southern waters around Santa Cruz Island, March and April, 1993- -2000. Northern waters Southern waters Depth (m) Location Mean Min Max Location Mean Min Max March sea surface temperature, as measured by satellite (PO.DAAC datai' 0 Block 686 14.5 12.8 15.7 Block 708 14.7 13.2 15.9 April temperature, measured at CalCOFI stations2 0 Station 83.42 13.6 11.6 16.7 Station 83.51 12.8 11.2 14.5 75 Station 83.42 9.9 9.3 11.2 Station 83.51 10.3 9.3 11.2 1 Measurements made on multiple nights per month of March (range of measured nights per month of March: 6-26 1. "Mean" is the overall average of the mean March temperatures; "Min" is the minimum of the mean values, "Max is the maximum, of the mean values. 2 One measurement made at each station at each depth per month of April (n = 8 for both depths). clearly delimited by the 100-m contour. The landings data, reported by fishing blocks, were much cruder in geographic scale and failed to catch this subtlety. The ground-truthing work conducted by aerial sur- veys indicated that detected light pixels are useful in estimating the number of squid vessels in operation. This result is consistent with examination of the fish- ery for the squid Illex argentinus in the southwestern Atlantic, where vessels use powerful lamps to attract the squid to lures (Waluda et al., 2002). In the latter fishery, analysis of images acquired by the DMSP-OLS satellites revealed a good fit between the recorded num- ber of vessels in operation on a given night and the number of light pixels detected (Waluda et al., 2002). In the present study, the fishery data showed a strong response to the 1997-98 El Nino event, which was one of the strongest events on record (Wolter and Timlin, 1998). Fishing effort and landings tended to peak in the Oct-Dec and Jan-Mar quarters before the 1997-98 El Nino. Both data series dramatically dropped during the 1997-98 El Nino and showed recov- ery afterwards. Squid abundance, measured as LPUE, also showed a pronounced drop and rapid increase in response to the El Nino. It is interesting to note that another index of market squid abundance, the occur- rence of squid beaks in the scat of sea lions, showed similar responses to earlier El Nino events (Lowry and Carretta, 1999). Squid beak occurrence dropped steeply during the strong 1983-84 El Nino, and increased afterwards. Beak occurrence also dipped and rose in response to a milder El Nino in 1992-93. Significantly, Lowry and Carretta (1999) examined southern Chan- nel Islands: Santa Barbara, San Clemente, and San Nicolas. Our present study reflects squid abundance primarily around northern Channel Islands (e.g., Santa Rosa, Santa Cruz, Anacapa). Taken together, these studies may indicate that El Nino exerts a bight-wide influence on squid abundance. We suggest that a strong El Nino event changes the reproductive conditions for the market squid in the -2.0-1 1 1 1 1 1 1 1 h 1992 1993 1994 1995 1996 1997 1998 1999 2000 Year Figure 4 Multivariate ENSO index iMEI) for the tropical Pacific (between 10CN and 10°Si, by month. Data were obtained from http://www.cdc.noaa.gov/~kew/MEI/table.html. Southern California Bight. With regard to spawning, the spawning population becomes less abundant on the traditional shallow-water spawning grounds. Research on a congener, the South African chokka squid (Loligo vulgaris reynaudii), points to possible environmental influences on spawning for loliginid squid (Roberts and Sauer, 1994). Off South Africa, a strong El Nino can lead to reduced upwelling and increased turbidity. In normal years, upwelling, presumably detected by the squid as an influx of cold water, may trigger spawning behavior (Roberts and Sauer. 1994). In El Nino years off South Africa, reduced upwelling and increased tur- bidity on the inshore spawning grounds are thought to force the spawners into deeper water, beyond the reach of the fishery (Roberts and Sauer, 1994). In a recent study, catch for the chokka squid increased with strong easterly winds, which caused upwelling, and decreased with increased turbidity (Schon et al., 2002). In the California Current System, upwelling decreases during strong El Nino events (Schwing et al., 2000). Upwelling 668 Fishery Bulletin 102(4) October 1992, 1993. 1994 V -O*^- Los Angeles A ^ March 1993, 1994, 1995 120VJ n9*w October 1995, 1996, 1997 March 1996, 1997, 1998 '"^. Los 34'" Nh . - ~ Angeles D • *C>^_ 120°W 119°W October 1998, 1999, 2000 120°W 119°W March 1999, 2000 119°W Figure 5 Location of fishing activity, as indicated by black areas, for the early ( October i and late (March l parts of the traditional squid fishing season in the Southern California Bight. 1992-2000. For each month, a multiyear composite image is shown. (A) October 1992, 1993. 1994. (Bi March 1993. 1994, 1995. (Cl October 1995, 1996, 1997. (Dl March 1996, 1997, 1998. (E) October 1998, 1999. (F) March 1999, 2000. in the Southern California Bight was reduced during the 1997-98 El Nino (Hay ward, 2000). It is not known how market squid adults respond to changes in water temperature or turbidity, or whether spawning fish shift to other habitats during El Nino events. A strong El Nino event can also alter feeding and developmental conditions for squid. During the 1997-98 El Nino, macrozooplankton abundance substantially decreased in the Southern California Bight and off Baja California (Lynn et al., 1998; Hayward, 2000; La- vaniegos et al., 2002). Food availability affects growth rates of loligind squid (Jackson and Moltschaniwskyj, 2001). Recently, Jackson and Domeier (2003) indicated lower growth rates for the market squid in the Southern California Bight during the 1997-98 El Nino. In the present study, fishing effort following the 1997-98 El Nino was generally below pre-El Nino lev- els. The subsequent high levels of catch in late 1999 and early 2000 may indicate that squid were in great abundance, thereby requiring less overall catch effort to meet market demand. A strong La Nina succeeded the 1997-98 El Nino (Lynn and Bograd. 2002; Schwing et al., 2002), with strong upwelling and high macrozoo- plankton abundance in the Southern California Bight by spring 1999 (Schwing et al., 2000; Hayward, 2000). Indeed, the high LPUE in the present study in late 1999 and early 2000 points to increased squid abundance in response to a more productive environment. Alterna- tively, one could argue that increased fishing efficiency. not increased squid abundance, resulted in high LPUE. One manifestation of higher fishing efficiency could be a contracted fishing range, where especially productive pockets are identified and targeted. An overall com- parison of fishing location in October and March before and after El Nino did not support this explanation: the total spatial extent of fishing activity was not greatly Maxwell et al.: Fishery dynamics of Loligo opalescens . 669 reduced in post-El Nino October or March. A noticeable concentration of fishing effort off the southern shore of Santa Cruz Island was evident in the post-El Nino period, however. The landings data may indicate that this southern shore, represented by blocks 708 and 709, was indeed productive. In the pre-El Nino period (1992-96), blocks 708 and 709 represented 3% of the landings in the bight. In the post-El Nino period (1999 to early 2000), these two blocks came to represent 12% of the landings. The spatial distribution of fishing activity appears to shift over the course of the squid fishing season. In the Southern California Bight, October and March mark the traditional beginning and end of the squid fishing season, respectively (Butler et al., 1999). In the present study, fishing activity along the Santa Rosa and Santa Cruz Islands moved largely to the southern shores by March, leaving the northern shores relatively unfished. This spatial shift may reflect change in local squid habitat or changes in the fishermen's behavior. As a rough indicator of habitat quality, water temperature did not consistently differ between the northern and southern waters around Santa Cruz Island in March and April, both at sea surface and at 75 meters depth. Wind conditions, on the other hand, change consider- ably from October to March. The northern shores of Santa Rosa and Santa Cruz lie on the rim of the San- ta Barbara Channel. Wind speed and wind stress are relatively low through the channel in the fall and early winter but increase significantly in March to remain high throughout the spring and summer (Winant and Dorman, 1997; Harms and Winant, 1998; Dorman and Winant, 2000). It remains unresolved whether the high winds in the Channel in March and April create ocean- floor turbulence and turbidity that discourage squid spawning (cf, Roberts and Sauer, 1994), or whether fishermen simply eschew the rocky Channel in favor of the southern shores of the islands. Although satellite remote sensing can generate a "neutral party" record of fishing effort, we note three caveats associated with satellite data. First, large sta- tionary sources of light, such as coastal cities, must be excluded when quantifying fishing vessel activity. The exclusion of urban light sources can result in under- estimating effort, because boats that work near large light sources can be excluded from analysis. We were concerned that an underestimation of effort along the mainland coast would explain this study's post-El Nino increase in LPUE. Landings data, however, may indi- cate that effort in coastal blocks actually declined after the 1997-98 El Nino. Coastal blocks accounted for 19% of the landings in the pre-El Nino years (1992-96), dropping to 11% of landings in the post-El Nino years (1999 to early 2000). Second, the spatial resolution of the satellite imag- es may be large enough to allow multiple boats to fit into one "pixel" of detected light. Thus, effort may be underestimated. Analysis of the ground-truthing fly- overs, however, did not indicate a strong interaction between boat aggregation and nightly fleet size. Boats may have indeed aggregated over the course of our study, but our analysis indicates that such aggregation was independent of nightly fleet size. In this case, the absolute values of estimated effort and LPUE would be underestimated across all dates. The relative values of effort and LPUE, however, will be only slightly affected within a time series; therefore we place confidence in our examinations of the temporal patterns of the ef- fort-based data. A third caveat is specific to the present study. The ground-truthing work occurred during a pe- riod of relatively low fishing effort (1999-2000). Future fly-overs during periods of greater effort will be useful in corroborating our observed relationship between fly- over and satellite data. The present study demonstrates that light detection by satellite remote sensing is useful for examining tem- poral and spatial patterns of fishing effort and popula- tion abundance, as measured by LPUE. Light detection by satellite has certain drawbacks, but these are not insurmountable. Importantly, geo-referenced satellite images provide an independent source of fishing effort, which can be feasibly integrated with environmental data through GIS analysis. With regard to market squid off California, satellite data can help provide fine-scale data on fishing location for this fishery's ongoing man- agement efforts4'5 (see also Mangel et al., 2002). Al- though mandatory shielding of the boat lights went into effect in May 2000, these lights are still detectable by the satellites (authors' pers. obs.). Recently, effort log- books have become mandatory for squid fishermen off California. This requirement points to a unique oppor- tunity to collect and corroborate fishery-dependent and independent measures of fishing effort. Acknowledgments We owe much gratitude to personnel of California's Department of Fish and Game for their assistance in the ground-truthing work. In particular, we thank the pilots Jeff Veal and Tom Evans, and the following aerial observers: D. Bergen, T. Bishop, S. Carner, D. Hanan, C. Kong, J. Kraus, A. Lohse, S. MacWilliams, D. Ono, M. Songer, J. Wagner, and E. Wilson. We also thank Paul Crone for collaboration on this project, Chris Reiss for extracting CalCOFI water temperature data. Rich Cosgrove for assistance with mapping, Kevin Hill for information about the Pacific Fisheries Management Council, and George Watters and anonymous review- ers for constructive comments. This project was funded by the California Department of Fish and Game and U.S. Department of Commerce (NOAA NESDIS Ocean Remote Sensing Program). 4 California Department of Fish and Game. 2003. Draft: Market squid fishery management plan. [Available from: Calif. Dept. Fish Game, 4949 Viewridge Avenue, San Diego, CA 92123.] 5 Maxwell, M. R., L. D. Jacobson, and R. Conser. Manuscript in review. 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Englewood Cliffs, NJ. 671 Abstract-In May 2001, the National Marine Fisheries Service (NMFS) opened two areas in the northwest- ern Atlantic Ocean that had been previously closed to the U.S. sea scallop I Placopecten magellanicus) dredge fishery. Upon reopening these areas, termed the "Hudson Canyon Controlled Access Area" and the "Vir- ginia Beach Controlled Access Area." NMFS observers found that marine turtles were being caught inciden- tally in scallop dredges. This study uses the generalized linear model and the generalized additive model fitting techniques to identify environmen- tal factors and gear characteristics that influence bycatch rates, and to predict total bycatch in these two areas during May-December 2001 and 2002 by incorporating environmental factors into the models. Significant factors affecting sea turtle bycatch were season, time-of-day, sea sur- face temperature, and depth zone. In estimating total bycatch, rates were stratified according to a combination of all these factors except time-of- day which was not available in fish- ing logbooks. Highest bycatch rates occurred during the summer season, in temperatures greater than 19°C, and in water depths from 49 to 57 m. Total estimated bycatch of sea turtles during May-December in 2001 and 2002 in both areas combined was 169 animals ( CV= 55.3 ), of which 164 ( 97% ) animals were caught in the Hudson Canyon area. From these findings, it may be possible to predict hot spots for sea turtle bycatch in future years in the controlled access areas. Magnitude and distribution of sea turtle bycatch in the sea scallop (Placopecten magellanicus) dredge fishery in two areas of the northwestern Atlantic Ocean, 2001-2002 Kimberly T. Murray Northeast Fisheries Science Center National Marine Fisheries Service 166 Water Street Woods Hole, Massachusetts 02543 E-mail address. Kimberly Murray19°C) 0.0000027(82.5) 0.0000052(62.1) 0.0000030(87.71 Low(<19°C) 0.0000002(99.5) N.C.E. 0.0000002(106.6) Mid-depth (49-57 ml High(>19°C) 0.0032018(64.9) 0.0061179(25.4) 0.0035838(57.2) Low(<19°C) 0.0002117(95.4) N.C.E. 0.0002371(98.3) Deep(>57m) High(>19°C) 0.0007578(73.8) 0.0014512(41.9) 0.0008485(80.5) Low(<19°C) 0.0000500(92.91 N.C.E. 0.0000560(103.8) Table 4 Total bycatch estimates by year and season with weighted CVs (%) N.C.E. =no commercial effort. Spring Summer Fall Total Hudson Canyon 2001 10(89.2) 50(61.5) 9(105.8) 69 2002 13(89.2) 78(61.5) 4(105.8) 95 Virginia Beach 2001 N.C.E. N.C.E. 5(105.8) 5 2002 0 0 N.C.E. 0 Totals 23 128 18 169(55.31 mated bycatch of turtles in the Virginia Beach area was five animals in 2001 and zero animals in 2002. Across both areas, the highest bycatches occurred in summer (128 turtles; 76%), followed by spring (23 tur- tles; U7( ) and fall (18 turtles; 10%) (Table 5). One hun- dred thirty-two (78%) (CV=49.6) sea turtles were caught in the mid-depth zone from 49 to 57 m, whereas 37 (22%) (CV=59.6) sea turtles were caught in waters deeper than 57 m. One-hundred fifty-eight (93%) (CV=51.2) sea turtles were caught in waters warmer than 19°C, and 11 (7%) (CV=74.9) in waters cooler than 19°C. Discussion Use of bycatch models Generalized linear and generalized additive models help to identify environmental variables or fishing practices that influence the probability of sea turtle bycatch. In estimating total mortality, bycatch rates can then be stratified according to these factors, reducing unexplained variability in the total estimate. More- over, understanding factors that lead to a high or low 678 Fishery Bulletin 102(4) Table 5 Total bycatch estimates by season, depth. and temperat jre strata in Hudson Canyon and Virginia Beach controlled access areas in 2001 and 2002 with 95<7r confidence intervals. Spring=May-Jun; Summer=Jul -Sep; Fall= =Oct-Dec. N.E.C = no commercial effort; N.O.=no observer coverage. Water depth Temperature 2001 2002 Total Spring Summer Fall Spring Summer Fall Shallow l<49 ml High(>19°C) 0 0 0 0 0 0 0 Low«19°Ci 0 N.O. 0 0 N.C.E. 0 0 Mid-Depth (49-57 mi High(>19°C) 6(0-13) 37(21-59) 2(0-5) 8(0-211 65(34-961 5(0-9) 123 Low(<19°C) 2(0-8) N.C.E. 4(0-191 1(0-2) N.C.E. 2i0-10i 9 Deep (>57 ml High(>19°C) 2(0-51 13(4-25i 2(0-51 4i0-lll 13(4-24) 1 (0-1) 35 Low«19°C) 0(0-11 N.C.E. 1 (0-6) 0(0-1) N.C.E. 1(0-2) 2 Total 10 50 9 13 78 9 169 probability of bycatch can motivate bycatch mitigation research. Finally, the ability to predict bycatch on the basis of explanatory variables allows one to examine the relative effectiveness of different management measures designed to reduce bycatch (Kobayashi and Polovina2). Ultimately this framework can improve the assessment of threats to turtles and broaden conservation options. Magnitude of bycatch During May-December in 2001 and 2002, an estimated 169 animals were captured incidentally by commercial sea scallop dredge vessels in two areas of the Mid- Atlan- tic Bight. Throughout the entire Mid-Atlantic Bight, the magnitude of bycatch was probably larger, particularly because the factors associated with the high bycatch rates were not specific to the controlled access areas. Of the 11 observed turtles measured for size, 9 (82%) were between 70-80 cm straight carapace length (the large juvenile stage). Stage class models indicate that the long- term survivability of loggerhead sea turtles is sensitive to mortality at this life stage (Crouse et al., 1987). Factors influencing bycatch The incidental capture of turtles occurs where there is overlap between fishing effort and turtle habitat. The elevated probability of turtle bycatch occurring in warm waters, during summer, at depths between 50 and 60 m is consistent with the habitat regime of loggerhead sea turtles in the Mid-Atlantic (Shoop and Kenney, 1992; Epperly et al., 1995; Coles and Musick, 2000). During the oceanic phase of their life cycle, sea turtles occupy habitats at specific temperatures or with bathymetric features that concentrate prey and other areas of enhanced productivity (Polovina et al.. 2000). In Mid-Atlantic waters, high aggregations of loggerhead sea turtes have been observed in the summer, in waters 22-49 m deep, at temperatures from 20° to 24°C (Shoop and Kenney, 1992). In the Hudson Canyon and Virginia Beach controlled access creas, the bycatch of sea turtles was associated with habitat conditions rather than gear characteristics. From these findings, it may be possible to predict future hotspots for sea turtle bycatch in the controlled access areas where fishing effort and sea turtles overlap in time and space. These hotspots may be centered over the portion of the Hudson Canyon where depths are between 50 and 60 m. after waters warm to 19 C. Because of the low amount of observer data in the Virginia Beach area, predicted bycatch rates for this area were based largely on conditions within the Hud- son Canyon area. Sea scallop fishing effort occurs year- round both north and south of the Hudson Canyon, and high concentrations of loggerhead sea turtles (de- termined from migratory patterns) exist in spring and fall from North Carolina to northern Maryland ( Shoop and Kenney, 1992). It is probable that the distribution of turtles and scallop fishing effort co-occur in other regions of the Mid-Atlantic, particularly south of the Hudson Canyon. The scallop dredge fishery in the Mid- Atlantic is a complex, dynamic system; there may be other factors influencing the bycatch of sea turtles in the fishery south of the Hudson Canyon that were not observed. However, without additional data on turtle interactions in these areas, it is unwise to extrapolate bycatch estimates beyond the scope of the data in this analysis. 2 Kobayashi. D. R., and J. J. Polovina. 2000. Time/area closure analysis for turtle take reductions. Appendix C, Environmental Impact Statement, FMP for Pelagic Fisher- ies of the Western Pacific, 44 p. NMFS Honolulu, Hawaii, 96822. Conservation management options Time and area closures Models of turtle migrations can be used to predict interactions with fisheries in time and space to maximize the efficiency of time and area Murray: Magnitude and distribution of sea turtle bycatch in the sea scallop dredge fishery 679 closures (Morreale, 1996). The results of this analy- sis indicate that bycatch rates are affected by season, depth, and sea surface temperature. Within certain months and depth zones, therefore, the time when sea surface temperature reaches a threshold level may be the time to trigger an area closure. For example, this type of management approach has been taken in the southeastern United States to regulate turtle bycatch in the large-mesh gill-net fishery.3 The timing of sea- sonally adjusted area closures is based upon analyzing sea surface temperatures in relation to the presence or absence of sea turtles throughout the area (Epperly et al., 1995; Epperly and Braun-McNeill4). In addition, temperature thresholds currently trigger area closures in the southern California driftnet fishery during El Nino conditions to prevent the incidental capture of log- gerhead sea turtles.5 Results from the present study can be used to help evaluate potential bycatch reduction under different management scenarios, given certain assumptions. For example, had the portion of the Hudson Canyon con- trolled access area between depths of 49 and 57 m been closed after surface waters reached 19°C in the summer (the stratum with highest bycatch), the closure would have reduced bycatch by 39%. For this estimate, it is assumed that surface temperatures remain above 19°C throughout the summer and drop below 19°C thereafter. Further, this bycatch reduction scenario also assumes that fishing effort shifts proportionately to the fall and spring season within the same depth zone and that bycatch rates remain the same as those that are cal- culated. Alternatively, fishing effort could shift within a season to shallow and deep depth zones if scallop catch-per-unit-of-effort were not affected. Under this assumption, bycatch would be reduced by 60'"< under the same time and area closure. However, unless there are concurrent reductions in fishing effort, bycatch reductions achieved by these measures could well be offset by increases in bycatch in other depth strata and seasons. Gear or fishing modifications Management actions to modify gear or fishing practices can be evaluated in a similar manner. For instance, this analysis indicates that bycatch rates are influenced by the time-of-day when dredges are in the water. Time-of-day was not used to stratify bycatch rates or to extrapolate total bycatch estimates because of limitations in the fishing effort data ( VTR records). If time-of-day had been incorporated into the bycatch model, the model would have predicted higher bycatch rates when dredges were set between 4 am and 4 pm (day tows). If the stratum with the highest bycatch rate (summer, high surface temperatures, and depths between 49 and 57 m), had been further strati- fied by time-of-day, the model would have predicted a bycatch rate of 0.008 sea turtles/dredge hauls during the day, and 0.002 turtles/dredge hauls during the night. If all the commercial vessels had been fishing during the day in this stratum (;? = 6352 dredges in 2001), the esti- mated bycatch would have been 51 turtles. If the vessels had been fishing during the night, the total estimated bycatch would have been 13 turtles. According to these rates and effort, restricting vessels to night-time tows between the hours of 4 pm and 4 am has the potential to reduce bycatch by 75% in this particular stratum. Although specific gear characteristics did not show a strong relationship to sea turtle bycatch in this analysis, further work should be conducted to evaluate whether specific gear characteristics could be modified to decrease bycatch. For example, the near significance with the model incorporating number of tickler chains (P=0.07) warrants further testing of this gear charac- teristic. Tickler chains cover the mouth of the dredge in a grid-like configuration with the vertical up and down chains. The number of chains on the bag and distance between the chains may help to prevent sea turtles from entering the dredge bag. This dredge configuration is currently being tested for sea turtle bycatch reduction in the Hudson Canyon area (DuPaul and Smolowitz6). Further research should also examine the behavior of sea turtles in relation to dredge gear for a more complete understanding of how and when turtles are entrapped. Sea turtles and scallop dredge interactions cannot be viewed in isolation from other gear types and conserva- tion measures. Some fisheries that co-occur with sea turtles may have an equal, if not greater, impact on turtles than do scallop dredges (e.g., the shrimp trawl fishery in the Gulf of Mexico (Henwood and Stuntz, 1987]). Changes in sea turtle abundance, or shifts in fishing effort, may increase the likelihood of encounters in both net and dredge fisheries. If environmental condi- tions associated with high bycatch rates in the Hudson Canyon and Virginia Beach areas are consistent across years, it may be possible to anticipate and deter future interactions from occurring. Acknowledgments I would like to thank Debra Palka and Marjorie Ross- man for help with analytical and statistical approaches to bycatch estimation. Andy Solow and Andy Beet at the Marine Policy Center, Woods Hole Oceanographic Insti- tute, also provided guidance in the statistical analysis. David Mountain provided invaluable help in acquiring 3 Final Rule. FR 67: 71895-71900. 3 December 2002. 4 Epperly, S. P. and J. Braun-McNeill. 2002. Unpubl. data. The use of AVHRR Imagery and the management of sea turtle interactions in the Mid-Atlantic Bight. NMFS Southeast Fisheries Science Center, Miami, Florida, 33149. 5 Final Rule, FR 68: 69962-69967, 16 December 2003. 6 DuPaul. W. P., and R. Smolowitz. 2003. Unpubl. data. Industry trials of a modified sea scallop dredge to minimize the catch of sea turtles. Virginia Institute of Marine Sci- ence, Gloucester Point, Virginia, 23062, and Coonamessett Farm, East Falmouth, Massachusetts, 02536. 680 Fishery Bulletin 102(4) sea surface temperature for data. Frederic Serchuk, Richa Marjorie Rossman, and Pau Fisheries Science Center all of the manuscript. Jeffrey S mous reviewers provided val peer review. Finally, I wish to collected data on interactions sea scallop dredge fishery. the Observer and VTR rd Merrick, Debra Palka, Rago at the Northeast provided initial reviews eminoff and two anony- uable comments during thank the observers who between turtles and the Literature cited Allen, L. K. 2000. Protected species and New England fisheries: an overview of the problem and conservation strategies. Northeastern Naturalist 7(41:411-418. Burnham, K. P. and D. R. Anderson. 2002. Model selection and multimodel inference: a prac- tical information-theoretic approach, 2nd ed., 488 p. Springer-Verlag, New York, NY. Cochran, W.G. 1977. Sampling techniques, 3rd ed., 428 p. John Wiley & Sons, New York, NY. Coles, W. C. and J. A. Musick. 2000. Satellite sea surface temperature analysis and correlation with sea turtle distribution off North Carolina. Copeia 2:551-554. Crouse, D. T., L. B. Crowder, and H. Caswell. 1987. 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Distribution and relative abundance of sea turtles caught incidentally by the U.S. pelagic longline fleet in the western North Atlantic Ocean, 1992-1995. Fish. Bull. 97:200-211. Appendix 1 Categorical variables examined in an analysis of factors affecting sea turtle bycatch in the sea scallop dredge fishery. Frequency of observed dredges ir each category is also shown. Number of Number of Number of Number of observed observed observed observed dredges in dredges in dredges in dredges in Hudson Virginia Hudson Virginia Variable Category Canyon 2001 Beach 2001 Canyon 2002 Beach 2002 Year 2001 or 2002 9493 520 8059 0 Season Spring = May and June 3919 0 1987 0 Summer = July, August, September 2719 0 3764 0 Fall = October, November, December 2855 520 2308 0 State in which Connecticut 199 0 595 0 scallops were landed Massachusetts 4925 0 5628 0 New Jersey 2849 0 740 0 Rhode Island 112 0 474 0 Virginia 1408 520 622 0 Frame width' Small = 3.0-3.9 m ( 10-13 ft) 560 0 443 0 category Medium = >3.9 m and <4.5 m (15 ft) 3987 122 3013 0 Large = <4.5 m-4.8 m ( 15-16 ft ) 4946 398 4603 0 Number of up and Code 1 = 0 chains 4256 520 2171 0 down chains used- Code 2 = 1-4 chains 4089 0 5378 0 Code 3 = >4 chains 1148 0 510 0 Number of tickler Code 1 = <2 chains 6890 520 4469 0 chains used3 Code 2 = >2 chains 2603 0 3590 0 Time-of-day Day = 4 am-4 pm 5514 346 4854 0 Night = 4 pm-4 am 3979 174 3205 0 Sea surface Hi = >19'C 3910 518 4883 0 temperature Low = <19°C 5583 2 3176 0 Depth Shallow = 40-<49 m (22-27 fmi 1089 42 782 0 Mid-Depth = 49-57 m (27-31 fin) 3371 280 3642 0 Deep = >57-88 m (31-48 fm) 5033 198 3635 0 ; Width of the dredge fra me. 2 Vertical chains attache i to the sweep on the bottom of the dredge that prevent rocks from entering the chain bag. Number of up and down chains were influenced by bottom type. 3 Horizontal chains attac hed to the sweep on the bottom of the dredge that help stir up contents of the sea bottom. Number of ti< kler chains were influenced by bottom t\ pe. 682 Abstract — Numerous studies have applied skeletochronology to sea turtle species. Because many of the studies have lacked validation, the applica- tion of this technique to sea turtle age estimation has been called into question. To address this concern, we obtained humeri from 13 known-age Kemp's ridley (Lepidochelys kempii) and two loggerhead (Caretta caretta) sea turtles for the purposes of examin- ing the growth marks and comparing growth mark counts to actual age. We found evidence for annual deposition of growth marks in both these spe- cies. Corroborative results were found in Kemp's ridley sea turtles from a comparison of death date and amount of bone growth following the comple- tion of the last growth mark (n=76). Formation of the lines of arrested growth in Kemp's ridley sea turtles consistently occurred in the spring for animals that strand dead along the mid- and south U.S. Atlantic coast. For both Kemp's ridley and loggerhead sea turtles, we also found a propor- tional allometry between bone growth • humerus dimensions) and somatic growth (straight carapace length i, indicating that size-at-age and growth rates can be estimated from dimen- sions of early growth marks. These results validate skeletochronology as a method for estimating age in Kemp's ridley and loggerhead sea turtles from the southeast United States. Validation and interpretation of annual skeletal marks in loggerhead (Caretta caretta) and Kemp's ridley (Lepidochelys kempii) sea turtles Melissa L. Snover Duke University Marine Laboratory 135 Duke Marine Lab Road Beaufort, North Carolina 28516 Present addresss: Pacific Fisheries Environmental Laboratory 1352 Lighthouse Ave. Pacific Grove, California 93950 E-mail address: melissa snover g noaa gov Aleta A. Hohn Center for Coastal Fisheries and Habitat Research National Marine Fisheries Service, NOAA 101 Pivers Island Road Beaufort, North Carolina 28516 Manuscript submitted 1 5 August 200,3 to the Scientific Editor's Office. Manuscript approved for publication ii June 2004 by the Scientific Editor Fish. Bull. 102:682-692 (2004 I. The basic tenet of skeletochronology is that bone growth is cyclic and has an annual periodicity in which bone for- mation ceases or slows before new, rel- atively rapid bone formation resumes (Simmons, 1992; Castanet et al„ 1993; Klevezal, 1996). This interruption of bone formation is evidenced within the primary periosteal compacta by histological features, which take two forms in decalcified and stained thin- sections. The most common form is a thin line that appears darker than the surrounding tissue, termed the "line of arrested growth" (LAG) (Castanet et al., 1977). The second, less-common form is a broader and less distinct line that also stains darker, referred to as an annulus (Castanet et al., 1977). Alternating with LAGs or annuli are broad zones that stain homogeneously light, and represent areas of active bone formation. Together, a broad zone followed by either a LAG or an annulus represents a skeletal growth mark (GM) (Castanet et al., 1993). To apply skeletochronology to a species, the annual periodicity of the GM must be validated. Validation studies are necessary not only to confirm the annual nature1 of the GM but also to identify and in- terpret anomalous LAGs. Anomalous LAGs that are a common problem in skeletochronology studies of reptiles and amphibians include double (Chin- samy et al., 1995; El Mouden et al., 1997; Guarino et al., 1998), splitting (Guarino et al., 1995; 1998; Coles et al., 2001), and supplemental (Guarino et al, 1995; Lima et al., 2000; Tren- ham et al., 2000) lines. In addition to anomalous LAGs, there are two other difficulties typical in skeletochronol- ogy studies; compression of LAGs at the periphery of the bone and resorp- tion of the innermost LAGs. In older animals the GMs are compressed at the outer periphery of the bone as a result of decreased growth. Francil- lon-Vieillot et al. (1990) term this phenomenon "rapprochement" and it is a problem when the LAGs become too close together to be differentiated — usually in the small phalangeal bones used in amphibian studies lEg- gert and Guyetant. 1999; Lima et al., 2000; Leclair et al.. 2000). In addition to anomalous and com- pressed LAGs, the loss of early GMs through endosteal resorption is an- other problem with skeletochronol- ogy. Although this does not present a problem with most amphibian species (Kusano et al., 1995; Castanet et al., 1996; Sagor et al., 1998), the prob- lem is extreme in skeletochronology studies of loggerhead (Caretta caretta; Klinger and Musick. 1995; Zug et al., 1995; Parham and Zug, 1997), green Snover and Hohn: Validation and interpretation of skeletal marks in Caretta caretta and Lepidochelys kempu 683 Table 1 Species and history of known -age sea turtles analyzed in this study. Sample Species History during captivity Age (yr) LK-1 Lepidochelys kempu Captive for first year, then released 5.0 LK-2 L. kempii Captive for first year, then released 6.5 LK-3 L. kempii Captive for first year, then released 4.5 LK-4 L. kempii Tagged and released after hatching 1.27 LK-5 L. kempii Tagged and released after hatching 1.70 LK-6 L. kempii Tagged and released after hatching 1.72 LK-7 L. kempii Tagged and released after hatching 2.37 LK-8 L. kempii Tagged and released after hatching 2.37 LK-9 L. kempii Tagged and released after hatching 3.25 LK-10 L. kempii Tagged and released after hatching 2.0 LK-11 L. kempii Tagged and released after hatching 2.75 LK-12 L. kempii Tagged and released after hatching 3.0 LK-13 L. kempii Tagged and released after hatching 4.25 CC-1 Caretta caretta Captive during entire life 29.4 CC-2 C. caretta Captive for first two years, then released 8.0 (Chelonia mydas; Zug and Glor, 1998; Zug et al., 2002) and Kemp's ridley (Lepidochelys kempii; Zug et al., 1997) sea turtles. In each of these studies, the authors used various protocols to estimate the number of lay- ers lost. Any protocol estimating the number of layers lost to resorption relies on the concept that the spatial pattern of the LAGs is representative of the growth of the animal. To confirm this assumption, researchers must establish a correlation between bone dimensions and body size (Hutton, 1986; Klinger and Musick, 1992; Leclair and Laurin, 1996). Two of the studies that have applied skeletochronology to sea turtles have demonstrated annual GMs in both juvenile (Klinger and Musick, 1992) and adult (Coles et al., 2001) loggerhead sea turtles. Numerous additional studies have applied skeletochronology to sea turtles. To date, the technique has been applied to loggerhead (Zug et al., 1986; Zug et al, 1995; Bjorndal et al., 2003), green (Bjorndal et al., 1998; Zug and Glor, 1998; Zug et al., 2002), Kemp's ridleys (Zug et al., 1997), and leatherback [Dermochelys coriacea) (Zug and Parham, 1996) sea turtles. What is needed for the appropriate application of skeletochronology to sea turtle species is additional validation of annual GMs and a guide to their interpretation. Furthermore, because resorption is a problem in sea turtle bones, the validation of a pro- portional allometry between bone and somatic growth is necessary to enable back-calculation. In this study, we address each of these issues for Kemp's ridley and loggerhead sea turtles by examining humeri from known-age animals. We analyzed each humerus without prior knowledge of the animal's age and we present the results of our analyses, including reinterpretations of bones for which we were incorrect in our age assessments. The purpose of this study was to use known-age samples both to validate the likeli- hood that GMs are annual and as a learning tool for the best guide to interpreting GM in wild animals. Materials and methods We obtained samples from two known-age loggerhead and 13 known-age Kemp's ridley sea turtles (Table 1). In addition, we collected samples from 240 wild logger- head and 262 wild Kemp's ridley sea turtles. With the exception of one loggerhead, CC-1, all of the sea turtles died in the wild and samples were retrieved from the carcasses. Sample CC-1 died in captivity. Sample preparation Zug et al. (1986) analyzed skeletal elements of the cra- nium and right forelimb of loggerhead sea turtles and determined that the humerus was most suited to skeleto- chronology studies. Therefore, we also used the humerus. Specimens arrived as either dried bones or whole flippers. For flippers, we dissected out the humerus, which v/as then flensed, boiled, and air-dried for at least two weeks. We cross-sectioned each humerus at a site just distal to the deltopectoral crest. At this site, the ratio of cortical to cancellous bone is highest (Zug et al., 1986), and the region immediately distal to the insertion scar of the deltopectoral muscle on the ventral side of the bone maxi- mizes that ratio (see Zug et al., 1986 for diagrams of the loggerhead sea turtle humerus). This site also provided a landmark that allowed us to section at equivalent sites on every humerus. We removed 2-3 mm thick sections at that site us- ing a Buehler® isomet low speed saw. This section was 684 Fishery Bulletin 102(4) fixed in 109c formalin then decalcified by using a com- mercial decalcifying agent (RDO, Apex Engineering Products Corporation, Calvert City, Kentucky). Time to decalcification varied with the size of the bone and the strength of the solution, usually between 12 and 36 hours. Following decalcification, 25-f.im thick cross-sec- tions were made by using a freezing-stage microtome. Sections were stained in Erlich's hematoxylin diluted 1:1 with distilled water (Klevezal, 1996) and mounted on slides in 100% glycerin. Known-age sea turtles We received the humeri from each of two captive, known- age loggerhead sea turtles after they died (Table 1). The first specimen, CC-1, was held in an outdoor tank during the summer months and inside a greenhouse during the winter months (this turtle was the same captive female noted in Swartz, 1997). The second, CC-2, was raised in captivity for two years then released from Panama City, Florida, into the Gulf of Mexico. For the Kemp's ridley sea turtles, we received humeri from 13 dead known-age animals (Table 1). The head- start Kemp's ridleys were raised in captivity for one year, then released as part of a binational program oper- ated jointly by state and federal U.S. agencies and the Instituto Nacional de la Pesca (INP) of Mexico (Klima and McVey, 1995). The coded-wire-tagged (CWT) Kemp's ridley sea turtles were tagged and released as hatch- lings. This tagging program is operated jointly by the U.S. National Marine Fisheries Service (NMFS) Galves- ton Laboratory and the INP of Mexico as a means of gaining a better understanding of the early life history of the Kemp's ridley sea turtle (Caillouet et al., 1997). Using the methods described previously, we prepared stained thin-sections from the humeri. Without prior knowledge of the animal's history, the number of visible LAGs was quantified for each bone and a minimum age estimated. Our age estimates were then compared to the age information available for each animal. Indirect validation of annual growth marks Peabody (1961) and Castanet et al. (1993) suggested that the correlation between the width of the last zone formed and the date of death provided an indirect means of vali- dating that deposition of the LAG occurs annually and at the same time of year for an individual population. We applied this method to 76 wild Kemp's ridley sea turtles for which humeri displayed between one and five LAGs. Each of these animals had stranded dead along the Atlantic coast between Maryland and North Carolina. Thin-sections were prepared of the humeri as described above. We quantified the width of the last zone formed by measuring the outside diameter of the whole section (D0) and the diameter of the last competed LAG (DL), between the lateral edges of the bone on an axis paral- lel to the dorsal edge. The amount of bone growth after the last LAG (D0-DL) was plotted against the Julian stranding date, with the assumption that stranding date approximated date of death. Least-squares linear regressions were fitted to the data. Validation of the relationship between LAG diameter and body size In order to relate GM diameters to somatic growth rates, there must be a constant proportionality between bone growth and somatic growth (Chaloupka and Musick, 1997). To address this proportionality, we took eight morphometric measurements of 240 wild loggerhead and 262 wild Kemp's ridley humeri, using digital calipers or a tape measure when dimensions were beyond the range of the calipers. Measurements of maximum length, longitudinal length, proximal width, distal width, delto- pectoral crest width, lateral diameter at sectioning site, ventral to dorsal thickness at sectioning site, and mass were recorded. We compared these measurements with the carapace length, measured as standard straight-line length (SCL) from the nuchal notch to the posterior end of the posterior marginal, using a least-squares linear regression. For mass, the data were natural-log trans- formed to form a linear regression. Results Known-age Kemp's ridley sea turtles Three Kemp's ridley sea turtles captive for one year and then released were recovered 4.5 to 6.5 years after hatching (Table 1). Sample LK-1 had minimal resorp- tion and four complete GMs, each comprising one zone followed by a LAG. An additional zone was seen at the periphery and the LAG that would complete this last GM was not yet visible at the outer edge of the humerus cross-section. From GM counts and death date, we esti- mated the age of this animal accurately at five years (Fig. 1). Sample LK-2 retained five completed and one incomplete GM; however, we observed a large area of resorption in the interior region of the cross-section that potentially obscured additional GMs. We aged this animal at a minimum of 5.5 years, the actual age being 6.5 years. Sample LK-3 displayed four completed GMs and one incomplete mark. Without prior knowledge of this animal's age, we estimated the age accurately at 4.5 years based on layer count and time of death. Ten of the Kemp's ridley sea turtle samples were tagged and released after hatching, and no time was spent in captivity (Table 1). Results from these ten re- covered animals allowed us the opportunity to study and interpret the early GM patterns in noncaptive ani- mals. The first year mark for Kemp's ridley sea turtles appeared to be a poorly defined annulus, as evidenced by LK-4 (Fig. 2A). In turtles greater than two years old, similar first year marks also appeared more or less distinctly (Figs. 2B and 3). Additional marks, which can only be interpreted as supplemental lines given the age of the animal, appeared between GM one and the outer edge of the bone in LK-6 (Fig. 2B) and LK-10. Specimens Snover and Hohn: Validation and interpretation of skeletal marks in Caretta caretta and Lepidochelys kempu 685 LK-7 and LK-8 were difficult to inter- pret and in our initial assessment we underestimated age by one year. In both of these samples, the LAG rep- resenting the end of the second GM was very close to the outer edge of the bone cross-section and was difficult to differentiate from the edge. Hence these samples were not counted in the initial assessment. Because both of these animals died in the fall, there would have been a full growing sea- son, and hence a growth zone, follow- ing the completion of the second GM. Both of these animals were recovered dead in Cape Cod, Massachusetts, during the fall of 1999 when record numbers of cold-stunned sea turtles stranded in that region. Humerus cross-sections from LK-9 through LK-13 (Fig. 3) showed poorly defined annuli at the end of the first GM — annuli similar to the poorly defined annulus in LK-4 (Fig.2A). Subsequent GMs in these humerus cross-sections contained well-defined LAGs. Without prior knowledge of these animals' history we accurately aged each of them from GM counts and stranding date. Specimens LK-9 through LK-13 demonstrated clearly that well-defined LAGs were depos- ited at the end of year two and in subsequent years, providing evidence that any lines between the year-one annulus and the year-two LAGs were supplemental. Known-age loggerhead sea turtles The first known-age loggerhead sea turtle, CC-1, was 29.4 years old. Eleven LAGs were discernible around the circumference of the bone cross- section (Fig. 4A), although the LAGs become too compressed on the lateral edges of the bone to be differentiated; hence counts were made on the ven- tral and dorsal edges.! Fig. 4). Trac- ing the LAGs from the lateral to the ventral edge of the bone, we observed that these LAGs at some point became bifurcating and splitting LAGs and we interpreted each branch as a separate LAG. An additional nine LAGs can still be seen within the resorption zone in most areas of the bone (Fig. 4B). On the dorsal side of the cross-section, at least four less-distinct LAGs or annuli could still be observed; these had been LAG-4 CAG"-3- E GM-' Figure 1 Image of a humerus cross-section from a headstart Kemp's ridley {Lepido- chelys kempii, LK-1) sea turtle. GM-1 refers to growth mark one; LAG-2, LAG-3, and LAG-4 refer to the lines of arrested growth ending growth marks two, three, and four. Curved dashed lines highlight GM-1 and the LAG. Black bar represents 1 mm in length. This specimen was 5.0 years old. Annulus ending GM-1 B /? Supplementa lines Annulus ending GM-1 Figure 2 Images of humeri cross-sections of two coded-wire-tagged Kemp's ridley sea turtles (L. kempii). GM-1 refers to growth mark one. Black bar repre- sents 1 mm for both images. (A) Specimen LK-4 was 1.27 years old. (B) Specimen LK-6 was 1.72 years old. 686 Fishery Bulletin 102(4) Annulus ending GM-1 3 "t 4 LAG-2 ' \ LAG-3LAG-4 Figure 3 Image of humerus cross-section from a coded-wire-tagged Kemp's ridleys (L. kempii). Black bar represent 1 mm in length. GM-1 refers to growth mark one; LAG-2, LAG-3, and LAG-4 refer to the lines of arrested growth ending growth marks two, three, and four. Curved black lines highlight LAGs or annuli. This specimen, LK-13, was 4.75 years old. resorbed in all other parts of the bone (Fig. 4C). There had been a great deal of remodeling within the bone and much of the inner portion of the bone had been resorbed. Summing all of these GMs, we gave a minimum age esti- mate of 24 years without prior knowledge of the history of the animal. The outermost 20 GMs contained well- defined LAGs that were spaced close together, whereas the four interior-most visible GMs contained LAGs or annuli that were spaced farther apart (Fig. 4). The number of layers completely resorbed was five. A second known-age loggerhead sea turtle, CC-2, was eight years old. We assigned a minimum age estimate of five years. Just outside of the resorption area was a series of three LAGs that were very close together (Fig. 5). In our initial analysis, we assumed that three LAGs so close together could not each be deposited an- nually and we interpreted the triple LAGs as a single LAG with an anomalous appearance. We re-evaluated this assumption after learning its history. The animal was in captivity for two years and then released at 42.7 cm SCL in October 1994. Counting back from the outside of the bone, the outermost of the triplet LAGs would represent spring 1996. Given this evidence, our best interpretation of this bone section was that the innermost of the triplets of LAGs indicated release and was therefore not an annual mark. The next LAG was likely deposited the following spring (1995) and was likely an annual mark. The third of the closely spaced LAGs likely represented spring 1996, indicating that the animal did not grow significantly in its first year in the wild (Fig. 5). Following the three closely spaced LAGs. there were four additional indistinct LAGs or annuli that represented the remaining years at large. The outermost of these was very close to the edge of the bone, indicating that the animal did not grow much, if at all, during the last summer of its life. Indirect validation of annual growth marks For Kemp's ridley sea turtles, there was a significant increase in the amount of bone deposited after the last LAG from 20 June to 30 November (Fig. 6). The LAGs near the outer edges of the bones were fully visible in strandings that occurred after 20 June. Earlier detec- tion of the outer LAGs was unlikely because a certain amount of bone formation must occur following the LAG before it can be discerned from the edge. There was not a significant relationship between bone growth and date from 1 December to 19 June. The slope of this regression was very close to zero (6 = -0.003). indicating no trend, either increasing or decreasing, in the amount of bone deposited during this time (Fig. 6). Validation of the relationship between LAG diameter and body size The regressions of the eight morphometric measure- ments of loggerhead and Kemp's ridley sea turtle humeri against SCL revealed high correlations between bone dimension and body size (Table 2). Most importantly for purposes of back-calculation, the lateral diameter at the sectioning site of the humerus i distal to the insertion scar of the deltopectoral muscle) and the body length of the animal was highly correlated. Snover and Hohn: Validation and interpretation of skeletal marks in Caretta caretta and Lepidochetys kempii 687 Discussion Validation of the annual nature of growth marks Our results supported annual deposition of GMs in log- gerhead and Kemp's ridley sea turtles. The headstarted and older CWT Kemp's ridley sea turtles in particular highlighted the likelihood of annual marks. These ani- mals displayed sharp and regularly spaced LAGs that were consistent with the actual ages of the animals. The results from the CWT Kemp's ridley sea turtles also emphasized the difficulties in interpreting early GMs. From these animals we concluded that in general Kemp's ridley sea turtles deposit a poorly defined annulus in their first year and well-defined LAGs starting with the end of the second year and in following years. For loggerhead sea turtles, only CC-2 spent any time in the wild. The number of GMs deposited after the animal was released (determined from the appearance of the anomalous triplet of LAGs) was consistent with the number of years for which the animal was at large, considering that the first mark was deposited at release. This indicated that not less than one GM was deposited per year, and that additional or supplemental LAGs or annuli indistinguishable from annual lines may be deposited under extreme conditions, such as at the time of release into the wild. Fortunately, in this case, these extreme conditions were not frequent enough to have a serious impact on age estimates. For the life-time cap- tive animal, CC-1, our estimated minimum age was five years shorter than the actual age of 29.4 years and clearly demonstrated that not more than one GM was deposited each year. Because of the relatively large size of the sea turtle humerus, in comparison to phalanges of amphibians, rapprochement did not appear to be a problem in our attempts to discern LAGs. This bone was similar in appearance to adult wild loggerhead and Kemp's ridleys sea turtles with rapprochement of the peripheral LAGs and resorption of most of the interior GMs. Although accurate age estimates cannot be made of these bones through skeletochronology, if rapproche- ment correlates to the timing of sexual maturity, counts of the compressed GMs can provide valuable informa- tion on postreproductive longevity and adult survival. This information can be combined with average age at reproductive maturation for piecing together the life history of sea turtles. Although our sample size for loggerhead sea turtles was very small (two), the size complements a tetracycline-injection study that previ- ously validated annual GMs for juvenile loggerhead sea turtles from Chesapeake Bay (Klinger and Musick, 1992). In addition, an adult loggerhead sea turtle from that same study stranded dead 8.25 years after in- jection and provided evidence of annual deposition of growth marks in adults (Coles et al., 2001). The indirect validation results for Kemp's ridley sea turtles highlighted the cyclic nature of bone growth; bone deposition increases from late spring through early summer to fall and no bone deposition occurs from De- cember to spring. From this information we inferred 10 11 B -9- -10- 11 =14/15= 16 12 13 17 19, 20 c -24- "./-23- .-22- J2.Y J20- Figure 4 Images of different portions of the humerus cross-sec- tions of CC-1 (Caretta caretta). Black bar represents 1 mm in length for all views. (A and Bl The outer edge of the bone is at the top of the photo. (C) The outer edge of the bone is towards the bottom of the photo. For all views, lines of arrested growth (LAGs) are labeled with numbers; low numbers represent the most recently deposited LAGs (near the outer edge of the bone) and higher numbers represent the earlier LAGs. that LAGs form annually in the spring for Kemp's rid- ley sea turtles that strand along the mid- to southeast U.S. Atlantic coast and that these LAGs are visible at the edges of the bones by late spring to early summer. 688 Fishery Bulletin 102(4) triple LAG ^> . >-'.c •* a C o y' ? f ^ 5 > Q J3 -j - ^ * a *•» '• ■•" h" ■ ■ ' \ ' ■-■> '¥> o " Figure 5 Image of a section of the humerus cross-section of CC-2 (C. caretta). Outer edge of bone is towards the bottom of the photo. Solid lines (upper left I highlight a series of triple lines of arrested growth (LAGs); curved dashed lines highlight the three diffuse LAGs. Black bar represents 1 mm in length. Most studied species of reptiles and amphibians deposit GMs within their bones (Castanet et al., 1993; Smirina, 1994). For some of these species, the annual nature of the GM has been validated (e.g.. Tucker, 1997; de Buffrenil and Castanet, 2000; Trenham et al., 2000). For others, it is consistent with their ecology that the marks must represent annual events (Castanet et al., 1993). Growth marks observed in loggerhead (Zug et al., 1986; Zug et al., 1995; Coles et al., 2001), Kemp's ridley (Zug et al., 1997), and green (Zug and Glor, 1998; Zug et al., 2002) sea turtles are similar in structure to those observed in other species of reptiles and amphibians. Drawing on previous studies of reptiles and amphibians, validation studies on sea turtles, and the evidence presented in this article, we assert that GM in bones of sea turtles are likely deposited primarily with an annual periodicity. Given these results, on the surface it seems contradic- tory that in two validation studies annual GMs could not be confirmed. For serpentine species, Collins and Rodda (1994) injected brown snakes with a fluorescent marker and kept them in captivity for one year under two different feeding regimes. Five or six GMs vary- ing in distinctness were identified beyond the fluores- cent marks in bone cross-sections. Statistical analyses showed that these marks may relate to shedding events. It is unclear if the GM pattern prior to captivity was similar to what was seen after the fluorescent mark. The forced feeding component of that study may have induced higher growth rates than would be found in nature, causing the shedding events to appear as his- tological marks in the bone. In a sea turtle study, Bjorndal et al. (1998) did not find GMs in the humeri of green sea turtle bones. They suggested that the tropical marine habitat of the study 5 -i LU 3 - 150 250 350 Julian date 450 550 Figure 6 Julian date of stranding plotted against the amount of bone deposited peripherally to the last LAG in Kemp's ridley sea turtles (L. kempii: n = 76). D0 represents the outside diameter of the humerus, DL represents the diameter of the last LAG. Julian dates on x-axis equate to 20 June through 19 June; therefore num- bers that are greater than 365 represent the Julian date plus 365. Solid lines represent linear regressions that were run separately for 6 months. 20 June to 31 November I filled squares) and 1 December to 19 June (open squares). The regression for the first six months was significant (P<0.006i and the regression for the second six months was not significant lP= 0.27 1. population (approximately 21°07'N) allowed for con- tinual activity and growth and inhibited GM forma- tion. However, GMs have been clearly demonstrated in green sea turtles from the coastal waters of Florida (approximately 29°N) (Zug and Glor, 1998) and Hawaii (approximately 22°N) (Zug et al., 2002). Other studies of reptiles and amphibians in tropical and warm tem- porate climates have reported distinct GMs in species that remain active year-round (i.e., do not hibernate or estivate) (Patnaik and Behera, 1981; Estaban et al., 1996; Guarino et al.. 1998). Interpretation of anomalous LAGs Although our sample sizes were small, especially for log- gerhead sea turtles, several characteristics were noted in the analyses of the samples that would affect how anomalous LAGs are interpreted. Three interpretations of double and bifurcating LAGs are provided. The first interpretation is that if double LAGs appear frequently in individual bones and throughout the sample, they likely indicate an ecology that has two growth cycles per year (Castanet et al., 1993). In this case the two LAGs are distinct from each other over the entire bone cross-section. This pattern was observed in the newt Triturus marmoratus living at a high altitude where the animals had both winter and summer dormancy periods (Castanet and Smirina. 1990; Caetano et al., 1985; Caetano and Castanet, 1993). The second inter- pretation of double LAGs is that they result from a brief Snover and Hohn: Validation and interpretation of skeletal marks in Caretta caretta and Lepidoche/ys kempn 689 Table 2 Regressions equations and statistics from correlations between dimensions of the humerus and notch-to-tip straight carapace length (SCL, cm) in loggerhead and Kemp's ridley sea turtles. All F statistics are significant at P<0.005. Humeral measurement Model equation SE slope Loggerhead sea turtles In =243) Maximal length (ML, mm) Longitudinal length ILL, mm) Proximal width (PW. mm) Deltopectoral crest width (DCW, mm) Site of sectioning width ISW, mm) Site of sectioning thickness ( ST, mm) Distal width (DW, mm) Mass(M, g) Kemp's ridley sea turtles (rc=262) Maximal length (ML, mm) Longitudinal length (LL, mm) Proximal width iPW, mm) Deltopectoral crest width (DCW, mm) Site of sectioning width ( S W, mm ) Site of sectioning thickness (ST. mm) Distal width ( DW, mm) Mass (M, g) SCL = 0.44xA/L + 5.97 SCL = 0.47xLL + 4.85 SCL = 1.06xPW + 7.31 SCL= 1.69xDCW + 6.04 SCL = 2.38xSW + 5.48 SCL = 4.13xST + 11.62 SCL = 1.28xZW+5.43 ln(SCL) = 0.30xln(M) + 2.94 SCL = 0.43xML + 4.69 SCL = 0.47xLL + 3.11 SCL = 1.12xPW+4.39 SCL = 1.69xDCW + 3.35 SCL = 2.48xSW + 2.74 SCL = 4.16xST+ 4.79 SCL= 1.36xDW+ 0.227 LNiSCL) = 0.30xLN(M) + 2.89 0.0064 4814 0.95 0.0064 5381 0.96 0.015 4857 0.95 0.026 4069 0.94 0.037 4110 0.94 0.080 2682 0.92 0.021 3684 0.94 0.0022 18905 0.99 0.0040 10970 0.98 0.0039 14772 0.98 0.010 12390 0.98 0.017 10200 0.98 0.033 5715 0.96 0.072 3306 0.93 0.013 11435 0.98 0.0023 16305 0.98 interruption of hibernation (Hemelaar and van Gelder, 1980). In this instance little bone deposition would occur and the layers would not be distinct from each other over the entire bone, thus giving the appearance of a bifurcating LAG (Hemelaar and van Gelder, 1980). The third interpretation of double or bifurcating LAGs is that they result from extreme decreased growth over the active period, which places annual LAGs very close to each other and in some cases they appear to merge (de Buffrenil and Castanet, 2000). With the first two interpretations, a double or bifur- cating LAG would be counted as one for the purposes of age estimation, whereas the third interpretation would necessitate counting each LAG or bifurcating branch separately. Coles et al. (2001) interpreted a bifurcat- ing LAG as one LAG in an adult loggerhead sea turtle that was recovered 8.25 years after it had been injected with oxytetracycline. In cross-sections of the humerus, Coles et al. (2001) reported seven LAGs following the tetracycline mark, six plus the bifurcating LAG. The animal was marked on 20 June 1989 and recovered dead on 22 September 1997. It is reasonable to assume that, as with Kemp's ridley sea turtles from the same region, the LAGs form in the spring, and Coles et al. (2001) showed that the oxytetracycline mark overlaid one of the LAGs — likely the LAG deposited in spring of 1989. Therefore, there should have been eight LAGs deposited after the tetracycline mark, not seven, each representing the spring of years 1990 through 1997. In this case, then, the bifurcating mark in this bone should be counted as two LAGs. Similarly, for splitting LAGs, where numerous thin- ner LAGs branch out from what appears to be one thick LAG, Francillon-Vieillot et al. (1990) examined different bones from the same animal and determined whether each thin LAG comprising splitting LAGs should be counted as one LAG. In our analysis of the adult log- gerhead sea turtle, CC-1, we observed several bifurcat- ing and splitting LAGs. each of which eventually split into two or more thinner LAGs. We counted each of the thin LAGs as one. Because the LAG count was close to the actual age of the animal, this interpretation ap- pears to have been appropriate for compressed LAGs in adult humeri. The question remains as to whether this is the ap- propriate interpretation for double or bifurcating LAGs in juveniles. Wild loggerhead growth rates have been monitored in an ongoing mark-recapture study in Pam- lico and Core Sounds in North Carolina (Epperly et al., 1995). Epperly et al. (1995) currently have 65 growth rates for 49 juvenile loggerhead sea turtles between 45.1 and 81.0 cm SCL at initial capture that were at-large for one year (±0.1 year). The mean annual growth rate for all of the animals is 2.09 cm/yr. However, of the 65 growth records, 11 of them displayed an annual in- crease of 0.3 cm or less in SCL (Braun-McNeill1). Hence it is not uncommon for juvenile loggerhead sea turtles to Braun-McNeill, J. 2004. Personal commun. Center for Coastal Fisheries and Habitat Research, National Marine Fisheries Service, NOAA, 101 Pivers Island Rd., Beaufort, NC 28516 690 Fishery Bulletin 102(4) grow little or not at all over the course of a year. Using the equation for width at sectioning site from Table 2, we found that the increase in bone diameter for these 11 animals was =0.13 mm or less, which places the LAGs very close together. Because it not uncommon for sea turtle to exhibit little or no growth over a year, LAGs spaced closely together very likely represent distinct years as also determined by de Buffrenil and Castanet (2000). Although the sample sizes are still small for a definitive answer, our results indicate that counting the LAGs individually is the correct interpretation of double or bifurcating LAGs in juvenile as well as adult loggerhead sea turtles. Similarly, our results indicate the same interpretation for double or bifurcating LAGs in juvenile Kemp's ridley sea turtles. The CWT Kemp's ridley sea turtles, samples LK-7 and LK-8, displayed LAGs near the outer edge of the bone and a small amount of bone was deposited after the LAGs. These animals were each 2.25 years old and had one-year marks visible in the humeri but no LAGs or annuli other than those at the periphery. Other CWT samples clearly indicated that LAGs are deposited at the end of the second GM. The indirect validation results demonstrated that LAGs were visible in bone tissue by late spring or early summer. It seemed that the LAGs at the outer edge of the LK-7 and LK-8 bones were the LAGs ending the second GM and that very little growth occurred over the subsequent growing season. Both of these animals were recovered as dead strandings resulting from a major cold stun event in Cape Cod, Massachusetts, in 1999; hence their growth rates may have been anomalous in their last year of life. Had these animals survived the cold stun event, they would have deposited a year-three LAG very close to year two, giving the appearance of a double or bi- furcating LAG. Another anomaly in skeletochronology, supplemen- tal lines, may form as a result of temporary stressful environmental events such as droughts. In support of this, Rogers and Harvey (1994) noted a supplemental line in 11 of 43 specimens of the toad Bufo cognatus, and in 10 of these animals the supplemental line was within a growth zone that corresponded to a drought year. Most skeletochronology studies that have noted the presence of supplemental lines have indicated that supplemental lines are easily identified as such because they are less distinct and do not appear around the entire circumference of the bone. In general, the same has been observed in sea turtles. Supplemental lines do appear but are generally easily differentiated from LAGs by appearance. An exception to this was the presence of supplemental marks in one- to two-year-old Kemp's ridley sea turtles. These marks were similar in appearance to the first year annuli. We were able to identify these marks as supplemental only by the observation of known-age animals. In addition, there appeared to be a supplemental line in CC-2 that rep- resented when the animal was released; hence, highly stressful events may cause the deposition of nonannual lines, but these events are likely to be relatively rare in wild turtles and not likely to interfere significantly with age estimations. Resorption of early growth marks The loss of the early GMs due to endosteal resorption and remodeling of the interior region of the bone is a lim- iting factor in the application of skeletochronology to sea turtles. From our findings, it was possible to accurately age juvenile Kemp's ridley sea turtles up to at least 5 years from GM counts and this may be true for other sea turtle species (e.g., Bjorndal et al., 2003), with the possible exception of the leatherback sea turtle (Zug and Parham, 1996). Because sea turtles have distinct life- cycle stages, we suggest that in order to age a population of sea turtles, one must acquire an ontogenetic series of samples spanning all sizes and stages. Average duration can be determined for each ontogenetic stage and the approximate age of older animals with extreme resorp- tion can be estimated. Because GM patterns appear to mimic somatic growth rates, once growth through each life-cycle stage is understood, backcalculation techniques can be used to estimate the number of layers resorbed. Conclusions For many species, skeletochronology is not a perfect method for age estimation. As GMs are histological expressions of variation in rates of osteogenesis (Casta- net et al., 1993). external factors and individual varia- tion will affect the appearance of the marks (Castanet et al., 1993, Esteban et al., 1996, Wave and Gregory. 1998). Endosteal resorption also serves to confound this technique and is the primary difficulty in the application of the technique to sea turtles. However, the evidence presented in the present study gives strong support to the concept that GMs are deposited on an annual basis in sea turtles and that the spatial pattern of the GMs correspond to the growth rates of the animal. The GMs therefore provide invaluable information on age and growth that cannot otherwise be easily obtained, and age determination by skeletocronology is valid and appropriate for the study of sea turtles. Acknowledgments We thank L. Crowder, S. Heppell, A. Read, and D. Rittschof for their valuable comments on earlier ver- sions of this manuscript. A. Gorgone. B. Brown and J. Weaver provided assistance with the preparation of the humeri. Most of the humeri were received through the Sea Turtle Stranding and Salvage Network, a coopera- tive endeavor between the National Marine Fisheries Service, other federal and state agencies, many academic and private entities, and innumerable volunteers. We especially thank R. Boettcher and W. Teas. In addition, humeri were received from F. Swartz at the Univer- sity of North Carolina-Chapel Hill Institute of Marine Snover and Hohn: Validation and interpretation of skeletal marks in Caretta caretta and Lepidochelys kempu 691 Science, B. 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Age and growth in leatherback sea turtles, Der- mochelys coriacea iTestudines. Dermochelyidae): a skel- etochronological analysis. Chelonian Conserv. Biol. 2:244-249. Zug, G. R., A. H. Wynn, and C. Ruckdeschel. 1986. Age determination of loggerhead sea turtles, Caretta caretta, by incremental growth marks in the skeleton. Smithsonian Institution, Contrib. Zool. 427. Washington D.C. 693 Abstract— Blue tCallinectes sapidus) (Portunidae), lady (Ovalipes ocella- tus) (Portunidae I, and Atlantic rock (Cancer irroratus) (Cancridae) crabs inhabit estuaries on the northeast United States coast for parts or all of their life cycles. Their distribu- tions overlap or cross during cer- tain seasons. During a 1991-94 monthly otter trawl survey in the Hudson-Raritan Estuary between New York and New Jersey, blue and lady crabs were collected in warmer months and Atlantic rock crabs in colder months. Sex ratios, male: female, of mature crabs were 1:2.0 for blue crabs, 1:3.1 for lady crabs, and 21.4:1 for Atlantic rock crabs. Crabs, 1286 in total, were sub- sampled for dietary analysis, and the dominant prey taxa for all crabs, by volume of foregut contents, were mollusks and crustaceans. The pro- portion of amphipods and shrimp in diets decreased as crab size increased. Trophic niche breadth was widest for blue crabs, narrower for lady crabs, and narrowest for Atlantic rock crabs. Trophic overlap was lowest between lady crabs and Atlantic rock crabs, mainly because of frequent consump- tion of the dwarf surfclam (Mulinia lateralis) by the former and the blue mussel (Mytilus edulis) by the latter. The result of cluster analysis showed that size class and location of capture of predators in the estuary were more influential on diet than the species or sex of the predators. The Hudson-Raritan Estuary as a crossroads for distribution of blue (Callinectes sapidus), lady (Ovalipes ocellatus), and Atlantic rock {Cancer irroratus) crabs Linda L. Stehlik Robert A. Pikanowski Donald G. McMillan James J, Howard Marine Sciences Laboratory Northeast Fisheries Science Center National Marine Fisheries Service, NOAA 74 Magruder Road Highlands, New Jersey 07732 E-mail address (for L L Stehlik): Linda Stehlika' noaa.gov Manuscript submitted 27 November 2000 to the Scientific Editor's Office. Manuscript approved for publication 4 May 2004 by the Scientific Editor. Fish. Bull. 102:693-710 (20041. The blue crab (Callinectes sapidus) (Portunidae), the lady crab {Ovali- pes ocellatus) (Portunidae), and the Atlantic rock crab (Cancer irroratus) (Cancridae) are the largest and most common brachyuran crabs inhabiting both estuaries and inner continen- tal shelves of the northeast coast of North America. The centers of abun- dance of these three species over- lap in estuarine and coastal waters from New York to Virginia, although their ranges along the northwest Atlantic coast are broad. The blue crab is nearly always an estuarine resident, except during its larval stages, and ranges from the waters off Nova Scotia to Argentina (Wil- liams, 1984). The northernmost estu- aries where the species is abundant enough for commercial harvest are in New Jersey and New York (Briggs, 1998; Stehlik et al., 1998). The lady crab is distributed from the waters off Prince Edward Island to those off Georgia but it is most numerous from Georges Bank to Cape Hatteras (Williams, 1984). The Atlantic rock crab (referred to as "rock crab" in this article) is distributed in waters from off Labrador to Florida but is most common in estuaries from Nova Scotia to Virginia (Williams, 1984; Stehlik et al., 1991). Seasonal migra- tions are common for all three spe- cies. Although Jonah crabs (Cancer borealis) are present on the continen- tal shelf, they are not included in the present study because they are rare within the Hudson-Raritan Estuary where our study was conducted. Physiological tolerances and habi- tat preferences of these crabs have been extensively studied. In eastern United States estuaries the blue crab occurs in shallow to deep, sandy to muddy estuaries and tributaries along marsh edges, and in seagrass (Van Engel, 1958; Milliken and Wil- liams, 1984; Hines et al., 1987; Wil- son et al., 1990; van Montfrans et al., 1991; Rountree and Able, 1992). In the colder portions of its range, it becomes less active at about 15°C (Leffler, 1972), and buries itself, with- out eating, when the temperature is <5°C (Auster and DeGoursey, 1994). It survives at 34°C (Leffler, 1972) and at salinities from 0 to 50 ppt (Guerin and Stickle, 1992). The lady crab is most common on sand substrates (Williams, 1984). It is present on the inner continental shelf from off Cape Cod to off the Carolinas throughout the year (Stehlik et al., 1991). Its tem- perature tolerance is unknown, but it does not survive in <21 ppt (Birchard et al., 1982). The rock crab's optimum temperature range for activity is 14- 22°C (Jeffries, 1966); thus the species avoids high summer temperatures. It is found on many substrates, such as sand, mud, bare rock, cobble, and algal beds. 694 Fishery Bulletin 102(4) The diet of the blue crab is generally mollusks, crabs, and fish, depending on crab size (Virnstein, 1977; Laughlin, 1982; Ryer, 1987; Hines et al„ 1990). The diet of the lady crab is mainly bivalves such as My a arenaria and Spisula solidissima, and some crustaceans (McDermott, 1983; Ropes, 1989; Stehlik, 1993). The rock crab consumes mollusks, small crustaceans, crabs, urchins, and fish (Scarratt and Lowe, 1972; Drummond- Davis et al., 1982; Hudon and Lamarche, 1989; Ojeda and Dearborn, 1991; Stehlik, 1993). In some of the aforementioned studies these crabs have been consid- ered opportunistic and as such may be competitors for the same prey taxa. However, differences in maximum body size, chela structure, and the presence or absence of swimming appendages among blue, lady, and rock crabs indicate that they may have differences in diet (Warner and Jones, 1976; Williams. 1984). Within the Hudson-Raritan Estuary, blue, lady, and rock crabs are all abundant, providing an opportunity to study partitioning of habitat and food resources by these species. The objectives of our study were to de- termine the temporal and spatial overlap of blue, lady, and rock crabs in this estuary and to differentiate the composition of their diets by the species, sex, and size of predators, and by location of collection. This study has potential practical applications. Re- source managers could use the results to consider when and where crabs depend upon certain locations to com- plete their life cycles, if dredging, filling, or sanctuaries were proposed. Dietary analysis of these crabs could indicate if they are a cause of mortality for young stages of commercially important species. For instance, the northern quahog (Mercenaria mercenaria) and the soft- shell clam 80% of the individuals are mature (produce viable eggs or sperm) as a separation boundary. Maturity in males is determined by dissec- tion or by allometric changes in growth of appendages (Hartnoll, 1978; Block and Rebach, 1998; de Lestang et al., 2003). Most female blue crabs in our study area and in Virginia had completed their pubertal molt and thus could reproduce by 12 cm CW (Van Engel, 1958; Fisher, 1999; Stehlik, unpubl. data). In Virginia, 80% of male blue crabs are mature by 11.9 cm (Van Engel, 1990). In lady crabs from the New York coast, nearly all males are mature at >6 cm, and females at about 5 cm (Briggs and Grahn3). In the middle-Atlantic portion of their range, male rock crabs mature at 5 cm (Haefner, 1976) and some females <5 cm bear eggs (Reilly and Saila, 1978). We chose the following CW boundaries for >80% maturity: blue crabs, >12 cm both sexes; lady crabs, 25 cm both sexes; and rock crabs, males ^5 cm and females ^4 cm. Data on blue, lady, and rock crabs from NEFSC (Northeast Fisheries Science Center, NOAA) fall bot- tom trawl surveys on the northeast United States con- tinental shelf, 1992-94, were used to expand the geo- graphical viewpoint of our study. The presence of each species in each tow was plotted to show distributions in a representative year. 1992. The plots were made with Surfer® (version 6, Golden Software Inc., Golden, CO). Methods on the trawl surveys are described elsewhere (Azarovitz, 1981). 3 Briggs, P. T.. and C. M. Grahn. 1996. Aspects of the fish- ery biology of the lady crab tOvalipes ocellatus) in New York waters, 8 p. An in-house paper. New York State Department of Environmental Conservation, 205 North Belle Mead Road, Suite 1, East Setauket, NY, 11733. 696 Fishery Bulletin 102(4) Foregut contents were analyzed as in Stehlik (1993). The foregut of each crab was removed and preserved in 7095 ethanol. After opening the foregut, we estimated fullness of the gut (from 0c/c to 100%) visually, and prey items were identified to the lowest possible taxon. The proportion of the total volume of the foregut contents con- tributed by each prey taxon was estimated visually — a less labor-intensive modification of the methods of Wil- liams (1981), Hyslop (1980), and Steimle et al. (1994). The volume of each prey taxon was multiplied by the percentage of gut fullness. Combining all foreguts, the volumes of prey taxa were listed in descending order. The top 12 prey categories on the list (with the excep- tion of "unidentified" and nonexclusive categories such as Mollusca) were selected for use in most of the subsequent analyses. Foreguts that did not contain prey in any of the 12 categories were dropped from numerical analyses. The dietary data were grouped in turn by predator species, sex, size class, and collection stratum, and the mean percentage volumes of each of the 12 mutually exclusive prey categories were calculated. For graphic representation of ontogenetic differences in diet, blue and rock crabs were grouped for convenience into 20- mm CW classes, and lady crabs were grouped in 10-mm CW classes because of their smaller size range. For numerical analyses, two maturity classes were used. We used Mann-Whitney tests to compare diets between sexes within predator species and between maturity stages within predator species. The test statistic was a chi square approximation. Group average cluster analysis was used to graph the separation of diets by species, sexes, maturity stages, and strata by using the 12 prey categories as dependent variables. A Bray-Curtis similarity matrix was gener- ated for each of the groupings, cluster analysis was performed by using Systat® (version 10. SPSS Inc., Chi- cago, IL), and dendrograms were generated by using the Bray-Curtis values as distance measures (Romesburg. 1984; Marshall and Elliott, 1997). A percent similarity level was chosen a posteriori that generated a reason- able number of classes. Analysis of similarity (ANOSIM) was used to test for statistical significance of dietary differences among predator species and for sexes within species. Analysis of dissimilarity (SIMPER) was used to determine which prey taxa contributed most to the differences between species pairs (Clarke and Warwick, 1994). Spatial, temporal, and trophic niche breadth and over- lap indices were calculated from the number per tow (1992-94) and diets (June 1991-June 1992) of each crab species and sex. Temporal niche and overlap were calculated by month for combined years. Female rock crabs were dropped from consideration of trophic niche overlap due to low sample size. Niche breadth (Colwell and Futuyama, 1971; Mar- shall and Elliot, 1997) is a measure of exploitation within a particular resource (for example, substrates or prey taxa within an estuary by a species). Niche breadth values are relative and can be compared only within one study. The highest value corresponds to the broadest niche, or to habitat or a diet generalist rather than to a specialist. Niche breadth (S) was calculated by the formula of Colwell and Futuyama (1971), and modified for measuring trophic niche breadth according to Hines et al. (1990): B = 1 / £( pk r from./ = 1 to n . where phl = Nkj I Yk ipki is the proportion of crabs of species k associated with resource state j)\ j = resource states (months, strata, diet cat- egories); n = number of resource states; Nkj = catch per tow of species k at resource state j; and Yk = catch per tow of species k over all resource states. When trophic niche breadth was calculated, Nh = total volume of diet category j consumed by preda- tor k ; Yk = total volume of all diet categories consumed by predator k. Niche overlap is a measure of the joint use of a resource by two species (Colwell and Futuyama, 1971). Niche overlap (CAl) between species h and i was calculated by the following formula (Colwell and Futuyama, 1971; Hines et al., 1990): C/,,=l-0.5(Xlp,,,-;V)fi-onV = lto». where ph/ and p„ are calculated in the same manner as Pk, above. This index ranges from 0 (no overlap) to 1 (complete over- lap) and is independent of sample size and differential resource availability (Eggleston et al.. 1998). Results Temperature and salinity Bottom water temperature in the study area followed a temperate seasonal cycle. The range during 1992-94 was from 0 to 26.6 C. Using the monthly mean tempera- ture below or above 10°C, and migration cycles of the crabs, we grouped the months into two seasons: winter (November through April) and summer (May through October). The mean temperature in the winter months 1992-94 was 5.5°C, and that for summer was 18.9°C. Temperature nearest the estuary mouth was usually a few degrees lower in summer months and higher in the winter months each year, compared with the average throughout the estuary. Bottom salinity in the study area ranged from 15.0 to 33.5 ppt. The majority of stations had salinities between Stehlik et al.: Distribution patterns of various crab species in the Hudson-Rantan Estuary 697 Table 1 Species, sex, number collected (n), and sex ratio (SR, ma e:female) of a 1 crab ; collecte d du ring the Hudson-Ra ■itan Estuary trawl survey, June 1991- December 1994. Mat urity boundaries are expla ned in the text For the subsample examined for stom- ach contents (June 1991- -June 1992), number i/i ), number of non-empty stomachs, and the mean an i range of carapace width (CW, mm) are presented. Crab species, SR(m:f) SR(m:f) stomach Subsample CW sex collected immature mature opened empty Mean (Range) Blue, male 2803 1:1.13 1:1.97 167 120 112 (35-185) Blue, female 4816 272 208 129 (21-169) Lady, male 14,903 1:1.30 1:2.12 173 124 60 (34-88) Lady, female 29.681 255 228 55 (30-89) Atlantic rock, male 15,503 4.65:1 21.43:1 400 281 92 (28-130) Atlantic rock, female 822 19 14 51 (29-80) Total 68,528 1286 975 25 and 30 ppt. Salinity decreased with distance from the bay mouth and in any one month, the difference in salinity between stations at the estuary mouth and those at the westernmost part of the study area was approximately 5-10 ppt. Catch by species, size, and sex From June 1991 through December 1994, more than 68,000 blue, lady, and rock crabs were caught in 1200 otter trawl tows (Table 1). Other mega-invertebrates in the tows included the northern moonsnail (Euspira heros), the horseshoe crab (Limulus polyphenols), the American lobster (Homarus americanus), the portly spider crab (Libinia emarginata), the flatclaw hermit crab tPagurus pollicaris), mud crabs (Xanthidae), and the sea star (Asterias sp. ) Catch per tow of crabs by size class increased as they became large enough to be retained by the mesh of the net (Fig. 2). Abundances of female blue and lady crabs in the study area were greater than those of the males. In rock crabs, males predominated (Table 1). Im- mature blue and lady crabs had sex ratios fairly close to 1:1 (male:female). Sex ratio in mature blue crabs, however, was 1:1.97, and in mature lady crabs, 1:2.12. In all sizes of rock crabs, sex ratio strongly favored males, particularly in mature crabs, in which the ratio was 21.43:1. Temporal and spatial variation in catch The maximum relative abundance of blue and lady crabs occurred during the warm months each year, whereas rock crabs were abundant only in the cold months (Fig. 3). Blue crabs were scarce in the otter trawls from January through May or June. We believe that many of them do remain in the study area, but are relatively inactive and are not accessible to otter trawls, as discussed below. Lady crabs migrated into the estu- ary in April and May and left in October and November. 15 10 5 0 a" ^ 20 o c a> cr a> r 10- Blue crabs r*r¥P ml I -^r I Lady crabs Male I Female IL 15- Rock crabs 10- n n I" r 5- i 0- ■■'Hi- II, 4 8 12 16 20 Carapace width (cmL Figure 2 Carapace width (cm) frequencies for male and female blue, lady, and rock crabs collected during the Hudson-Raritan Estuary trawl survey, June 1991-December 1994, by percent frequency of the total catch of each species. 698 Fishery Bulletin 102(4) Rock crabs migrated into the estuary in November and gradually left during April, May, and June. Hundreds of soft and postmolt male rock crabs were caught each winter in the study area (Fig. 4). The high- est numbers of molting rock crabs were collected each December and January, and almost all of these crabs had completed molting by February. Very few molting or postmolt blue or lady crabs were caught. The relative abundances of the three species varied by stratum (Fig. 5, A-C; Fig. 6). Blue crabs of both sexes were caught mainly in strata near river mouths (strata 1. 2, and 6), in the Chapel Hill and Raritan channels (strata 8 and 9) in summer, but mainly in stratum 6 and in the channels in winter. Lady crabs were widely distributed and were caught throughout the study area, including the outer strata close to the ocean. Male rock crabs were most frequently collected in and near the channels and in strata 1 and 6, whereas female rock crabs were sparsely scattered throughout the study area. 6 8 10 12 2 4 6 8 10 12 1991 1992 4 6 8 1993 10 1; 4 6 8 10 12 1994 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 1: 1991 1992 1993 1994 10 12 2 4 6 8 10 12 1991 1992 6 8 10 1 j 1993 4 6 8 10 12 1994 O Figure 3 Catch per unit of effort (number/towl of blue, lady, and rock crabs by month, graphed with mean bottom temperatures. June 1991-December 1994. Stehlik et al.: Distribution patterns of various crab species in the Hudson-Rantan Estuary 699 Foregut fullness The total number of blue, lady, and rock crab foreguts examined was 1286. Foregut fullness varied by month in blue and rock crabs. The average fullness of blue crabs was 1% by volume from January through April, and 34% for the rest of the year. Ovigerous blue crabs (/? =27) averaged 40% full. Lady crabs' average full- ness was 41% during the months when they were pres- ent. The average fullness of rock crabs was 30% in all months when they were present; a minimum occurred in January when fullness was 7%. Of 419 rock crabs exam- ined, intermolt crabs (rc=293) were 33% full, premolt crabs (n = 9) were empty, soft crabs (n=22) were empty, and postmolt crabs 1/2 = 95) were 20% full. Some rock crabs in the late postmolt stage were full even though their chelae were not completely calcined. Diet composition The number of crabs containing food was 975, and they consumed 44 identifiable taxa (Table 2). Most of the mollusks preyed upon were <15 mm in shell length. The crabs consumed were mud crabs (Xanthidae) and juvenile stages of other Anomura and Brachyura. When foreguts were only partially full, well-digested remains of prey frequently could be identified by pieces of shell or opercula, mandibles (for shrimp), or chela tips and carapace fragments (for crabs) (Elner et al., 1985). Rec- ognizable prey taxa were grouped into 12 mutually exclusive categories (Table 3), which contributed 80.1% of the volume of all prey. The prey category "CRABS" represented pooled fragments of all crabs except Pagu- 120 100 80 60- 40 20 0 V Papershell ■ Soft = Premolt Hard 8 9 10 11 12 1 3 4 5 6 1991 1992 Figure 4 Number of rock crabs at each molt stage by month. 1991-92. Figure 5 Catch per unit of effort of (Al blue, (B) lady, and (Cl rock crabs, sexes combined January 1992-December 1994, mapped by the midpoint of each tow. ridae and Xanthidae. Crabs containing prey in one or more of the 12 categories numbered 713. Differences in diet by predator species, sex, and size Although the three predator species shared most prey taxa, there were differences in the proportions of the taxa consumed (Fig. 7). Mann-Whitney tests comparing diets of sexes within each species showed only two sig- nificant differences out of 36 comparisons. After cluster analysis upon the 12 prey types by species, sex, and size class (immature and mature), the resulting den- drogram showed that diets were most similar between size classes within a species (Fig. 8). Female rock crabs were not included because of their small sample size. When the diets of the three species were compared by analysis of simi- larity (ANOSIM) they were found to be different (P=0.067>, but the data were extremely variable and not normally distributed. No signifi- cant differences were found between sexes within species and we there- fore pooled sexes within species. Pairwise comparisons of the spe- cies were performed by analysis of dissimilarity (SIMPER). Four taxa contributed significantly to the dif- ference in diets of the first pair: the bivalves M. edulis and M. lateralis were more important in the diets of lady crabs, and Xanthidae and CRABS, were more important in the diets of blue crabs. The diets of blue crabs and rock crabs were significantly different in four taxa: CRABS and M. lateralis were more important for blue crabs, and M. edulis and Xanthidae for rock crabs. 700 Fishery Bulletin 102(4) Rock crab Hudson-Rantan Estuary New York " qp ° © ° ,0 rf> None o 1-9 o 10-49 O 50-99 O 100-249 O 2 250 Figure 5 (continued) The diets of lady crabs and rock crabs were significantly different in two taxa: M. lateralis for lady crabs and M. edulis for rock crabs. Within the crab size ranges sampled adequately by our gear, we found some ontogenetic differences in di- ets (Fig. 9). Notably, amphipods and shrimp were con- sumed by smaller sizes of all three predators. Certain mollusks, such as N. trivittatus and the Atlantic jack- knife clam (Ensis directus), increased in occurrence in foreguts with increasing crab size. Smaller lady crabs primarily fed upon M. lateralis, but larger ones broad- ened their diets to include other mollusks such as slip- persnails (Crepidula spp.) and M. edulis. Blue and rock crabs exhibited two peaks in consumption of M. edulis: the foreguts of small crabs contained recently settled mussels, whereas those of large crabs contained shell fragments and meat of larger mussels. Xanthidae and Paguridae, small in body size, were eaten mostly by intermediate-size predators. Mann-Whitney tests showed that amphipods were the only prey significantly different (P<0.01) between maturity classes for all three crab species. Spatial variability in diets Cluster analysis of the diets by species and stratum defined six groups at 50% similarity (Fig. 10). Group A consisted of lady and rock crabs caught at oceanward Stehlik et al.: Distribution patterns of various crab species in the Hudson-Raritan Estuary 701 Blue crab, winter E3 Males ■ Females 1 12 4 5 6 7 8 9 Blue crab, summer fll 12 3 4 5 J 6 7 8 9 Lady crab, summer 12 3 4 5 6 l DO 80 60 40 20 0 Rock crab, winter n q_q_ gj 6- 4 2 0 Rock crab, summer ^R-n I 2mlA m 3 4 Strata of Hudson-Raritan estuary Figure 6 Catch per unit of effort for male and female crabs by seasons and strata, January 1992-December 1994. Note differences in scale among j axes. 100 Blue Lady Rock m FISH □ XAN HI PAG Hi CRAB ^ SHR ■ AMPH mi MUL i MYT m ENS □ CREP g§ NASS ■ POLY Figure 7 Percent volume of prey (of the 12 chosen categories) of blue, lady, and rock crabs from the Hudson-Raritan Estuary, including all strata and size classes, June 1991-June 1992. n=713. Codes for prey taxa are from Table 3. Rock_M_Imm Rock_M_Mat Blue_M_Imm Blue_F_Mat Blue_M_Mat Blue_F_Imm Lady_M_Mat Lady_F_Mat Lady_M_Imm Lady_F_Imm i 1 1 1 1 1 1 1 1 100 90 80 70 60 50 40 30 20 Percent similarity in diet Figure 8 Cluster analysis dendrogram of similarities of the diets of species, sexes (M=male. F=female), and matu- rity stages (Imm=immature, Mat = mature) of blue, lady, and rock crabs, including all seasons and strata, June 1991-June 1992. Female rock crabs were not included because of the small sample size. 702 Fishery Bulletin 102(4) Table 2 Percent frequency of occurrence (ci FRE) and percent volume (%VOL ) of prey of blue, lady. and rock crabs collected du "ing the trawl survey, June 1991-June 1992. Dashes mean that the dietary item was not found in any stomachs of that crab species. "Unid." means unidentified: "other' means uncommon identified taxa not listed below. Blue crab Lady crat Rock crab r£FRE <7rVOL 7rFRE ^VOL %FRE rrVOL Number of nonempty foreguts 328 352 295 Plant material 1.5 <0.1 2.8 0.3 4.4 0.1 Hydrozoa 0.6 <0.1 1.4 <0.1 1.4 0.1 Mollusca. unid. 6.7 0.9 3.4 0.9 12.3 14.2 Bivalvia, unid., other 12.2 1.4 11.1 1.6 7.8 4.6 Anadara transversa — — 0.3 0.1 — — Ensis directus 2.4 0.8 9.9 4.8 8.2 6.2 Lyonsia hyalina — — 0.3 <0.1 — — Mercenaria mercenaria 0.3 <0.1 0.3 <0.1 — — Mulinia lateralis 9.3 13.6 43.5 33.1 6.8 1.4 Mya arenaria — — 0.3 0.4 — — Mytilus edulis 19.6 14.3 13.9 9.8 28.7 27.3 Nucula proximo 3.4 0.4 5.1 0.6 0.7 0.1 Petricola pholadiformis 1.2 0.1 2.3 0.8 — — Pitar morrhuanus — — 0.3 <0.1 — — Spisula solidissima — — 1.7 0.5 — — Tellina agilis 4.6 1.5 9.4 2.1 1.4 0.2 Gastropoda, unid., other 6.4 0.7 4.0 0.2 1.0 0.1 Crepidula fornieata, eonvexa 8.6 2.5 5.4 2.8 0.3 <0.1 Crepidula plana 0.6 <0.1 0.3 <0.1 — — Nassarius obsoletus 1.5 1.2 0.3 0.2 0.3 <0.1 Nassarius trivittatus 20.8 6.8 15.6 4.6 0.3 <0.1 Naticidae — — 0.6 <0.1 — — Rictaxis punctostriatus — — 0.9 0.1 0.3 <0.1 Cephalopoda 0.3 0.8 0.6 0.3 — — Polychaeta, unid., other 2.4 0.2 4.3 0.3 2.7 0.9 Glyceridae — — 0.9 0.3 — — Hydroides dianthus — — 0.3 <0.1 — — Nephtyidae — — 0.9 0.3 — — Nereidae 1.5 0.3 1.7 0.1 0.7 0.3 Pherusa afftnis — — — — 0.3 0.1 Pectinaria gouldii 2.4 0.8 15.9 2.4 0.7 <0.1 Polynoidae — — 0.3 <0.1 1.4 0.1 Insecta 0.3 <0.1 — — — — Crustacea, unid., other 4.9 0.6 3.4 0.3 5.8 1.4 Amphipoda, unid., other 2.8 0.6 7.7 1.4 1.4 0.2 Ampelisca sp. 1.5 0.6 6.8 1.3 0.7 0.4 Corophium sp. 0.6 0.2 1.7 0.7 — — Gammarus sp. — — 3.1 3.3 — — Mysidacea — — 0.3 <0.1 — — Caridean shrimp, unid.. other 0.6 <0.1 2.0 0.1 0.7 <0.1 Crangon septemspinosa 2.8 0.6 6.0 2.3 3.1 2.0 Crabs unid., other' 17.7 7.4 8.8 2.7 11.3 4.2 Callinectes sapid us 0.3 <0.1 0.6 0.1 1.4 1.8 Cancer irroratus 1.5 1.0 2.3 0.5 2.0 1.4 Libinia sp. 0.9 0.6 0.9 0.6 1.0 0.5 continued Stehlik et al.: Distribution patterns of various crab species in the Hudson-Rantan Estuary . 703 Table 2 (continued) Blue ci ab Lady crab Rock crab ^FRE %VOL <*FRE WOL <7tFRE -2VOL Crabs unid., other1 (cont.l 0.6 <0.1 2.0 0.1 0.7 <0.1 Ovalipes ocellatus 3.4 3.6 0.6 0.2 1.0 1.0 Pagurus longicarpus 2.1 1.5 1.7 0.6 — — Pagurus sp. 8.0 4.3 5.4 2.3 1.7 0.1 Xanthidae 21.1 20.8 15.9 10.6 21.2 18.4 Fish remains and scales 2.1 0.9 3.4 0.7 6.4 3.7 Inorganic debris, sand, mud 0.9 <0.1 0.9 0.2 0.7 0.1 Shell hash 2.4 1.5 — — — — Human-made objects 4.0 <0.1 4.8 <0.1 1.7 <0.1 Unid. organic matter — 9.2 — 5.3 — 9.0 Mytilus byssus 1.8 <0.1 2.0 <0.1 3.1 0.2 Table 3 Twelve mutually exclusive prey categories that contributed 80^ of the prey volume of all crabs examined. Codes are used in Figures 7 and 9. "Other" means uncommon identified taxa. CODE Category Identifiable species NASS mud snails CREP slipper shells ENS razor clam MYT blue mussel MUL dwarf surfclam POLY Polychaeta AMPH Amphipoda SHR shrimp CRAB crabs PAG hermit crabs XAN mud crabs FISH fish, fish scales Nassarius trivittatus, N. obsoletus Crepidula fornicata, C. convexa, C. plana Ensis directus Mytilus edulis Mulinia lateralis all all Crangon septemspinosa, unid., other Libinia sp.. Cancer irroratus. Ovalipes ocellatus, Callinectes sapidus, crab unid. and others excluding Paguridae or Xanthidae Pagurus acadianus, P. longicarpus, unid., other Xanthidae: Dyspanopeus sayi, unid., other all outer strata (4, 5, and 7) that consumed large quanti- ties of M. edulis. Clumps of recently settled and larger mussels were frequently collected in trawl nets in these strata. Group B contained crabs from Gravesend Bay, (stratum 6 ) that ate primarily M. edulis and M. lateralis. Group C contained crabs caught in the siltier southern strata and nearby channel (strata 1, 2, and 9) that con- sumed mainly M. lateralis, M. edulis, and CRABS. Group D consisted of rock crabs collected at inner strata (2, 3, and 8) that fed primarily upon E. directus and Xan- thidae. Ensis directus was most common in diets in the northern sandier strata (strata 3, 5, 6, and 7). Groups E and F consisted of lady and rock crabs that consumed mainly M. lateralis. Four species-stratum combinations did not cluster with any groups. Temporal, spatial, and trophic niche breadth and overlap Niche breadth and overlap were calculated for both sexes of the three crab species (Table 4). Lady crabs of both sexes had the narrowest temporal niches (3.896 and 4.592), reflecting their presence in the estuary strictly in warm months. The temporal niche breadth of female blue crabs (8.187) was greatest, reflecting their year-long presence in the study area, even in the cold months when many males remain in rivers. The temporal overlaps of 704 Fishery Bulletin 102(4) Blue 10° crab Lady crab Rock crab Percent volume 40-59 60-79 80-99 100-119 120-139 140-159 160-185 100 tfiQfiS tssss FISH □ XAN m PAG ggg CRAB ^ SHR ■ AMPH on MUL □ MYT ESS ENS □ CREP B NASS ■ POLY IffiS FISH □ XAN m PAG \g% CRAB m SHR ■ AMPH m MUL □ MYT ss ENS □ CREP HI NASS ■ POLY 100 £3 FISH □ XAN m PAG gg CRAB m SHR ■ AMPH he MUL □ MYT us ENS □ CREP n NASS ■ POLY 40-59 60-79 80-99 100-119 120-139 Carapace width classes (mm) Figure 9 Percent volume of prey I of the 12 categories I by 10- or 20-mm size classes of blue, lady, and rock crabs, all seasons and strata. Codes for prey taxa are from Table 3. male and female lady crabs with male rock crabs were the lowest in the matrix (0.149 and 0.186). The spatial niche breadths of lady crabs were larg- est (7.320 and 7.324) (a result of their nonaggrega- tive distribution throughout the study area), whereas the other two species tended to aggregate in certain locations, particularly in or near channels. Female rock crabs also had a broad spatial niche, although they were caught much less frequently than the other groups. Spatial overlap was highest within species, particularly between male and female lady crabs (0.908). Stehlik et al.: Distribution patterns of various crab species in the Hudson-Raritan Estuary 705 Predator/ stratum Lady Lady Rock Rock Rock Rock Blue Lady Blue Lady Blue Lady Blue Blue Rock Blue Rock Rock Rock Lady Lady Rock Lady Lady I 1 1 1 1 1 1 1 1 1 100 90 80 70 60 50 40 30 20 10 Percent similarity in diet Figure 10 Cluster analysis dendrogram of the diets of blue, lady, and rock crabs from the nine strata of the Hudson-Raritan Estuary. The vertical line at 50% similarity defines groups A-F. 42.00 40 00 - 38.00 36.00 - Trophic niche breadth was greatest in male and fe- male blue crabs (5.234 and 6.563) and male lady crabs (6.166) (Table 4). It was narrowest for female rock crabs, but sample size was low. Overlap was highest within species: blue crab males and females (0.819), and lady crab males and females (0.861). Overlap was lowest between lady and rock crabs, sexes combined (0.427 i. Discussion Temporal and spatial overlap within the estuary The scatter plots (Fig. 5) and spatial niche overlap indices indicate substantial likelihood of co-occurrence and encounter among blue, lady, and rock crabs in the Hudson-Raritan Estuary. However, the species were not all active in the study area at the same time. Seasonal migration and winter torpor are two mechanisms that, at times, prevent interspecies encounters. Rock crabs had low temporal overlaps with blue and lady crabs because when rock crabs migrate in from the coastal ocean, lady crabs migrate out and blue crabs become less active and sometimes bury themselves. Although otter trawling does not adequately sample buried blue crabs, commercial crab dredgers catch large numbers of overwintering blue crabs from December through March in and near the Raritan and Chapel Hill channels (Stehlik et al., 1998). Temporal overlap between blue crabs and lady crabs was fairly high because of their co-occurrence in the ■fr it Z^r^*-^" A Qa"" '\ O -76.00 -74.00 -72.00 -70.00 -68.00 -66.00 Figure 11 Presence of blue, lady, and rock crabs at stations from the fall 1992 bottom trawl survey, (Northeast Fisheries Science Center, Woods Hole, MAl. Each point represents presence at a station. Occurrence inside the estuaries! boxed symbols) was derived from the literature cited in this article. warm months. It was expected that intra-estuarine spa- tial separation might minimize contact between these species because they are reported to prefer different substrates. The blue crab is known to occupy a variety of substrate types, including sand, mud, and submerged vegetation (Milliken and Williams, 1984; Wilson et al. 1990), whereas the lady crab is primarily collected on sand (Williams and Wigley, 1977). The lady crab bur- ies itself in sand more readily than in mud (Barshaw and Able, 1990) and it is able to forage more efficiently in sand than in sand-gravel or sand-shell substrates (Sponaugle and Lawton, 1990). However, as shown in Figures 5 and 6, lady crabs were not confined to sandy strata but were most abundant on the fine-grained sedi- ment strata 1, 2, and 9. The pattern of seasonal estuarine use by blue and lady crabs is not unique to the Hudson-Raritan Estuary. Other estuaries in which the two Portunidae are abun- dant in summer months but uncommon in winter are Barnegat Bay, NJ (Milstein et al., 1977; pers. observ. ), Delaware Bay (Winget et al., 1974), and Chesapeake Bay (Haefner and Van Engel, 1975). Rock crabs undergo seasonal migrations from coastal waters into and out of estuaries, but the timing differs by latitude. In Canada, the Gulf of Maine, and northern Massachusetts, rock crabs are much more abundant in immediate coastal waters, estuaries, and in the inter- tidal zone in warmer months (Krouse, 1972; Scarratt and Lowe, 1972). Rock crabs are more numerous in Narragansett Bay, Rhode Island, in warmer months (Jeffries, 1966; Clancy4). Juveniles are present inside Clancy, M. 2002. Personal commun. Boston University, College of General Studies, Division of Natural Science, Boston, MA, 02115. 706 Fishery Bulletin 102(4) Table 4 Niche breadth and overlap for temporal, spat al, and ti -ophic dimensions among blue, lady, and rock crabs. For temporal and spatial niches, all crabs (of all sizes) collected in 1992- 94 are included. For trophic analyses, only the crabs containing one or more of the 12 prey categories were included. Female rock crabs were size. not included in trophic overlap because of the small sample Number Mean Niche of crabs CW, mm breadth Overlap matrices Temporal niche {n =12 months, 1992-94) BCF LCM LCF RCM RCF Blue crab male (BCM) 2191 125 6.104 BCM 0.854 0.604 0.618 0.297 0.463 Blue crab female (BCF) 3483 129 8.187 BCF 0.649 0.576 0.376 0.564 Lady crab male (LCM) 11883 62 4.592 LCM 0.894 0.186 0.417 Lady crab female (LCF) 25312 61 3.896 LCF 0.149 0.339 Rock crab male (RCM) 14530 85 6.764 RCM 0.479 Rock crab female (RCF) 778 51 5.782 Spatial niche (ra=9 strata, 1992-94) BCF LCM LCF RCM RCF Blue crab male 2191 125 3.927 BCM 0.757 0.720 0.703 0.675 0.474 Blue crab female 3483 129 5.343 BCF 0.679 0.685 0.746 0.641 Lady crab male 11883 62 7.324 LCM 0.908 0.677 0.616 Lady crab female 25312 61 7.320 LCF 0.678 0.638 Rock crab male 14530 85 5.447 RCM 0.763 Rock crab female 778 51 7.191 Trophic niche in=12 prey categories, 1991-92) BCF LCM LCF RCM Blue crab male 84 111.0 5.234 BCM 0.819 0.570 0.580 0.576 Blue crab female 139 127.9 6.563 BCF 0.629 0.651 0.609 Lady crab male 98 59.4 6.166 LCM 0.861 0.437 Lady crab female 200 54.9 4.655 LCF 0.417 Rock crab male 181 88.8 4.139 Rock crab female 11 53.7 2.620 Trophic niche, sexes combined Ui = 12 prey categories, 1991-92) LC RC Blue crab (BO 223 121.6 6.250 BC 0.628 0.623 Lady crab ( LC ) 298 56.4 5.140 LC 0.427 Rock crab ( RC ) 192 86.8 4.008 that bay all year (Reilly and Saila, 1978). In contrast, in Delaware Bay and Chesapeake Bay they occur in coastal waters and estuaries mainly in colder months (Winget et al., 1974; Haefner and Van Engel, 1975; Haefner, 1976). Our data showed that rock crabs in the Hudson-Raritan Estuary conform to the pattern of migration typical of the latter southern bays. A crossroads or overlap in distribution of the three crab species is more evident when a broader area on the continental shelf from Cape Cod to Cape Hatteras is con- sidered. Crab presence was plotted by using data from the fall 1992 continental shelf trawl survey 12 cm over a two-year period was 2.6:1 (Meise and Stehlik, 2003). In the Hudson-Raritan Estuary, female lady crabs a5 cm outnumbered males 2:1. Many of these females were ovigerous and therefore estuarine use may be related to reproduction. We were unable to locate pub- lished reports of lady crabs or other Ovalipes spp. mat- ing locations, single-sex migrations, or locations of lar- Stehlik et al.: Distribution patterns of various crab species in the Hudson-Raritan Estuary 707 val release, any of which might be a reason for the use of the estuaries by female lady crabs. The rock crabs that enter the estuary were predomi- nantly males, and many females may never enter the estuary. Males use the estuary to molt, and possibly to avoid predators offshore. In comparison, on the north- west Atlantic continental shelf, the sex ratio in winter dredge collections was 1:2.2 males:females (Stehlik et al., 1991). Feeding periodicity Food consumption in crabs is affected by daily and seasonal cycles, temperature changes, reproductive rhythms, and molt (Warner, 1977; Stevens et al, 1982; Ryer, 1987; Mantelatto, 2001). In our study area, blue crabs ate little when inactive during the winter months, as reported above. Choy (1986) reported less feeding during egg-brooding in Portunidae, but in our study we found that fullness was about 40% in both egg-bearing and non-egg-bearing females in summer. A lack of feed- ing before and during molt, until calcification has suf- ficiently progressed, is typical of crabs (Warner, 1977). Empty stomachs in premolt and soft rock crabs in our study supported this observation. Diet composition We found that in the Hudson-Raritan Estuary, the most important prey items of blue crabs by volume were Xanthidae, then the mollusks M. edulis and M. late- ralis, whereas only 2% of the prey volume was from cannibalism. In contrast, small blue crabs are of major importance in the diets of large blue crabs in Florida (Laughlin, 1982) and Maryland (Hines et al., 1990). and cannibalism is the source of more than 75% of the mortality of juveniles near estuarine shores (Hines and Ruiz, 1995). The major targets of cannibalism, early instars or molting juveniles, may be more abundant in rivers adjacent to our study area (Meise and Stehlik. 2003). The diets of rock crabs in estuarine and coastal Canada and Maine usually contained a larger num- ber of prey categories than did the diets in the pres- ent study (Scarratt and Lowe, 1972; Drummond-Davis et al.. 1982; Hudon and Lamarche, 1989; Ojeda and Dearborn, 1991). These northern studies were done on rock, boulders, cobble, sand, and algal beds, where the diversity of habitats within a study area may offer a larger assortment of potential prey than the soft-bottom habitat of our estuary. In the Hudson-Raritan Estuary, juveniles of commer- cially or recreationally harvested species were rarely consumed by the three species of crabs. Among mol- lusks, M. arenaria and M. mercenaria were scarce in crab stomachs, perhaps because other taxa such as M. lateralis, N. trivittatus, and Xanthidae provided abun- dant prey. The other commercially important species eaten by crabs was the blue crab juvenile, but infre- quently as mentioned above. Differences in diet among species, sexes, and size classes of predators Our data did not support our hypotheses, based on exist- ing studies, that blue, lady, and rock crabs would have different diets as a consequence of their species-specific body and chela structures. Blue and lady crabs (unlike rock crabs) swim, allowing them a greater foraging area than rock crabs. Chela structure affects the type and size of prey that can be crushed (Vermeij, 1978; Seed and Hughes, 1995; Behrens Yamada and Bould- ing, 1998). In Portunidae. the long chelae (in relation to their CW) have short muscle fibers better suited to quick grabbing than to prolonged crushing (Warner and Jones, 1976; Seed and Hughes, 1997). The chelae of Cancridae are monomorphic (same characteristics left and right sides), have relatively short, stout teeth, and close relatively slowly because of their muscle fibers (Warner and Jones, 1976). Chela crushing force (New- tons), measured with a force transducer, is positively correlated with chela height and thickness (Govind and Blundon, 1985; Block and Rebach, 1998). Although the chela structures of blue and rock crabs are quite different, the chelae of mature rock crabs (9-13.5 cm CW) generate crushing forces comparable to those of cutter and crusher chelae of mature male blue crabs (12-16 cm) (Govind and Blundon, 1985). Chela crushing force in mature blue and rock crabs is likely to be more than sufficient for successful foraging upon all but the largest prey (Block and Rebach, 1998) and may not be a major determinant of diet. In fact, crabs often prey upon small or young bivalves rather than on large sizes, perhaps because the latter require more handling time and may damage chelae (Juanes. 1992; Seed and Hughes, 19951. Because Portunidae swim and have more versatile chelae, they may be ex- pected to have broader trophic niches than Cancridae. In our study, blue crabs had the broadest trophic niche, lady crabs had an intermediate trophic niche, and rock crabs had the narrowest trophic niche. We found no significant differences in diet by sex within species. Sexual dimorphism within a crab spe- cies accelerates after puberty (Hartnoll. 1978), but our study included many immature crabs. Some experiment- ers using force transducers found no significant differ- ence in crushing force between the sexes of blue crabs of a broad size range (Blundon and Kennedy, 1982; Seed and Hughes, 1997), but in blue crabs >135 mm, males produced significantly more force than females (Eggleston, 1990). Sexual dimorphism is found in chela length, but not chela height, in lady crabs (significantly- different slopes of CL/CW by regression; Stehlik, un- publ. data). Carapace width and the proportion of chela height to carapace width are positively correlated with crushing force, which makes it possible for larger crabs to con- sume larger, harder-shelled mollusks or crustaceans (Hartnoll, 1978; Block and Rebach, 1998). The larg- est lady crabs do not grow to the carapace widths or chela lengths of mature blue crabs; therefore the force 708 Fishery Bulletin 102(4) of their chelae cannot match those of blue crabs. As they grow, Cancridae and Portunidae undergo shifts in diet, and may be divided into ontogenetically distinct trophic units (Laughlin, 1982; Stevens et al., 1982: Stoner and Buchanan, 1990; Rosas et al.. 1994). In our study, larger crabs dropped amphipods and shrimp from their diets, but otherwise only minor changes occurred in prey identity and relative volumes of prey taxa among size classes (Fig. 9). An interesting ontoge- netic shift was in the size of prey eaten: small crabs ate small individuals of prey taxa, such as M. edulis, and Xanthidae, and large crabs ate large individuals of the same taxa. Thus in our study the influence of physical structure upon diet was greater as body size increased within a species than among species. Spatial variability and overlap in diets The three predators were scattered throughout the cluster diagram of diet among strata of the estuary (Fig. 10), yet crabs from inner and outer groups of strata usually clustered separately. We concluded that loca- tion influenced diet more than did predator identity. The inner, outer, and channel strata differ in depth, sediment type, currents, and mean temperature, and therefore in benthic and epibenthic prey assemblages. Our results support the concept that these species are mainly opportunistic in diet, as was suggested for blue crabs (Laughlin, 19821, and rock crabs (Hudon and Lamarche, 1989). The Hudson-Raritan and other nearby coastal and estuarine areas from Long Island Sound to Chesapeake Bay are crossroads where blue, lady, and rock crabs share space and resources. Acknowledgments We thank those who helped design and carry out the Hudson-Raritan Estuary trawl surveys, especially Stuart Wilk, Anthony Pacheco, and Eileen MacHaffie. We also thank Fred Farwell, Sherman Kingsley, and the NOAA Corps captains and crew. Suellen Fromm was instru- mental in obtaining data from NEFSC trawl surveys. We thank the scientists who shared their opinions and unpublished data. 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Habitat use by juvenile blue crabs: a comparison among habitats in southern New Jersey. Bull. Mar. Sci. 46:105-114. Winget, R. R., D. Maurer, and H. Seymour 1974. Occurrence, size composition and sex ratio of the rock crab, Cancer irroratus Say and the spider crab. Libinia emarginata Leach in Delaware Bay. J. Nat. Hist. 8:199-205. 711 Abstract — As nearshore fish popu- lations decline, many commercial fishermen have shifted fishing effort to deeper continental slope habitats to target fishes for which biological information is limited. One such fish- ery that developed in the northeastern Pacific Ocean in the early 1980s was for the blackgill rockfish (Sebastes melanostomits), a deep-dwelling (300-800 mi species that congre- gates over rocky pinnacles, mainly from southern California to southern Oregon. Growth zone-derived age esti- mates from otolith thin sections were compared to ages obtained from the radioactive disequilibria of 210Pb, in relation to its parent, 226Ra, in otolith cores of blackgill rockfish. Age esti- mates were validated up to 41 years, and a strong pattern of agreement supported a longevity exceeding 90 years. Age and length data fitted to the von Bertalanffy growth func- tion indicated that blackgill rockfish are slow-growing (A' = 0.040 females. 0.068 males I and that females grow slower than males, but reach a greater length. Age at 509c maturity, derived from previously published length-at- maturity estimates, was 17 years for males and 21 years for females. The results of this study agree with gen- eral life history traits already recog- nized for many Sebastes species, such as long life, slow growth, and late age at maturation. These traits may undermine the sustainability of black- gill rockfish populations when heavy fishing pressure, such as that which occurred in the 1980s, is applied. Radiometric validation of age, growth, and longevity for the blackgill rockfish (Sebastes melanostomus) Melissa M Stevens Allen H. Andrews Gregor M. Cailliet Kenneth H. Coale Moss Landing Marine Laboratories 8272 Moss Landing Road Moss Landing, California 95039 E-mail address ((or A. H Andrews, contact author): andrewsiS'mlml calstate.edu Craig C Lundstrom Department of Geology University of Illinois— Urbana Champaign 255 Natural History Bldg. 1301 W.Green Street Urbana, llmois 61801 Manuscript submitted 9 June 2003 to the Scientific Editor's Office. Manuscript approved for publication 18 June 2004 by the Scientific Editor. Fish. Bull. 102:711-722 (20041. The blackgill rockfish (Sebastes mela- nostomus) is a deep-water rockfish that is found mainly along the conti- nental slope between 300 and 800 m depth off central and southern Cali- fornia (Moser and Ahlstrom. 1978; Cross, 1987; Williams and Ralston, 2002). Although not as heavily tar- geted in relation to other commercially important rockfish species, a directed commercial fishery for blackgill rock- fish has existed since the mid-1970s, beginning off southern California (Point Conception area) and spreading northward ( Monterey area) as stocks of other heavily fished rockfishes declined (Butler et al., 1999). Using acoustic sonar and set nets, the com- mercial fleet was able to catch large aggregations of previously unexploited blackgill rockfish. Landings peaked in 1983 with 1346 metric tons (t) caught coast-wide, but declined over the next decade, presumably because of the dis- appearance of the large concentrations that could be located with acoustical gear (Butler et al., 1999). In 2001, 141 t were reportedly landed along the entire west coast (PacFIN1) — less than half of the allowable catch (343 t; NOAA, 2001) for blackgill rockfish that year. The first stock assessment of black- gill rockfish was made by Butler et al. (1999). One objective of this as- sessment was to determine age and growth characteristics, which were then applied to estimate age-at-ma- turity, natural mortality, and stock biomass. Using conventional aging methods (i.e., otolith increments), we estimated that blackgill rockfish live at least 87 years and reach full (100%) maturity from 13 to 26 years for females, and from 13 to 24 years for males. Although such estimates are useful and should be considered whenever available, validation of the age-estimation procedure is needed to be certain of accurate age estimates (Beamish and McFarlane, 1983; Campana, 2001). Inaccurate age de- terminations in some cases have led to overharvesting of stocks such as Pacific ocean perch (Sebastes alutus) 1 PacFIN (Pacific Fisheries Information Network). 2002. Commercial fisher- ies landing data. http://www.PacFIN. org. [Accessed 9 August 2002]. 712 Fishery Bulletin 102(4) and orange roughy (Hoplostethus atlanticus; Beamish, 1979; Archibald et al., 1983; Mace et al., 1990). These historical examples of fishery collapses necessitate that age validation be achieved before age and growth infor- mation is applied to management. In the last decade, radiometric age validation has been applied successfully to over 20 species of rock- fishes and other marine teleosts (Burton et al., 1999; Kastelle et al., 2000; Andrews et al., 20021. The most common technique uses the disequilibria between two radioisotopes, radium-226 (226Ral and lead-210 (210Pb), present in the otolith (Bennett et al., 1982; Smith et al., 1991). Radium-226 is a naturally occurring radioisotope and calcium analogue that is incorporated from the surrounding seawater into the aragonitic crystalline matrix of fish otoliths. Radium-226 decays through a series of short-lived radioisotopes to 210Pb. Because the half-lives of these isotopes are known, the ratio of activity between them (210Pb:226Ra) gives a measure of elapsed time since the initial incorporation of 226Ra into the otolith (Campana et al., 1990). Radium-226 decays very slowly (a 1600 year half-life) in relation to 21"Pb (a 22 year half-life), allowing the activity ratio of these radioisotopes to build into secular equilibrium (1:1 ra- tio; Smith et al., 1991). Based on this relationship (also referred to as ingrowth), the 210Pb: 226Ra activity ratio is suitable for age determination in fishes up to 5 half- lives of 210Pb, or approximately 120 years of age (An- drews et al., 1999b; Campana. 2001). This approach is therefore ideally suited to the blackgill rockfish, whose longevity has been estimated at almost 90 years (Butler et al., 1999). The objectives of this study were 1) to estimate age from otolith growth zone counts, 2) to describe growth, and 3) to validate the annual periodicity of growth zones used to estimate longevity for the blackgill rock- fish with the radiometric aging technique. An ancillary objective was to create a reliable predictive relationship between average otolith weight and estimated age for use as a timesaving tool in the management of this species. Growth zones quantified in sectioned otoliths were used to estimate age, and growth was described by using the von Bertalanffy growth function. Final age estimates were directly compared to radiometric ages to evaluate agreement between the two methods and ulti- mately were used to validate age estimation procedures, age-at-maturity, and longevity for this species. Materials and methods Approximately 1210 blackgill rockfish sagittal otoliths were available for this study. Otoliths were collected by National Marine Fisheries Service (NMFS) person- nel from commercial vessels in 1985 at ports along the California coastline (Long Beach to Fort Bragg), and during NMFS research surveys from 1998 to 2000 from central California to the Oregon-Washington border. Thirty-two juvenile blackgill rockfish, collected from spot prawn traps along the central California coast, were provided by Robert Lea of the California Department of Fish and Game (CDFG). Fish total length iTL; cm or mm), catch area (port or geographic location), and otolith weights (right and left. 1985 samples only) were pro- vided. Otoliths were first considered for age estimation i sectioning!, and the remainder were reserved for radio- metric analysis. Otolith weights (left and right, male and female) were measured to the nearest milligram and compared with £-tests to determine if significant differences in mass existed between sides or sexes. Estimation of age and growth Based on previous aging studies and the need to con- serve samples for radiometric analysis, approximately 310 otoliths (25% of the collection) were assumed to be sufficient for age estimation. The left otolith from 5 to 30 fish, depending upon the number available in each 50-mm size class (ranging from 100 mm to 600 mm), was randomly chosen by using a basic resampling tool. Otoliths were thin-sectioned and mounted onto glass slides. Approximately 50 otoliths were damaged in the sectioning process, leaving 260 otoliths available for age estimation. Sections were viewed by three readers under magnifi- cation (25 and 40x) with transmitted or reflected light. Each reader obtained age estimates by inspecting all available growth axes, choosing the most discernible axis, and reading it three times consecutively. A growth zone (here termed an "annulus") was defined as one pair of translucent (winter-forming) and opaque (summer- forming) bands. A final age, based on each reader's most confident estimate, was chosen. Precision between and within readers was compared by using average percent error (APE; Beamish and Fournier, 1981 1, index of pre- cision (D) and coefficient of variation (CV; Chang, 1982). Percent agreement among readers was also calculated. Reader 1 (author) determined the final age estimate for each section as described in Mahoney (2002). Ages that could not be confidently resolved (through re-examina- tion or discussion) were removed from analysis. Length and age estimates for males, females, and sex- es combined were fitted to the von Bertalanffy growth function (VBGF). A small portion of juvenile samples (/j =16) were included in each function. Because there was strong agreement between facility aging techniques (MLML and NMFS, La Jolla. Butler et al. 1999), ad- ditional aged samples were added to strengthen the VBGF and age prediction models (ra=119). Estimates of age at first, 50%, and 100^ maturity were calculated by inserting existing size at maturity data (Echeverria. HIST ) into the VBGF and solving for age (t). Age prediction, age group determination, and core extraction Campana et al. 1 1990 ) was the first to circumvent the assumption of constant 226Ra uptake throughout the life of the fish by eliminating younger growth layers from adult otoliths, leaving just the oldest layers of Stevens et al.: Radiometric validation of age, growth, and longevity of Sebastes melanostomus 713 otolith growth (i.e., the core, representing the first few years of life). Radium-226 is present at such low activity levels, however, that many otolith cores from fish of a similar age and same sex must be pooled to acquire the mass of material needed for detection (-0.5 to 1 gram; Andrews et al., 1999a, 1999b). Because we possessed a limited number of blackgill rockfish otoliths (-1200), an age prediction model was created to conserve otolith material for radiometric analysis. It was appropriate to assume from the results of Francis (2003) that within- sample heterogeneity with respect to otolith age and mass growth rate was negligible in the core material. To determine age groups for radiometric analyses, final ages for fish whose otoliths were sectioned, along with their corresponding average otolith weight (left and right, «=2), were used to predict age for the re- maining fish in the collection. Several parameters were regressed to determine a predictive relationship be- tween average otolith weight (henceforth termed "oto- lith weight") and estimated age (i.e., section age). The following regressions were compared to estimated age by using Kruskal-Wallis (nonnormal) ANOVA: 1) oto- lith weight (to the nearest 0.001 g), 2) otolith weight and fish length (to the nearest 1 mm), and 3) otolith weight plus otolith length (to the nearest 0.001 mm) multiplied by otolith weight (as an interaction term). A power function was also investigated but did not result in a better fit than that provided by a simple linear regression (either log-transformed or normal). A paired sample (-test and student's (-test for slopes were used to determine if a significant difference existed between male and female otolith weight, and between male and female otolith weight-to-age regressions, respectfully. The final regression equations were applied to the aver- age otolith weight for all individual remaining fish to obtain a predicted age. Age groups were created if there was sufficient otolith material from fish of the same sex and of a similar predicted age. The predicted age range for each group was kept as narrow as possible while permitting enough material for analysis; approximately 25 to 50 otoliths were needed at a target core weight of 0.02 g. Fish that had both oto- liths intact (not sectioned or broken) were preferred to reduce the number of fish for each radiometric sample. To better insure sample conformity, 90% confidence intervals with respect to fish length and otolith weight were used to eliminate from each group dissimilar fish that may have varied significantly from predicted age. In addition to this discriminating technique, groups were further confined by capture year and location. Only samples caught in the same year and similar geo- graphic location (based on the majority of port locations within 300 miles) were included in the same group. Core size was determined by viewing several whole juvenile blackgill rockfish otoliths with estimated ages between 1 and 7 years. The first annulus was deter- mined to be approximately 2 mm wide, and a 3-year-old otolith was measured at 3 mm wide, 4 mm long, and 1 mm thick, and having a weight of 0.02 g. These dimen- sions were chosen as the target core size because a core of this size could be easily extracted, yet was young enough to minimize the possible error associated with variable 226Ra uptake in the first few years of growth. Otoliths from adult fish were ground down to the tar- get core size with a lapping wheel and 80- to 120-grit silicon-carbide paper. Otoliths from selected juveniles, if older than age 3 (core size), were also ground to the target core size. Radiometric analysis The radiometric analysis was conducted as described in Andrews et al. (1999a, 1999b). Because previous studies have revealed extremely low levels of 210Pb and 226Ra in otolith samples, trace metal precautions were employed throughout sample cleaning and processing (Bennett et al., 1982; Campana et al., 1990; Andrews et al. ,1999a). Acids were double distilled (GFS Chemicals", Powell, OH) and all dilutions were made using Millipore- filtered Milli-Q water (18 MQ/cm). Samples were thoroughly cleaned, dried, and weighed to the nearest 0.0001 g prior to dissolution. Whole juvenile otoliths groups were analyzed first to determine if exogenous 210Pb was a significant factor, and to determine baseline levels of 226Ra activity. Because of the low-level detection problems associ- ated with (beta) /3-decay of 2lnPb, the activity 210Pb was quantified through the autodeposition and (alpha) a-spectrometric determination of its daughter proxy, polonium-210 (210Po, half-life=138 days; Flynn, 1968). In preparation for 210Po analysis, samples were dissolved in acid and spiked with a calibrated yield tracer, 208Po, estimated to be 5 times the activity of 210Po in the oto- lith sample. Polonium isotopes from the sample were autodeposited onto a purified silver planchet (A.F Mur- phy Die and Machine Co., North Quincy, MA) held in a rotating Teflon™ holder over a 4-hour period (Flynn, 1968). The activity of 208Po and 210Po on the planchets was measured with ion-implant detectors in a Tennelec (Oak Ridge, TN) TC256 or-spectrometer interfaced with a multichannel analyzer and an eight channel digital multiplexer. Counts were recorded with Nucleus'-' soft- ware (Nucleus Personal Computer Analyzer II, The Nu- cleus Inc., Oak Ridge, TN) on an IBM computer. Counts measured over periods that ranged from 28 to 50 days accumulated from 160 to 919 total counts. Lead-210 ac- tivity, along with uncertainty, was calculated in a series of equations that corrected for background and reagent counts, as well as error associated with count statistics and procedure (pipetting error, yield-tracer uncertainty, etc; Andrews et al., 1999a). The remaining sample was dried and conserved for 226Ra analysis. Determination of 226Ra employed an elemental sepa- ration procedure followed by isotope-dilution thermal ionization mass spectrometry (TIMS) as described in Andrews et al. (1999a, 1999b). The sample was spiked with a known amount of 22sRa yield tracer estimated to produce a 226Ra:228Ra atom ratio close to one. The samples were dissolved in strong acid and dried re- peatedly (~90-100°C) until the sample color was bright 714 Fishery Bulletin 102(4) white, indicating that most organic material had been removed. A three-step elemental separation procedure was used to remove calcium and bari- um, elements that interfere with the detection of radium in the TIMS process. This involved pass- ing the samples through three cation exchange columns, two containing a slurry of BioRad AGE 50W-X8 resin (first and second column), and one containing EiChroM Sr® resin (third column). The samples were introduced to a highly acidic medium within the columns, which separated the elements according to elution characteristics (An- drews et al., 1999b). Radiometric age for each group was determined by inserting the measured 210Pb and 22BRa activi- ties into the secular equilibrium model (Smith et al., 1991) and correcting for the elapsed time be- tween capture and autodeposition. Because these activities were measured from the same sample, the calculation was independent of sample mass (Andrews et al., 1999a, 1999b). Propagated uncer- tainty associated with the final 210Pb activity was based on count statistics, and procedural error and uncertainty for the final 226Ra activity was based on procedural error and an instrumental TIMS analysis routine (Wang et al., 1975; An- drews et al.. 1999b). The combined errors were used to calculate high and low radiometric ages. Accuracy of age estimates Measured 210Pb:226Ra activity ratios for each age group, along with their total sample age (predicted age + time since capture), were plotted with the expected 210Pb:226Ra growth curve. Each age group range was widened by multiplying the minimum and maximum age in the range by the age estimate CV, which was determined from the variability in age estimates among three readers. Agreement between the measured ratio with respect to esti- mated age and the expected ratio (ingrowth curve) provided an indication of the age estimate accuracy. Radiometric age was compared to the average pre- dicted age for each group by using two tests: 1) a paired sample £-test to determine if a significant difference existed between the two age estimates for the groups and 2) predicted age was plotted against radiometric age and the correlation was compared to a hypothetical agreement line (slope of 1) by using r-tests for slope and elevation. Results - __ — - mZ&toSEL \ 1 mm jjK, yjmiffZ- iTm pp>*^^ i : Figure 1 Three images of a blackgill rockfish (Sebastes melariostomus) otolith section viewed with transmitted light at 25x magni- fication (top), 40x (center), and 80x magnification (bottom). This section was aged most consistently as 90 years under a microscope, but because of finer digital resolution and contrast, the section pictured can be aged as high as 102 years. Estimation of age and growth Growth zones observed within otolith sections of most blackgill rockfish were difficult to interpret. Distinction of the first annulus was often ambiguous, and the band- ing pattern during the first several years (1 to -10) of growth was, in some sections, wide and inconsistent. After approximately 8 to 12 growth zones, the zone width transitioned to a narrower zone, which became extremely compressed after 20-40 growth zones. In some sections, these older zones were beyond optical resolution, whereas in others they were remarkably clear (Fig. 1). Stevens et al.: Radiometric validation of age, growth, and longevity of Sebastes melanostomus 715 Table 1 Comparison of von Bertalanffy growth function pai ameters for this studv and Butler et al. (1999 ; in parentheses), for com- bined sexes, females. and males. All lengths are total lengths (mm). Note that the sample size for females and males does not sum to 332 because the same juvenile samp es Ul-. = 16) were used for each sex, and onlv once for combined sexes. N.R.= not reported Combined sexes Females Males L_ (mm) 509(524') 548(554') 448(467') 95% CI 491-528 (N.R.) 520-576 (N.R.i 434-462 (N.R.) k 0.045(0.040) 0.040(0.040) 0.068(0.060) 95% CI 0.038-0.052 0.033-0.047 0.058-0.078 *o -4.86 (-5.02) -4.49 (-4.66) -2.37l-2.98) 95% CI -6.60 to -3.12 -6.30 to -2.67 -3.55 to -1.19 /! 332(335) 181 (98) 167(78) r2 0.81(0.79) 0.87(0.90) 0.87(0.92) 1 Total ler gths from some samples in Butler et al., 1999 were estimated from fork length < FL in mmi by using an equation from Echeverria and Lenarz 1984). The most consistent axis in the otolith section for which confident interpretations could be made was along either the sulcus ridge, or along the dorsoventral margin. Final age estimates were resolved for 197 fish, or approximately 76% of the 260 successfully sectioned otoliths. Agreement among readers was relatively low: approximately 24% of age estimates were within ±1 year, 61% were within ±5 years, and 87% were within ±10 years. The mean difference in age estimates be- tween readers was 2.9 ±4.0 years. Among the three readers, APE was 10.7%, D was 8.4%, and CV was 14.6%. Average percent error, D, and CV estimates were comparable within readers; reader 1 APE was 5.2%, D was 4.1%, and CV was 7.0%. The two oldest fish to be aged were a 90-year-old male (450 mm TL) collected in 1999 and an 87-year-old female (546 mm TL) collected in 1985. Both individuals were caught south of Point Conception, California. The VBGF fitted to age and length data resulted in distinct growth curves for male and female blackgill rockfish (Fig. 2). This difference is also represented by non-overlapping confidence intervals with respect to the primary VBGF parameters (LM, k; Table 1). The growth coefficient, k, ranged from 0.040 (±0.007, fe- male) to 0.068 (±0.010, male), and asymptotic length was 448 ±14 mm for males to 548 ±28 mm for females. The asymptotic length for females was 32 mm less than the largest female fish sampled (580 mm TL), and for males, was 74 mm less than the largest male sampled (522 mm TL). The fit for all three functions was satis- factory (r2=0.81, 0.87; Table 1, Fig. 2). Estimated ages at first, 50%, and 100% maturity, derived from insert- ing published estimates of length-at-maturity (Echever- ria, 1987) into the growth model for each sex, were 15, 21, and 22 years for females and 13, 17, and 28 years for males (Table 2). Table 2 Age at maturity estimates, in years, for ma blackgill rockfish (95'i confidence intervals theses). Maturity estimates were derived published estimates of length at maturity Bertalanffy growth function. e and female are in paren- by inserting into the von First 50% 100% maturity maturity maturity Females 15(12-22) 21(16-31) 22(17-33) Length at maturity' ( mm ) 300 350 360 Males 13(11-15) 17(14-20) 27(22-35) Length at maturity' (mm) 290 330 390 ' Echeverria 11987). Age prediction, age group determination, and core extraction A paired sample r-test indicated that there was a sig- nificant difference between male and female average otolith weight (f=4.54, P<0.001), and a student's r-test for slopes indicated a significant difference between male and female average otolith weight-to-age regressions (rm, = 1.967, ?=2.87, P<0.05). Therefore, male and female age estimates and regressions were treated separately. There was no statistical difference between regres- sions involving fish length and average otolith weight (Kruskal-Wallis one-way ANOVA on ranks, #=4.834, P=0.089). A simple linear regression, with average oto- lith weight as the independent variable and estimated 716 Fishery Bulletin 102(4) age as the dependent variable, was sufficient to pre- dict age. Log normalizing the regressions to stabilize the variance in older age estimates was unsuccessful (Cochran's test: «=0.05, 36 df, C=0.4748, P=0.486). The final regressions are given in Figure 3. Fourteen age groups based on the predicted ages of unsectioned otoliths were chosen. These groups consist- 700 600 500 400 ■2> 300 200 100 o males X females female Female age = 548(1 - e-°<*°('*««»)t ^=181 (/=0.87) Male age = 448(1 - e"0068!'*237!), n=167 (/=0.87) 10 20 30 40 50 60 Otolith section age (yr) 70 80 90 100 Figure 2 Blackgill rockfish (Sebastes melanostomus) von Bertalanffy growth functions plotted for males and females. Observed and expected values, as well as the parameters of the equations, are given. Note that the same juvenile samples (/; = 16) were included in both male and female equations. 100 - males: 90 - y=108.24x-2.65 r2=0.83, n=151 " 80 - , females females: ♦ J^- y=93.803x + 0.175 ' males . ^^ 70 - f 60" I 50 - m CO a 4°- < 30 - 20 - r2=0.85, n=165 ' ♦ * ■ l^' ^ ■ 0 9 m, _^^° •» " ■ • • ^^^r? ■ males 10 - • females 0 'T "' 1 1 1 1 1 1 1 1 1 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900 Average otolith weight (g) Figure 3 Predictive relationship between average otolith weight and estimated age for blackgill rockfish (Sebastes melanostomus). These regression equations were used to predict the age of fish whose otoliths were reserved for radiometric analysis. ed of four juvenile groups, and five male and five female adult groups I Table 3). Fish lengths ranged from 82 mm to 580 mm TL, and predicted age ranged from 1 to 69 years. The number of otolith cores per age group ranged from 11 to 59, representing 7 to 32 fish per group. Total sample weight for each age group ranged from 0.4649 g to 1.6424 g. Whole otolith weight ranged from 0.041 to 0.842 g, and average individ- ual core weight for the adult age groups ranged from 0.025 g to 0.028 g. The process of extracting the core inadvertently destroyed some otoliths in the grinding pro- cess, leading to smaller samples for some groups. Radiometric analysis Radiometric analysis of all age groups (n = 14) resulted in the successful determination of 210Pb activity for all samples, and lim- ited success for 226Ra (Table 4). Activities of 210Pb increased, as expected, fivefold from juvenile to adult age groups, and ranged from near 0.01 dpm/g for the juvenile samples to over 0.05 dpm/g for the oldest age groups. Error associ- ated with these measurements ranged from 3.7 to 9.2 % (Is). The detection of 226Ra activity was met with some technical difficul- ties. Because of poor radium recovery, radium measure- ments were unreliable in three samples and radium was lost in four samples. Therefore, an average of the reliable 226Ra measurements was used because of the rel- ative consistency of levels measured in these samples (0.0643 ±0.0035 dpm/g, n=l). The use of a single estimate for 226Ra activ- ity was acceptable prior to refinement of the technique (Andrews et al., 1999b). Cal- culated 210Pb:226Ra ratios increased as expected from 0.172 to 0.845 and 0.912 for the oldest groups (Table 4). Age estimate accuracy Radiometric ages were in agreement with predicted ages, as evidenced by concor- dance of 210Pb:226Ra activity Stevens et al.: Radiometric validation of age, growth, and longevity of Sebastes melanostomus 717 Table 3 Summary data for 14 pooled otolith age groups of blackgill rockfish. The age range and sample weight of each age group was based on the age prediction model and otolith availability. Groups were confined by year of capture, and for the 1985 samples, by port location. Mean total length (±1 standard deviation) of individuals per group is provided, along with the number offish and otoliths, total sample weight, and average core weight. Sample number Age group (yr) Sex Capture year Mean length ±cjiTL mm) Number of fish,' number of otoliths Sample weight (g) Avg. core weight (g) BG1 1-3 Juvenile 1998 154+26 7, ll2 0.4649 0.042 BG2 4 Juvenile 1998 200 ±8 10, 82 1.1687 0.065 BG3 4-5 Juvenile 1999 217 ±9 15, 192 1.6630 0.088 BG4 1-7 Juvenile 2000 119 ±37 25, 362 0.7854 0.022 BG5 29-31 Female 1985 400 ±20 25,46 1.2510 0.027 BG6 26-28 Male 1985 379 ±19 22.35 0.8866 0.025 BG7 11-17 Female 1998 276 ±20 22,33 0.9018 0.027 BG8 39-41 Female 1985 458 ±22 31,53 1.3332 0.025 BG9 48-54 Male 1985 459 ±21 25,48 1.2491 0.026 BG10 60-69 Female 1985 525 ±30 19,30 0.8254 0.028 BG11 19-23 Male 1998 329 ±16 21,39 1.0313 0.026 BG12 56-59 Female 1985 502 ±28 13.25 0.6989 0.028 BG13 39-41 Male 1985 428 ±24 31,59 1.6424 0.028 BG14 42-47 Male 1998 423 ±26 32,54 1.4267 0.026 1 Both otoliths were not available for every fish chosen. - Whole j jveni e otoliths. Table 4 Summary of radiometric results for pooled otolith age groups. Samples are listed in order of increasing age-group range. Activi- ties are expressed as disintegrations per minute, per gram (dpm/g). Radium-226 activity was averaged among samples with low- analytical error (<10%; n=l) and was determined to be 0.0643 (±0.0035) dpm/g. This value was then applied to all samples to gain an estimate of 226Ra activity and radiometric age. Agreement between radiometric age and predicted age was qualified by the degree of overlap between the two age ranges. Radiometric age incorporates the time between capture and analysis. Sample 210Pb activity 210pb.226Ea Radiometric Radiometric Predicted age Average Age range number (dpm/g) ±%s1 activity ratio age (yr) age range (yr) group range2 age3 (yr) agreement^ BG1 0.0154 ±8.6 0.234 7.1 5.4-8.7 0-3 2 Exceeds BG2 0.0124 ±6.7 0.193 5.5 4.3-6.5 4-5 4 Overlaps BG3 0.0118 ±5.5 0.184 5.5 4.5-6.4 4-6 4.5 Overlaps BG4 0.0111 ±9.2 0,172 6.0 5.3-6.7 0-8 3.5 Encompasses BG7 0.0300 ±5.6 0.467 18.0 15.2-21.4 9-19 14 Overlaps BG11 0.0276 ±5.8 0.430 15.7 13.2-18.7 16-26 21 Overlaps BG6 0.0440 ±4.7 0.684 22.3 16.2-30.3 22-32 27 Overlaps BG5 0.0439 ±4.4 0.683 22.1 16.2-30.3 25-35 30 Overlaps BG13 0.0481 ±3.8 0.749 29.3 21.8-40.4 33-47 40 Overlaps BG8 0.0494 ±4.0 0.769 32.1 23.7-45.1 33-47 40 Overlaps BG14 0.0499 ±3.8 0.777 45.8 37.3-59.1 36-54 45 Overlaps BG9 0.0560 ±3.7 0.871 50.7 35.8-85.1 41-62 51 Encompasses BG12 0.0586 ±4.7 0.912 62.9 40.7-undef. 48-67 57 Encompasses BG10 0.0543 ±4.4 0.845 44.8 31.6-71.6 51-79 65 Overlaps ' Error calculation based on the standard deviation of 210Pb activity (Wanget al., 1975). 2 Predicted age range was extended by 14.6% of coefficient of variation (CV) associated with growth-zone-derived age estimates. 3 The average predicted age of each radiometric age group. 4 Definition of terms: Exceeds = radiometric age range is greater than predicted age range; Overlaps = radiometric age range partially agrees with predicted age range; Encompasses = radiometric age range was in agreement with predicted age range. 718 Fishery Bulletin 102(4) in otolith cores with expected ingrowth curves through time (Fig. 4). Of the 14 pooled otolith groups, three had radiometric age ranges that fully encompassed the pre- dicted age range, ten resulted in overlapping age ranges, and one exceeded predicted age (Table 4). In addition, radiometric ages were in close agreement with predicted ages in a direct comparison (r2=0.88; Fig. 5). Further Ntests indicated no significant difference in slope (£=1.92, P=0.092) or elevation U=0.163, P=2.201) between the regression and a hypothetical agreement line (slope of 1), confirming the close agreement of radiometric age and predicted age. Discussion Estimation of age and growth The growth pattern present in otoliths of blackgill rock- fish was often difficult to interpret. Complications inher- ent to the growth pattern were the following: obscure growth zones up to age 10-15 (the ages when the otolith begins to thicken laterally), rapid transition to slower growth, conflicting or ambiguous growth patterns, and poor resolution of extremely compressed zones in old-age fish. Irregular patterns may have led to enumeration of false growth zones (checks), and the compression of the outer layers may have concealed growth zones present in older fish. This finding has been consistent among previ- ous studies of rockfishes I Chilton and Beamish, 1982). 1.2 1 1 1.0 0.9 0.8 0.7 06 0.5 04 0.3 0.2 0.1 0.0 4^ ^ =2 i Expected Ingrowth Curve a Juvenile age groups • Female age groups ■ Male age groups 20 40 60 Sample age (yr) Figure 4 Measured 210Pb:226Ra ratio plotted against total sample age (mean predicted age plus time since capture! of blackgill rockfish iSebastes melanostomus), with respect to the expected 210Pb:226Ra activity ratio. Horizontal error bars represent the predicted age range (based on age prediction model extended by 14.6% CV). Vertical error bars represent high and low activity ratios based on the analytical uncertainty associated with 2lnPb and 226Ra measurements. Because of the difficulty involved in interpreting growth patterns, aging of blackgill rockfish otoliths involved a high degree of individual subjectivity, as evidenced by the relatively low precision (D=8.4%) and high varia- tion (CV=15%) between readers. However, there were some remarkably clear otoliths and for these we were highly confident of age estimates (Fig. 1). Overall, 87% of between-reader age estimates were within 10 years, emphasizing that although the method of interpretation of growth can be imprecise, it provides a reasonable indication of the growth characteristics and longevity of this species. The von Bertalanffy growth parameters for male and female blackgill rockfish appear to indicate that blackgill rockfish possess distinct patterns of growth (Table 1). Female blackgill rockfish exhibited a slower growth rate than males up to approximately 25-30 years of age (Fig. 2). At this point, the male growth rate slows and approaches an asymptotic length of 448 mm, but females continue to grow in length, reaching an asymptotic length of 548 mm. This trend of slower growing, but ultimately larger females has been ob- served in other slope-dwelling Sebastes species, such as the darkblotched (S. crameri; Rogers et al., 20001, and splitnose (S. diploproa; Wilson and Boehlert. 1990) rockfishes. For both sexes, the growth coefficient is low (k = 0. 040-0. 068) when compared to shallower-dwell- ing (50-200 m) rockfishes, such as the greenstriped (S. elongates, 0.10-0.12; Love et al., 1990) and widow (S. entomelas, 0.20-0.25; Williams et al., 2000) rock- fishes, but very similar to other deep-dwell- ing, long-lived species, such as the short- spine thornyhead (Sebastolobus alaseanus, £ = 0.020; Cailliet et al., 2001), yelloweye (S. ruberrimus, £ = 0.046; Andrews et al., 2002), and bank (S. rufus, £ = 0.041; Cailliet et al., 2001) rockfishes. Previous maturity estimates for blackgill rockfish (7-9 yr males, 6-10 yr females; Echeverria 1987). based on whole otolith counts, were much lower than estimates ob- tained from section ages in the present study (Table 2). Maturity estimates from our study support those derived by Butler et al. (1999). largely because the aging protocol was the same between facilities. Although our growth model included some age estimates (37%) from Butler et al. (1999), our results further confirm age at maturity (Table 2). Compared to other species of the genus, blackgill rock- fish have a late maturity that resides at the upper end of the range for rockfishes (Cail- liet et al., 2001; Love et al., 2002). Extraordinarily old ages in average-size fish exhibited by the blackgill rockfish should not be dismissed as an anomaly. In this study the oldest blackgill rockfish was a 90-year-old male (aged as high as 102 years) that was 450 mm TL. This fish was 160 mm less than the maximum reported 100 Stevens et al.: Radiometric validation of age, growth, and longevity of Sebastes melanostomus 719 0 length (Love et al., 2002). Accord- ing to an experienced rockfish age and growth researcher, "some of the oldest specimens [rockfish] are rarely the largest (lengthwise), and most, if not all, are males." (Munk2) The reasons for this age- length pattern are beyond the scope of this study, but the impli- cations for stock dynamics and management are that it is worthy of further consideration. Age prediction, age-group determination, and core extraction The use of otolith weight as a proxy for age has benefits over conven- tional otolith aging methods by reducing cost, increasing sample size, and allowing greater objectiv- ity (Boehlert, 1985; Pawson, 1990; Fletcher, 1991; Pilling et al., 2003). In this study, predicting ages from otolith weight increased the number of unsectioned otoliths that could be used in the radiometric analysis, but the prediction model also ampli- fied the uncertainty associated with estimates of age. especially in older fish. The variance around the regression line increased with otolith weight, and log normalizing the data did not eliminate this problem. Older predicted ages, there- fore, were more uncertain than younger ages (Fig. 3). Although limited to a specific otolith weight range, the prediction model presented here may provide managers with a more efficient and less costly way to investigate the age structure of blackgill rockfish stocks. In an ideal study, otoliths from the entire estimated age range for blackgill rockfish would be available in the sample set. Otoliths from fish with predicted ages greater than 70 years, however, were not present in our study in sufficient numbers to allow age determination by radiometric methods. This was so, even though more than half of the 1200 otolith pairs obtained for ourstudy were sampled directly from commercial fishing vessels in 1985 along the coast of central and southern Cali- fornia, where the bulk of the fishery occurred. Because fishermen often target adult aggregations, the absence of these older individuals may be an indication that the population had already experienced depletion of older age classes at the time of sample collection, particularly if natural mortality is thought to be low for most rock- 1:1 line Regression y=0.823x + 2.534 r =0.89 20 40 60 Predicted age (yr) 80 100 Figure 5 Direct comparison of mean predicted age and radiometric age for 14 pooled otolith age groups for blackgill rockfish (Sebastes melanostomus). A regres- sion of the data points and 1:1 line of agreement are included for comparison. Horizontal error bars represent the error associated with age estimation (average predicted age multiplied by 14.6% CV), plus the standard error of the regression (Is) used to predict age for radiometric samples. Vertical error bars represent high and low radiometric age estimates based on the analytical uncertainty associated with 210Pb and 22fiRa measurements. fishes (Bloeser3). However, it is possible that the largest, oldest fish are naturally rare, even at the start of an intensive commercial fishery. Knowledge of blackgill rockfish pre-exploitation stock structure and population dynamics would help to elucidate which (depletion of older age classes or a natural situation of low numbers of older fish) is the more likely scenario. Radiometric analysis In previous studies the analytical uncertainty of 226Ra was the limiting factor in radiometric age determina- tion (Andrews et al., 1999a). Typically, TIMS determi- nation of 226Ra reduces error to less than 1-3% of the determined value, but technical difficulties (improperly mixed nitric acid) led to poor recovery and loss of radium in seven samples. The remaining seven samples were deemed reliable because of relatively high radium recov- ery, longer run times, and low analytical uncertainty as determined by the TIMS analysis routine. The 226Ra activity determined for these samples was consistent 2 Munk. K. 2002. Personal commun. Alaska Department of Fish and Game, P.O. 25526, Juneau, AK 99802. 3 Bloeser, J. A. 1999. Diminishing returns: the status of West Coast rockfish, 94 p. Pacific Marine Conservation Council, P.O. Box 59, Astoria, OR 97103. 720 Fishery Bulletin 102(4) enough that we could assume that 226Ra activities were similar among all samples and that use of an average was valid (0.0643 [±0.0035] dpm/g). This approach is acceptable because 226Ra activities measured in pre- vious radiometric studies on Pacific rockfishes were relatively constant. For example, the activity of cored yelloweye rockfish (S. ruberrimus) otoliths had a mean 226Ra activity of 0.0312 (±0.0026) dpm/g (n=18; Andrews et al., 2002), and the rougheye rockfish (S. aleutianus), another deepwater species (to 730 m; Love et al., 2002), had a similar otolith core 226Ra activity averaging 0.065 (±0.003) dpm/g (Kastelle et al., 2000). Accuracy and uncertainty of ages estimates Radiometric activities measured in blackgill rockfish otoliths generally agreed with expected activity ratios for 210Pb and 226Ra (Fig. 4), confirming the validity of growth-zone-derived age estimates. In addition, a direct comparison between radiometric age and predicted age resulted in a strong agreement (r2=0.89; Fig. 5), which was further supported by slope and elevation tests that revealed no significant difference from a 1:1 agreement line. The most critical sources of error involved in age estimation, prediction, and radiometric age determina- tion were the following: 1) age estimate uncertainty, 2) regression error associated with predicted ages, and 3) analytical uncertainty associated with the radiometric aging technique (TIMS and a-spectrometry). Conven- tional aging techniques are inherently subjective (Boe- hlert, 1985; Campana, 2001) and thus create uncertain- ty associated with an estimated age. This uncertainty is transferred to the prediction model, where the natu- ral variability associated with individual otolith weight must also be considered. For most samples, however, the error bars either overlapped or were in contact with the agreement line (Figs. 4 and 5), further confirming the concordance of radiometric age with predicted age. Implications for management When considering the longevity of rockfishes for which a maximum age has been reported (Munk, 2001; Cail- liet et al., 2001), a longevity exceeding 90 years places the blackgill rockfish within the top 2Q(7c of long-lived rockfishes. There is a trend for rockfishes that may indicate that longevity increases as maximum depth of occurrence increases, and physiological adaptations to the environmental conditions of deep-sea living could provide an explanation (Cailliet et al., 2001). The con- firmed longevity and the maximum depth of occurrence (~800 m) for the blackgill rockfish provide further sup- port for this concept. Longevity in the rockfishes has been central to its evolutionary success in relation to other marine teleosts. The suite of life history characters implicit with a long lifespan (slow adult growth, late age-at-maturity, low adult natural mortality) represent a "slow and steady" adaptive strategy, whereby the energy allocated towards individual growth is prolonged, eventually contributing to greater fecundity (due to larger size at maturity) over the lifespan of the individual. This reproductive strategy serves to propagate genetic material across several generations, as well as to diffuse the effect of mortality associated with each reproductive event (Lea- man, 1991). In this sense, longevity may act to buffer the species against short-term (El Nino) and long-term environmental change (Pacific Decadal Oscillations), and the stochasticity inherent in the Pacific Ocean system (Moser et al., 2000). In the absence of fishing pressure, the genetic con- tribution of a slow-growing, longer-lived species may be more conserved in the collective species' gene pool i Munk2 1. In the presence of fishing pressure, however, this "slow and steady" adaptation may be detrimental (Musick, 1999). Although modeling fish populations for the purpose of management typically involves some or all of these parameters, the focus is often on deter- mining sustainable biomass and this approach largely ignores the unknown effects of changes in age struc- ture due to removal of the oldest individuals from the population (Craig, 1985), as well as a loss of genetic diversity that could prevent full recovery of severely depleted populations (Hauser et al, 2002). Given the current depressed condition of many heavily fished rock- fish stocks, species-specific life history characteristics, such as longevity, growth rate, and age-at-maturity estimates, should be given thorough consideration in the development of an effective management strategy. Management regulations that account for these charac- teristics, such as a limited fishing season, or designa- tion of harvest refugia (Yoklavich, 1998), would provide a stronger basis for conservation and sustainability of the resource. Acknowledgments We wish to thank John Butler, Don Pearson, and Cindy Taylor of the Southwest Fisheries Science Center, Mark Wilkins, Jerry Hoff, Waldo Wakefield, and Bob Lauth of the Alaska Fisheries Science Center, and Bob Lea of CDFG for donating specimens for this study. Mary Yoklavich (NMFS). Di Tracey and Larry Paul (NIWA, New Zealand), Kristen Munk (ADFG), Don Pearson (NMFS), Tom Laidig (NMFS), and Steve Campana (DFO, Canada) provided valuable insight into black- gill rockfish growth patterns. Patrick McDonald of the Oregon Department of Fish and Wildlife aged otolith sections. Pete Holden at the University of California, Santa Cruz, measured radium in the refined samples using TIMS. The comments and suggestions of three anonymous reviewers were greatly appreciated. This work was supported by the National Sea Grant College Program of the U.S. Department of Commerce's National Oceanic and Atmospheric Administration under NOAA Grant number NA06RG0142, project number R/F-182. through the California Sea Grant College Program; and in part by the California State Resources Agency. Stevens et al.: Radiometric validation of age, growth, and longevity of Sebastes melanostomus 721 Literature cited Andrews, A. H., G. M. Cailliet, and K. H. Coale. 1999a. 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Distribution and co-occurrence of rockfishes (family: Sebastidael over trawlable shelf and slope habi- tats of California and southern Oregon. Fish. Bull. 100:836-855. Wilson. C. D.. and G. W. Boehlert. 1990. The effects of different otolith ageing techniques on estimates of growth and mortality for the splitnose rockfish, Sebastes diploproa, and canary rockfish, S. pmniger. Calif. Fish Game 76:146-160. Yoklavich, M. (ed). 1998. Marine harvest refugia for West Coast rockfish: a workshop, 162 p. NOAA-Tech. Memo. NMFS-SWFSC-255. 723 Abstract— Larval and juvenile devel- opment of finescale menhaden (Bre- voortia gunteri) is described for the first time by using wild-caught indi- viduals from Nueces Bay, Texas, and is compared with larval and juvenile development of co-occurring gulf menhaden (B. patronus). Meristics. morphometries, and pigmentation pat- terns were examined as development proceeded. An illustrated series of finescale menhaden is presented to show changes that occurred during development. For finescale menhaden, transformation to the juvenile stage was completed by 17-19 mm standard length (SL). By contrast, transfor- mation to the juvenile stage for gulf menhaden was not complete until 2.3- 25 mm SL. Characteristics useful for separating larval and juvenile fines- cale menhaden from gulf menhaden included 1) the presence or absence of pigment at the base of the insertion of the pelvic fins; 2) the standard length at which medial predorsal pigment occurs; 3) differences in the number of dorsal fin ray elements; and, 4) the number of vertebrae. Descriptions of larval, prejuvenile, and juvenile finescale menhaden (Brevoortia gunteri) (family Clupeidae), and comparisons to gulf menhaden (B. patronus) James M. Tolan Texas Parks and Wildlife Department 6300 Ocean Dr., NRC 2501 Corpus Christ!, Texas 78412 E-mail address: James Tolan @tpwd. state. tx. us David A. Newstead Center for Coastal Studies Texas A&M University-Corpus Christi 6300 Ocean Dr., NRC 3216 Corpus Christi, Texas 78412 Manuscript submitted 9 September 2003 to the Scientific Editor's Office. Manuscript approved for publication 14 June 2004 by the Scientific Editor. Fish. Bull. 102:723-732 (20041. Finescale menhaden {Brevoortia gun- teri Hildebrand), one of three recog- nized species of menhaden (Reintjes, 1969; Hettler. 1984) found in the Gulf of Mexico, occurs in the northern and western Gulf of Mexico, from Chande- leur Bay, Louisiana, to Campeche Bay, west of Punto Morros (McEachran and Fechhelm, 1998). Despite their common occurrence in coastal and estuarine waters along the Texas and Mexico coasts (Simmons, 1957; Helher, 1962; Hoese, 1965; Whitehead, 1985; Castillo-Rivera and Kobelkowsky, 2000), their early development has not been described. Early develop- ment of gulf menhaden (B. patronus Goode), on the other hand, has been well described (Suttkus, 1956; Hettler, 1984; Ahrenholz, 1991). In coastal waters of the western Gulf of Mexico, finescale menhaden are spatially and temporally sym- patric with gulf menhaden (Castillo- Rivera et al., 1996). Gulf menhaden are found throughout the northern gulf from Florida Bay to Campeche Bay. Yellowfin menhaden \B. smithi Hildebrand) are found in the eastern gulf from the Mississippi River Delta to Cape Lookout, North Carolina, and co-occur with finescale menha- den only in its extreme western range (Dahlberg, 1970; Hoese and Moore, 1977 ). A large amount of hybrid intro- gression occurs between gulf and yel- lowfin menhaden, although finescale hybrids (either finescalexgulf menha- den or finescalexyellowfin menhaden) have not been reported (Ahrenholz, 1991). Both finescale and gulf menhaden are estuarine-dependent species in- habiting shallow nursery areas for their early development (Gunter, 1945; Shaw et al., 1985; Castillo-Ri- vera and Kobelkowsky, 2000). Gulf menhaden are intermittent or multi- ple spawners (Christmas and Waller, 1975; Lewis and Roithmayr, 1981), and adults move offshore in late sum- mer and early fall. Spawning off the coast of Texas is protracted, and the spawning season begins at the end of August and continues through April (Shaw et al.. 1985). Estuarine immi- gration of gulf menhaden ranging in size from 10 to 32 mm TL has been observed from late October through April (Copeland, 1965: Gallaway and Strawn, 1974; Allshouse, 1983). In Nueces Bay, the greatest densities of gulf menhaden larvae are seen from late February to early May, and the peak immigration of 19-26 mm TL individuals occurs from late April and early May (Newstead, 2003). Fines- cale menhaden spawn in estuarine or nearshore areas (Gunter, 1945; Sim- mons, 1957) and their spawning sea- son has been reported from November to March (Ahrenholz, 1991). Hellier (1962) reported 25-mm-TL specimens taken from the Upper Laguna Madre 724 Fishery Bulletin 102(4) on the lower Texas coast during February, and Gunter (1945), Simmons (1957), and Hoese (1965) have reported postlarval finescale menhaden from the middle and low- er Texas coasts from January to May. Gulf menhaden have received considerable attention in fishery science because of their large population sizes and resulting ecological and economic importance in the northern Gulf of Mexico (Nelson and Ahrenholz, 1981; Smith, 19911, whereas finescale menhaden are less numerous and not directly sought by any recognized fishery (Ahr- enholz, 1991). Our study describes for the first time the development of postflexion (late larval), prejuvenile, and juvenile finescale menhaden. Materials and methods squares regression techniques (SigmaPlot, version 5.0, SPSS Inc., Chicago, IL) in order to graphically illustrate any development differences between the two species. Increasing ratios (BD/SL, CP/SL, and EYE/SL) were described with an exponential rise-to-maximum equation: y = a(l -bx ), whereas, the decreasing ratio of PAL/SL measurements were described with a exponential decay equation: y = ae -bx In both equations, y = body proportion ratio; .r = SL: a = intercept; and b = SL specific exponential rate of change. A total of 170 wild-caught finescale menhaden larvae and juveniles were used to describe early development. All specimens came from ichthyoplankton collections in Nueces Bay, Texas (27.87°N, 97.5TW), during May and June 2003. Individuals were collected in the tidal channels of Nueces Delta with a side-mounted push net (60-cm ring net, 0.505-mm mesh). For comparison, 357 wild-caught gulf menhaden larvae and juveniles col- lected during May and June of 1999, 2000, and 2002 from two nearby stations outside the delta (less than 1.5 km away), in addition to the tidal channel collections of 2003. were also studied. All individuals were initially fixed in either 10% formalin or 95% ethanol and trans- ferred to fresh 95% ethanol after 48 hours. Pigment patterns were recorded and specimens of fin- escale menhaden were illustrated. Gulf menhaden were not illustrated because the figures in Hettler (1984) are adequate. Morphometries Body measurements were made to the nearest 0.1 mm with an ocular micrometer fitted to a dissecting micro- scope. All individuals collected were postflexion, preju- venile, or juvenile stage as defined in Leis and Rennis (1983), and standard length (SL) was measured as the distance from the tip of the snout along the midline to a vertical line through the posterior edge of the hypural plate. All lengths are SL unless otherwise noted. Defini- tions and other terms are as follows: BD = body depth; vertical depth at the pectoral sym- physis. CP = caudal peduncle; horizontal distance from the posterior edge of the dorsal fin base to the pos- terior edge of the hypural plate. EYE = eye diameter; horizontal distance between the anterior and posterior edges of the fleshy orbit. PAL = preanal length; distance from the tip of the snout to the origin of the anal fin. measured along the midline. Ratios of these four body proportion measurements in relation to SL were fitted by means of nonlinear least Meristics Each specimen was examined to determine whether scale formation had been initiated, and a total count of ventral scutes for specimens in which they were suf- ficiently developed was obtained. A total of 37 finescale and 48 gulf menhaden from the 2003 collections were cleared and stained according to Potthoff ( 1984) and used for fin-ray and vertebrae counts. Because of the dif- ficulty in accurately counting myomeres in transforming clupeids (Hettler, 1984; Ditty et al., 1994), we chose to count total vertebrae and use the number of postdorsal and preanal vertebrae instead of postdorsal and preanal myomeres as a potential diagnostic character. Fin-ray counts included dorsal, anal, and caudal fins (both prin- cipal and procurrent rays). Results Morphological development Finescale menhaden larvae were first collected at 9.7 mm and ranged to 22.5 mm as transforming juveniles (Fig. 1). Transformation from the larval to the juvenile form began around 14 mm and was completed by around 20 mm (Fig 2). Ratios of body depth, caudal peduncle, and eye diameter all increased in relation to standard length as larvae grew, whereas snout-to-anal length decreased i Table 1). The decrease in snout-to-anal length reflected the transformation from the elongate fusiform shape of the larvae to the laterally compressed deep-bodied shape of the juvenile. Scales began to form at around 15 mm on the caudal peduncle region and progressed forward along the ventral and lateral surfaces towards the dorsal sur- face. None of the individuals examined had the enlarged and fringed median scales preceding the dorsal fin, which are an adult characteristic of the genus Brevoor- tia. Ventral scutes also began forming around 15 mm, and the full complement of 27-31 scutes (McEachran and Fechhelm, 1998) was found by 19 mm. Gulf menhaden ranged from 11.7 mm as larvae to 40.4 mm juveniles. For gulf menhaden, body depth. Tolan and Newstead: Larval and |uvenile development of Brevoortia gunten 725 Table 1 Proportional measurements in development. relation to standard length (SL) used to describe finescale menhaden {Brevoortia gunteri) larval Length class (mm, SL) Number of specimens Body depth: SL Preanal length: SL Caudal peduncle: SL Ey 3 diameter: SL <11.0 1 0.100 0.804 0.256 0.054 11.1-12.0 2 0.108 0.798 0.259 0.059 12.1-13.0 5 0.119 0.778 0.251 0.059 13.1-14.0 15 0.139 0.764 0.263 0.064 14.1-15.0 28 0.136 0.756 0.269 0.062 15.1-16.0 25 0.168 0.735 0.281 0.074 16.1-17.0 23 0.208 0.711 0.302 0.082 17.1-18.0 17 0.220 0.705 0.311 0.085 18.1-19.0 3 0.239 0.700 0.327 0.080 19.1-20.0 2 0.219 0.700 0.304 0.078 >20.1 1 0.318 0.690 0.304 0.093 caudal peduncle, and eye diameter ratios all similarly increased in relation to standard length as larvae grew, whereas snout-to-anal length decreased (Table 2). Scale initiation in gulf menhaden was not seen until 19 mm, and ventral scutes did not begin forming until around 18 mm. The full complement of scutes (28-32 scutes; McEachran and Fechhelm, 1998) was seen by around 25 mm. No enlarged median dorsal scales were noted from the gulf menhaden individuals examined. With little overlap in the 15-20 mm size range (see Fig. 1) and a limited number of juvenile-size finescale menhaden (SL>20 mm), it was not possible to effectively separate finescale and gulf menhaden morphometrically on the basis of BD:SL, PAL:SL, CP:SL, and EYE:SL ratios (Fig 3). By 25 mm, proportional body measure- ments had become nearly constant for gulf menhaden whereas body measurements were still changing for finescale menhaden even though they appeared to be fully transformed. For a fish of given size, finescale menhaden typically had a greater body depth, a shorter preanal length, and a greater caudal peduncle length than gulf menhaden. Meristic features No recently hatched or preflexion finescale menhaden were examined and all postflexion individuals followed the fin development sequence identified for other clupe- ids (Houde et al., 1974: Hettler, 1984; Ditty et al., 1994). The caudal and dorsal fins are first to develop, followed by the pelvic fins, whereas the pectoral fins are the last to fully develop even though the pectorals are the first fins to form as nonrayed buds. Only vertebrae and dorsal-fin ray counts were useful in separating finescale and gulf menhaden, because most other meristics over- lapped (Table 3). Finescale menhaden had fewer total vertebrae ( = 43 vs. 46) and fewer dorsal-fin rays (median 125 100 75 B. gunteri n=170 50 25 § o O J" ^^ 25 50 S. patronus n=357 75 100 — i i i i 5 15 25 35 45 Body length (mm) Figure 1 Length-frequency histograms for Brevoortia gunteri and B. patronus derived from ichthyoplankton col- lections in Nueces Bay, Texas, during May and June (1999-2003). value = 18 vs. 21) than gulf menhaden. Postdorsal and preanal vertebrae also showed a high degree of overlap between the two species (Table 3). The forward move- ment of the anal fin in relation to the dorsal fin was most evident in fully transformed gulf menhaden, and the number of postdorsal-preanal vertebrae decreased from 4 to -3. The relative placement of the anal fin also 726 Fishery Bulletin 102(4) i'liiiiiiin'illftiiiiumi 13.7 mm 5.0 mm Figure 2 Developmental stages of Brevoortia gunteri. iAi Postflexion larva, 13.7 mm. (B) Postflexion larva, 15.0 mm. (C) Transforming larva, 17.2 mm. (D) Transforming larva, 19.0 mm. (Ei Transformed juvenile, 23.9 mm. Table 2 Proportional measurements relative to standard length (SL ) used to describe gulf menhaden {Brevoortia patronus) larval development. Length class Number of Body depth: Preanal length: Caudal peduncle: E ve diameter: (mm, SL) specimens SL SL SL SL <14.0 1 0.086 0.829 0.229 0.061 14.1-15.0 1 0.119 0.786 0.238 0.061 15.1-16.0 3 0.136 0.764 0.261 0.063 16.1-17.0 4 0.136 0.764 0.261 0.063 17.1-18.0 5 0.144 0.749 0.257 0.065 18.1-19.0 13 0.169 0.733 0.265 0.067 19.1-20.0 26 0.183 0.725 0.274 0.074 20.1-21.0 22 0.176 0.735 0.271 0.068 21.1-22.0 17 0.236 0.719 0.282 0.084 22.1-24.0 9 0.286 0.709 0.298 0.095 24.1-26.0 2 0.337 0.730 0.313 0.103 26.1-29.0 11 0.352 0.730 0.299 0.098 29.1-32.0 4 0.356 0.740 0.308 0.100 >32.1 3 0.380 0.736 0.294 0.097 Tolan and Newstead: Larval and |uvenile development of Brevoortia gunten 727 gSSgSSgHJfMISHp Figure 2 (continued) Table 3 Meristies in finescale menhaden, Brevoortia gunteri, (37 specimens) and in gulf menhaden. B. patronus, (48 specimens). Median values are given in parentheses. B. gunteri B. patronus Number in full complement Meristic B. gunteri B. patronus Caudal-fin rays Principal (dorsal) 10 10 10-11 (ventral l 9 9 9-10 Procurrent (dorsal) 7-9(8) 8-9(8) 8-9 (ventral l 7-8(7) 5'-8(7) 7-8 Dorsal-fin rays 17-20 ( 18 ) 16'-23(21) 17-20 21-23 Anal-fin rays 18-24(22) 20-23(21) 20-25 18-22 Vertebrae 43-44(43) 40'-46(46) 42-44 45-46 Postdorsal and preanal vertebrae 0-3(2) -32-4(l) 2-43 1 B. patronus larva SL = 11.7 mm. 2 B. patronus fully transformed individual SL = 40.4 mm. 3 Postdorsal and preanal myomere counts in larvae 10-15 mm SL (Ditty et al., 1994) 728 Fishery Bulletin 102(4) A 0.4 - ° s^20 mm) are silvery in color over most of the body. The head, back, and dorsal and cau- dal fins are all pigmented. In preservation, two dark, slash-like pigments spots were present on the posterior lateral body above and below the urostyle. In nearly all other aspects, juvenile finescale menhaden closely 730 Fishery Bulletin 102(4) resembled adults by this size. No humeral spots were noted in the two individuals examined. Discussion Finescale menhaden larvae resemble the larvae of other clupeids (Houde and Fore, 1973; Jones et al., 1978; McGowan and Berry, 1983; Hettler, 1984: Ditty et al., 1994) in having elongate, slender bodies, light pigmen- tation, and a small head lacking spines. They have a long, straight gut, often with striations along the hind- gut, posteriorly placed dorsal and anal fins, and the vent is always posterior to the dorsal fin base (Jones et al., 1978). Hettler (1984) discussed the separation of individual species of Brevoortia, and Ditty et al. (1994) presented a synopsis of characters to separate clupeid larvae (<15 mm) based on meristic, morphometries, and pigmentation. Finescale menhaden have 43-44 vertebrae, whereas gulf menhaden have 44-46. Yel- lowfin menhaden are reported to have 45-47 vertebrae (Houde and Swanson, 1975). The number of vertebrae, which should approximate the number of myomeres in larvae much smaller than those collected in this study, in conjunction with pigment differences have been shown to be useful in separating clupeid species com- plexes (Ditty et al., 1994). In the western gulf, counts of 43-44 vertebrae (=myomeres) would separate finescale menhaden from other clupeid larvae such as Sardinella aurita (45-47 vertebrae; Ditty et al., 1994), Etrumeus teres (48-50 vertebrae; Fahay, 1983), and Opisthonema oglinum (45-46 vertebrae; Richards et al., 1974). Spe- cies from the western gulf with similar vertebral counts (Harengula jaguana, 39-42; Houde et al., 1974; and Jenkinsia lamprotaenia, 39-42; Powles, 1977) can be distinguished from finescale menhaden by their larger PAL:SL ratio (>85% at 15 mm for Harengula vs. <85l7c for finescale menhaden. Table 1) and fewer anal rays (13-14 for Jenkinsia vs. 18-24 for finescale menhaden, Table 3). Although vertebral counts were used suc- cessfully in distinguishing finescale menhaden from gulf menhaden, the time necessary to clear and stain larvae makes this method impractical for distinguishing between large numbers of menhaden. In larval and prejuvenile stages, finescale and gulf menhaden are morphologically very similar. Propor- tional body measurements overlapped too greatly to reliably distinguish the two species. Only the presence of medial predorsal pigment prior to transformation, stellate melanophores at the pelvic fin base, and the size at transformation were useful characters in distin- guishing the two species. Hettler (1984) noted that gulf menhaden lack paired melanophores in the predorsal region until initiation of transformation. Pigment at the pelvic fin base appears to be a diagnostic character for the small-scale menhadens because Houde and Swan- son (1975) also reported a similar feature in yellowfin menhaden as small as 12.3 mm. Although the presence of this pigment at the pelvic fin base is proposed to be diagnostic for the small-scale menhadens (present study), Hettler's (1984) illustration of a 16.5-mm gulf menhaden shows this pigment. Pigmentation descrip- tions for developing gulf menhaden have not specifi- cally addressed melanophores at the pelvic fin insertion (Hettler, 1984). Finescale menhaden transform at a smaller size (17-19 mm) than any of the other Gulf of Mexico menhadens. Gulf menhaden did not complete transformation until around 25 mm, which is in agree- ment with the reported lengths of 25-28 mm for both laboratory reared and wild-caught individuals ( Suttkus. 1956; Hettler. 1984). Yellowfin menhaden reach trans- formation at an intermediate size (20-23 mm; Houde and Swanson, 1975). Even as adults, finescale menhaden very closely re- semble gulf menhaden (Hoese and Moore. 1977) and few reliable characters effectively separate them. Only the absence of striations on the margin of the operculum, a single humeral spot (with no hint of trailing spots along the lateral margins I, and more scale rows (60-77 in fi- nescale vs. 36-50 in gulf menhaden; Hoese and Moore, 1977; McEachran and Fechhelm, 1998) distinguish fin- escale from gulf menhaden. All other meristics overlap greatly; i.e., counts of dorsal-fin rays, anal-fin rays, pectoral-fin rays, pelvic-fin rays, gill-raker counts, and ventral scutes. Although externally similar, significant differences in internal structure between finescale and gulf menhaden have been documented. Finescale men- haden have fewer branchiospinules and shorter inter- mediate gill rakers than gulf menhaden and, as such, filter mainly zooplankton from the water column; gulf menhaden, in contrast, feed primarily on phvtoplankton and detritus (Castillo-Rivera et al., 1996). Based on length-frequency differences seen between the two species (Fig. 1), the reported spawning season for finescale menhaden along the middle Texas coast could be extended to late May. We still do not know of characters that would distinguish finescale menhaden eggs, and yolksac. preflexion, and flexion larvae from other species in the genus Brevoortia. In order to fully describe the development of finescale menhaden, labo- ratory spawning and rearing experiments are needed to fully describe these early-life stages. Houde (1973) presented relatively simple rearing techniques that al- low descriptions of the developmental stages of larval fish (from egg through transformation of larvae to the juvenile stage). These methods have been used success- fully for Atlantic, gulf, and yellowfin menhaden, and presumably, finescale menhaden could be reared with these same techniques if their eggs could be obtained. The rearing of finescale menhaden would also allow the effectiveness of the proposed pigment characters used to separate finescale menhaden from gulf menhaden to be tested. Acknowledgments This work was performed with funding from the Coastal Bend Bays & Estuaries Program under contract 0203. The Texas Parks and Wildlife Department, Coastal Tolan and Newstead: Larval and |uvenile development of Brevoortia gunteri 731 Studies Program, Resource Protection Division provided additional interagency cooperation in the form of equip- ment and field logistical support. This manuscript was improved by comments from two anonymous reviewers. Literature cited Ahrenholz, D. W. 1991. Population biology and life history of the North American menhadens, Brevoortia spp. Mar. Fish. Rev. 53(41:3-19. Allshouse, W. C. 1983. The distribution of immigrating larval and post- larval fishes into the Aransas-Corpus Christi Bay complex. M.S. thesis, 118 p. Corpus Christi State Univ., Corpus Christi, Texas. 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Description of eggs, larvae, and early juveniles of gulf menhaden, Brevoortia patronus, and comparisons with Atlantic menhaden, B. tyrannus, and yellowfin menhaden, B. smithi. Fish. Bull. 82:85-95. Hoese, H. D. 1965. Spawning of marine fishes in the Port Aransas, Texas, area as determined by the distribution of young and larvae. Ph.D. diss., 144 p. Univ. of Texas, Austin, TX. Hoese, H. D., and R. H. Moore. 1977. Fishes of the Gulf of Mexico: Texas, Louisiana, and adjacent waters. 327 p. Texas A&M Univ. Press, College Station. Houde, E. D. 1973. Some recent advances and unsolved problems in the culture of marine fish larvae. Proc. World Mari- culture Soc. 3:83-112. Houde, E. D., and P. L. Fore. 1973. Guide to identify eggs and larvae of some Gulf of Mexico clupeid fishes. Fla. Dep. Nat. Resour. Mar. Res. Lab. Leafl. Ser. 4 (part 1, no. 23). 14 p. Houde, E. D., W. J. Richards, and V. P . Sakensa. 1974. Descriptions of eggs and larvae of the scaled sar- dine, Harengula jaguana. Fish. 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FAO, Rome. 733 Abstract-The stomach contents of the minimal armhook squid iBerryteuthis anonychus) were examined for 338 specimens captured in the northeast Pacific during May 1999. The speci- mens were collected at seven stations between 145-165°W and 39-49°N and ranged in mantle length from 10.3 to 102.2 mm. Their diet comprised seven major prey groups (copepods, chaetognaths, amphipods. euphausi- ids, ostracods, unidentified fish, and unidentified gelatinous prey) and was dominated by copepods and chaeto- gnaths. Copepod prey comprised four genera, and 86% by number of the copepods were from the genus Neo- calanus. Neocalanus cristatus was the most abundant prey taxa, composing 50% by mass and 35% by number of the total diet. Parasagitta elegans (Chaetognatha) occurred in more stomachs (47%) than any other prey taxon. Amphipods occurred in 19% of the stomachs but composed only 5% by number and 3% by mass of the total prey consumed. The four remaining prey groups (euphausiids, ostracods. unidentified fish, and unidentified gelatinous prey) together composed <29c by mass and <1% by number of the diet. There was no major change in the diet through the size range of squid examined and no evidence of cannibalism or predation on other cephalopod species. Diet of the minimal armhook squid (Berryteuthis anonychus) (Cephalopoda: Gonatidae) in the northeast Pacific during spring Kazuhisa Uchikawa National Research Institute ot Far Seas Fisheries 5-7-1 Shimizu-Ondo Shizuoka, 424-8633, Japan E-mail address stomyctS affrc go ip John R. Bower Northern Biosphere Field Science Center, Hakodate Branch Hokkaido University 3-1-1 Minato-cho, Hakodate Hokkaido 041-8611, Japan Yasuko Sato Department of Agriculture, Forestry and Fisheries, Nngata Prefecture Agriculture Affairs Division Shlnko-cho Nngata 950-8570, Japan Yasunori Sakurai Graduate School of Fisheries Sciences Hokkaido University 3-1-1 Minato-cho, Hakodate Hokkaido 041-8611, Japan Manuscript submitted 2 September 2003 to the Scientific Editor's Office. Manuscript approved for publication 29 June 2004 by the Scientific Editor. Fish. Bull. 102:733-739 12004). The squid family Gonatidae plays an important role in the ecosystems of the North Pacific. In the Sea of Okhotsk, the annual production of gonatid squids is more than half that offish production (Lapko, 1996), and in the western and central Bering Sea, gonatid production is thought to exceed that of the dominant fish fami- lies (Radchenko, 1992). In the sub- arctic North Pacific, the gonatids are an important link in the pelagic food web iBrodeur et al., 1999). To better understand the food web in the North Pacific and the processes influencing the production of gonatid squids in this region, information is needed on the feeding behavior of these squids. The minimal armhook squid iBerry- teuthis anonychus) (also known as the "smallfin gonate squid" [Roper et al., 1984]) is a small gonatid (maximum mantle length = 150 mm) distributed mainly in the northeast Pacific (Rop- er et al.. 1984: Bower et al., 2002). It is a major prey for fishes, squids, sea- birds, and marine mammals (Ogi et al., 1980; Pearcy et al., 1988; Pearcy, 1991; Kuramochi et al., 1993; Pearcy et al., 1993; Ohizumi et al., 2003) but is not targeted by any fishery. Despite the importance of B. anonychus in the food web of the subarctic North Pa- cific, the only published reports on its feeding behavior are two abstracts in the Russian literature (Lapshina, 1988; Didenko, 1990). In this arti- cle, we provide further information on the feeding behavior of B. anony- chus by describing the diet of a wide size range of squid collected from the northeast Pacific during late spring. Methods Berryteuthis anonychus was collected during a United States National 734 Fishery Bulletin 102(4) 65°N 60°N 55°N 50°N 45 N 40° N 35°N 180 W "I T 170°W 160:W 150'W 140 W 130"W 120 W Figure 1 Sampling stations in the northeast Pacific where Berryteuthis anonychus were collected during May 1999. Numbers indicate station numbers. Marine Fisheries Service (NMFS) survey of salmon in the northeast Pacific (Carlson et al., 1999; Bower et al., 2002). Samples were collected during 6-17 May 1999 at seven stations between 145-165°W and 39-49°N (Fig. 1). At each station, a midwater trawl modified to fish at the surface was towed for 1 hour. The trawl was 198 m long and had hexagonal mesh in its wings and body, and a 1.2-cm mesh liner was used in the codend. Trawling speeds were 7-9 km/h, and the average net dimensions while fishing were 16 m vertical spread and 45 m horizontal spread. Squid samples were frozen on board to -20°C and preserved in 50% isopropyl alcohol in the laboratory. The mantle length (ML) of each squid was measured to the nearest 0.1 mm, and each squid was weighed to the nearest 0.01 g. The stomach contents of 338 squid (167 males, 144 females, 27 undetermined) ranging in ML from 10.3 to 102.2 mm (Fig. 2) were examined un- der a stereomicroscope. A total of 359 squid were col- lected during the survey (Bower et al., 2002), but 21 of these specimens were either damaged or lost, and thus excluded from our analyses. Most prey items were frag- mented; therefore prey identification was usually based on diagnostic body parts as described in Brodsky (1950), Miller (1988), Baker et al. (1990), and Vinogradov et al. (1996). and by comparison with zooplankton specimens collected in the same area. The prey items were counted and weighed to the nearest 0.01 mg. These wet mass measurements presumably underestimated the initial wet masses because mass loss occurs in invertebrate samples preserved in isopropyl alcohol (e.g., Howmiller, 1972), and it was assumed all prey taxa were equally affected by the preservation. The numbers of individuals of each 6CH 50- 40- Z 30- 20- 10-| 0 n = 338 10 20 30 40 50 60 70 80 90 100 Mantle length (mm) Figure 2 Length-frequency distribution for Berryteuthis anonychus. prey taxon were estimated from the numbers of prey parts, such as copepod mandibles, amphipod heads and chaetognath seizing hooks. Because of the difficulty in distinguishing the copepods Neocalanus plumchrus and N. flemingeri, they were grouped as a single taxon, N. plumchrus+flemingeri. Some calanoid copepods that could not be identified to genus level were identified as either a "specialized form" or a "generalized form"; characters of the specialized form included appendages that were greatly enlarged or strongly developed with chelae, spines on the posterior corners of the terminal thoracic segment, Uchikawa et al.: Diet of Berryteuthis anonychus in the northeast Pacific during spring 735 Table 1 Numbers of Berryteuthis anonychus stomachs with iden- tifiable prey remains. without identifiable remains, and without remains from the northeast Pacific. Station num- bers refer to those shown in Figure 1 With Without Station identifiable identifiable Without no. remains remains remains Total 1 25 0 0 25 2 29 19 45 93 3 33 0 2 35 4 12 1 2 15 5 51 9 6 66 6 44 18 16 78 7 26 0 0 26 Total 220 47 71 338 and an asymmetrically swollen genital segment. The gen- eralized form included calanoid copepods of the Calanus type that did not share any of these characters. A stomach-contents index (SCI, %) was calculated as SCI=(wet mass of total stomach contents/wet body mass)xl00. For each prey taxon, the percentages by number (N) and wet mass (WM) of the total prey, and the percentage frequency of occurrence (F) were de- termined. An index of relative importance (IRI) was calculated for each prey taxon as IRf = Ft x lNt+ WMt) (Pinkas et al., 1971), where i denotes the taxon. The IRI for each major group of prey taxa was then standard- ized to '/dRI (Cortes. 1997): n %IRI, = 100 x IRI, I £ IRIt , where /; is the total number of groups collected. Copepod mandible size is directly related to the cara- pace length of several calanoid copepods in the North Atlantic (Karlson and Bamstedt, 1994); therefore man- dible width was used as an indicator of relative prey size to compare copepod prey size with squid mantle size. A total of 87 mandibles were measured from the stomachs of 10 squid measuring 29-102 mm ML. Results Of the 338 stomachs examined, 267 (79%) contained prey, and 220 (65% ) contained identifiable prey (Table 1). Individual SCI values ranged from 0% to 8.0% (station mean = 1.0%). SCI values varied significantly among sampling times (Kruskal-Wallis test, P<0.001), and the two highest SCI values occurred in the afternoon and just after sunset (Fig. 3). The diet of B. anonychus comprised seven major prey groups and was dominated by copepods (A?=70%, 3.0-1 T t h> 2.5- { 1 2.0- o 15" CO 1.0- 263 -15 0.5" 66 _87 78 1 1 1 1 1 1 1 1 1 6 8 10 12 14 16 18 20 22 24 Time of day (h) T Sunrise Sunset Figure 3 Mean stomach contents index (SCI) of Berrvteuthis anonychus collected in the northeast Pacific during May 1999 at different times of day. SE = standard error of the mean. Numbers indicate squid sample size for each sampling. WM=85%, F=74%, %IRI=87%) and chaetognaths (N=24%, WM=11%, F=48%, <7dRI=129c) (Table 2). The five other prey groups (amphipods, euphausiids, ostra- cods, unidentified fish, and unidentified gelatinous prey) each had a 9cIRI value <1%. Copepod prey comprised four genera, and 86% by number of the copepods were from the genus Neocala- nus. Neocalanus cristatus was the most abundant prey taxa. composing 50% by mass and 35% by number of the total diet. The three Neocalanus taxa (Neocalanus spp., N. plumchrus+flemingeri, and N. cristatus) com- posed 85% by mass and 68% by number of the diet. Neocalanus cristatus was identified based on the pres- ence of the head crest, which develops at the C5 copepo- dite stage (Brodsky, 1950). Thus, this taxon comprises only the C5 and C6 stages, and possible members of the Neocalanus spp. taxon include N. plumchrus, N. flemingeri, and earlier stages (C1-C4) of N. cristatus. Squid >60 mm ML fed mainly on Neocalanus crista- tus (2V=39%, WM=53%, F=50%) and Neocalanus spp. (iV=29%, WM= 31%. F=40%), whereas those <60 mm ML fed mainly on Neocalanus spp. (AT=43%, WM=53%, P=29%) and Neocalanus plumchrus+flemingeri (N=8%, WM=10%, F=14%), and consumed few C5-C6 Neocala- nus cristatus (N=4%, WM=4%, F=6%). The mandible size of copepod prey showed a clear positive relationship with ML (Fig. 4), indicating that the squid fed on larger copepods as the squid grew. Taxa from other copepod genera (i.e., Candacia, Metridia, and Pleuromamma) composed 0.5% of the total prey number and 0.1% of the total wet mass (Table 2). Parasagitta elegans, the only identified chaetognath, occurred in more stomachs (47%) than any other prey taxon and in 58% of the stomachs from squid >60 mm 736 Fishery Bulletin 102(4) Table 2 Prey items identified from stomach contents of Berry teu this anonychus collected in the northeast Pacific during May 1999. %IRI: standardized index of relative importance. %IRI values in parentheses are those for <60 mm ML and >60 mm ML squid. Fre- quency of occurrence was calculated from the number of stomachs containing food. "- means prey taxon was not present in stomachs. Number Wet mass Frequency of %IRI Taxon (%) (%) occurrence (%) (<60 mm ML, >60 mm ML) Copepoda 70.2 85.3 74.2 86.5(80.9,84.81 Candacia columbine 0.2 0.1 1.9 Candaeia sp. <0.1 <0.1 0.4 Metridia paeifica 0.2 <0.1 2.2 Neoealanus cristatus 35.0 50.4 23.2 Neocalanus plumchrus+flemingeri 3.1 1.8 12.4 Neoealanus spp. 30.0 32.3 33.3 Pleuromamma spp. 0.1 <0.1 1.9 Calanoida (generalized form) 0.5 0.3 4.9 Calanoida (specialized form) 0.1 0.1 0.4 Unidentified Calanoida 0.9 0.3 14.2 Unidentified Copepoda 0.1 0.1 2.6 Chaetognatha 23.9 10.8 47.6 12.4(18.1, 13.9) Parasagitta elegens 23.8 10.7 47.2 Unidentified Chaetognatha 0.1 0.1 1.1 Amphipoda 4.6 2.5 19.1 1.0(1.0. 1.3) Hyperia medusarum 0.8 0.9 2.2 Themisto paeifica 2.5 0.9 7.5 Unidentified Hyperiidae 0.4 0.5 0.7 Unidentified Physocephalata <0.1 <0.1 0.4 Unidentified Hyperiidea 0.7 0.2 7.5 Unidentified Amphipoda 0.1 <0.1 1.9 Euphausiacea 0.5 0.9 4.5 <0.1 (<0.1,0.1) Euphausia paeifica <0.1 0.4 0.5 Thysanoessa sp. <0.1 <0.1 0.4 Unidentified Euphausiacea 0.5 0.5 3.7 Ostracoda <0.1 <0.1 1.1 <0.1 (<0.1,— ) Unidentified fish <0.1 0.8 0.4 <0.1 (— , <0.1) Unidentified gelatinous prey <0.1 <0.1 0.4 <0.1 (<0.1,— ) Unidentified Crustacea 0.1 <0.1 1.1 Unidentified material 0.6 0.1 18.7 Neocalanus plumchrus and N. flemingeri were grouped as a si ogle taxon tN. plumchrus+flemingeri) because of d i f ri c l Ity in distinguishing these species in partly digested materials. Neocalanus cristatus comprises stages C5 and C6 only. Neocalanus spp. = N. cristatus (stages Cl- C4 ). N. plumchrus, and N. flemingeri. Calanoida (specialized form! = unidentified individuals with markedly enlarged appendages. strongU deve loped chel ae, a spine on the posterior corner of the terminal thoracic segment, or asymmetrically swollen genital segments. Calanoida (generalized form) = unidentifi ?d Calanus-type individuals that share none of the characters of the special zed form. ML. P. elegans was the third most abundant prey taxon, composing 24% by number and 11% by mass of the total diet (Table 2). Amphipods (mainly Themisto paeifica and Hyperia medusarum) were consumed by 199r of the squid but composed only 5% by number and 3% by wet mass of the total prey consumed. The four other prey groups combined composed <2re by mass and <1% by number of the diet. There were no major changes in %IR1 val- ues through the size range of squid examined (Table 1) and no evidence of cannibalism or predation on other cephalopod species. Uchikawa et al.: Diet of Berryteuthis anonychus in the northeast Pacific during spring 737 0, KM > i — i~i — ■ i ■ — r-1 — i ■ i — ■ i ■ — r-1 — i 20 30 4(1 50 60 70 80 90 100 110 Mantle length (mm) Figure 4 Relationship between mandible width of copepod prey and mantle length of Ber- ryteuthis anonychus. Discussion The diet of Berryteuthis anonychus collected in the north- east Pacific during May was dominated by calanoid copepods and chaetognaths. During early July in this area, B. anonychus larger than those examined in the present study (ML: 75-127 mm vs. 10-102 mm) fed on a wider variety of prey, including primarily calanoid copepods, hyperiid amphipods, pteropods, and euphau- siids (Lapshina, 1988). Possible causes for this change in diet include seasonal change in prey availability and an ontogenetic change in the squid's ability to capture prey. The zooplankton composition in the upper 150 m of the subarctic North Pacific is highly seasonal. Neocala- nus copepods, the major prey of B. anonychus, dominate the epipelagic zooplankton community during spring and early summer (Mackas and Tsuda. 1999). They then descend from the upper layer to spend the late summer, autumn, and early winter at 400-2000 m, well below the depth range of B. anonychus (0-200 m; Nesis, 1997). As a result of this ontogenetic descent, the upper ocean zooplankton biomass decreases greatly, and the community is then dominated by a different group of species. This group includes euphausiids (Mackas and Tsuda, 1999), which are consumed by more B. anony- chus in July (28%; Lapshina, 1988) than in May (5%; present study). Other prey that show a large increase in frequency of occurrence between May and July are amphipods (19% in May, 52% in July) and pteropods (0% in May, 40% in July). Oceanic squids such as B. anonychus generally feed on small crustaceans as juveniles and then shift their diet to larger fish and other cephalopods as they grow (Rodhouse and Nigmatullin, 1996). We observed no such ontogenetic shift within the size range examined, but copepod prey size was found to increase with growth. These data are consistent with those for other squids in that prey size increases during development (Nixon, 1987; Hanlon and Messenger, 1996). Most gonatids undergo ontogenetic vertical descent (Roper and Young, 1975; Nesis, 1997), and a clear shift in the diet can ac- company this habitat shift (e.g., as seen in Berryteuthis magister; Nesis, 1997). Nesis (1997), however, suggested that B. anonychus does not undergo ontogenetic descent; therefore no such habitat-change-related shift in diet would be expected to occur in this species. Highest feeding intensities were recorded in the after- noon and just after sunset, which would indicate that B. anonychus feeds both day and night. Such a feed- ing scenario is supported by the high overlap in depth distributions of B. anonychus (day: 50-200 m, night: 0-150 m; Nesis, 1997) and its main prey, Neocalanus cristatus; during spring, N. cristatus occurs mainly at 50-150 m, and like the other Neocalanus species, shows no evidence of diel vertical migration (Mackas et al., 1993). Therefore B. anonychus and N. cristatus occupy nearly the same depth range both day and night. The chaetognath Parasagitta elegans was the third most abundant prey taxon and was consumed by more squid than any other taxon. Parasagitta elegans forms an important fraction of the springtime macrozooplank- ton community in the North Pacific (Brodeur and Ter- azaki, 1999) and inhabits mainly the epipelagic layer (0-200 m) (Kotori, 1976; Terazaki and Miller, 1986); therefore predation on P. elegans could also occur both day and night. Another gonatid squid. Gonatus mado- kai, has also been found to prey on Parasagitta sp. (Kubodera and Okutani, 1977). There was no evidence of cannibalism, which com- monly occurs in many gonatids, particularly Berryteu- this magister and Gonatopsis borealis (Lapko, 1996; Nesis, 1997). Cannibalism in squids appears to occur less frequently when prey are abundant (Shchetinnikov, 1992; Santos and Haimovici, 1997), as is the case in the North Pacific during spring. In addition, at nearly every station sampled, squid of a small size range were collected (Bower et al., 2002); therefore it seems that op- portunities for intercohort cannibalism were limited. The large stock size of B. anonychus in the North Pacific (Nesis, 1997) and its importance in the diet of higher predators may indicate that the food chain from copepods through squids and these higher predators is an important trophic pathway in the pelagic food web of the Subarctic Pacific during spring. The large seasonal- ity in zooplankton composition in the upper 150 m may indicate that these trophic pathways will show similar seasonal variations. Acknowledgments We thank the late H. Richard Carlson for providing us with squids collected during the May 1999 NMFS salmon survey aboard the FV Great Pacific. We also thank Chingis Nigmatullin and the late Kir Nesis for translating two Russian abstracts into English, H. Sugi- 738 Fishery Bulletin 102(4) saki and M. Terazaki for helping identify prey, K. Ichige for helping in the laboratory, and the three anonymous reviewers of the manuscript. Literature cited Baker A. de C, B. P. Boden, and E. Brinton. 1990. A practical guide to the euphausiids of the world, 96 p. Natural History Museum, London. Bower, J. R., J. M. Murphy, and Y. Sato. 2002. Latitudinal gradients in body size and maturation of Berry tetuhis anonychus (Cephalopoda: Gonatidae) in the northeast Pacific. Veliger 45:309-315. Brodeur, R. D., and M. Terazaki. 1999. Springtime abundance of chaetognaths in the shelf region of the northern Gulf of Alaska, with observa- tions on the vertical distribution and feeding of Sagitta elegans. Fish. Oceanogr. 8:93-103. Brodeur, R., S. McKinnell, K. Nagasawa, W. Pearcy, V. Radchenko, and S. Takagi. 1999. Epipelagic nekton of the North Pacific subarctic and transition zones. Prog. Oceanogr. 43:365-397. Brodsky, K.A. 1950. Calanoida of the far eastern seas and Polar basin of the U.S.S.R. Dokl. Akad. Nauk. SSSR. 35:1-442. USSR Israel Program Scient. Transl. (1967), Jerusalem. Transl. No. TT-65-51200, 440 p. Carlson, H. R., J. M. Murphy, C. M. Kondzela, K. W. Myers, and T Nomura. 1999. Survey of salmon in the northeastern Pacific Ocean, May 1999. North Pacific Anadromous Fish Commission, Document 450, 37 p. Natl. Mar. Fish. Serv., Juneau, AK. Cortes, E. 1997. A critical review of methods of studying fish feeding based on analysis of stomach contents: application to elasmobranch fishes. Can. J. Fish. Aquat. Sci. 54:726- 738. Didenko, V. D. 1990. Prospects of fishery for the squid Berryteuthis anonychus in the north-eastern Pacific. In "Vth all- USSR conference on commercial invertebrates, Minsk- Naroch. Abstracts of Communications," p. 82-83. [In Russian. I VNIRO Publishing, Moscow. Russia. Hanlon, R. T. and J. B. Messenger. 1996. Cephalopod behaviour, 232 p. Cambridge Univ. Press, Cambridge, UK. Howmiller, R. P. 1972. Effects of preservatives on weights of some common macrobenthic invertebrates. Trans. Am. Fish. Soc. 101:743-746. Karlson, K., and U. Bamstedt. 1994. Planktivorous predation on copepods. 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Science Publishers, Lebanon, NH. 740 Abstract— The relative abundance of Bristol Bay red king crab (Para- lithodes camtschaticus) is estimated each year for stock assessment by using catch-per-swept-area data col- lected on the Alaska Fisheries Sci- ence Center's annual eastern Bering Sea bottom trawl survey. To estimate survey trawl capture efficiency for red king crab, an experiment was con- ducted with an auxiliary net (fitted with its own heavy chain-link foot- rope I that was attached beneath the trawl to capture crabs escaping under the survey trawl footrope. Capture probability was then estimated by fitting a model to the proportion of crabs captured and crab size data. For males, mean capture probability was 727 at 95 mm (carapace length), the size at which full vulnerability to the survey trawl is assigned in the current management model: 84. 1^ at 135 mm, the legal size for the fish- ery; and 939c at 184 mm, the maxi- mum size observed in this study. For females, mean capture probability was 707c at 90 mm, the size at which full vulnerability to the survey trawl is assigned in the current manage- ment model, and 777 at 162 mm, the maximum size observed in this study. The precision of our estimates for each sex decreased for juveniles under 60 mm and for the largest crab because of small sample sizes. In situ data collected from trawl- mounted video cameras were used to determine the importance of various factors associated with the capture of individual crabs. Capture probabil- ity was significantly higher when a crab was standing when struck by the footrope, rather than crouching, and higher when a crab was hit along its body axis, rather than from the side. Capture probability also increased as a function of increasing crab size but decreased with increasing footrope distance from the bottom and when artificial light was provided for the video camera. Capture probability of a survey trawl for red king crab (Paralithodes camtschaticus) Kenneth L. Weinberg Alaska Fisheries Science Center National Marine Fisheries Service, NOAA 7600 Sand Point Way N.E Seattle, Washington 98115 E-mail address ken Weinberg gnoaa gov Robert S. Otto Kodiak Fisheries Research Center National Marine Fisheries Service, NOAA 301 Research Court Kodiak, Alaska 99615 David A. Somerton Alaska Fisheries Science Center National Marine Fisheries Service, NOAA 7600 Sand Point Way N.E. Seattle, Washington 98115 Manuscript submitted 5 September 2003 to the Scientific Editor's Office. Manuscript approved for publication 28 April 2004 by the Scientific Editor. Fish. Bull. 102:740-749(2004) Regulations limit the annual har- vest of Bristol Bay red king crab (RKC; Paralithodes camtschaticus) to males >135 mm in carapace length1 (6.5 inches carapace width), and the size of the harvest is dependent upon the estimated biomasses of mature males and females. For stock assess- ments of RKC, area-swept abundance estimates are determined from the data from annual eastern Bering Sea (EBS) bottom trawl surveys conducted by the National Marine Fisheries Ser- vice, Alaska Fisheries Science Center (AFSC), and these estimates are used as input into a length-based assess- ment model (Zheng et al., 1995) to compute the total allowable catch for each annual fishing season. It is assumed with the current as- sessment model that all male RKC >95 mm and all female RKC >90 mm within the path of the survey trawl (wingtip to wingtip) are captured. This assumption seems reasonable because the survey trawl uses a small diameter footrope designed to stay close to the bottom and red king crab are quite large. However, video pho- tography taken following the 2000 EBS survey revealed that a consider- able number of large (>90 rami RKC pass under the footrope of the survey trawl. To assess the potential impact of escaping crab on the calculation of crab biomass, we conducted an ex- periment to estimate the size-related capture efficiency of the standard survey bottom trawl for Bristol Bay RKC. In this experiment, crab pass- ing beneath the survey trawl were subsequently captured with an aux- iliary net that was attached under- neath and behind the footrope of the survey trawl (Engas and Godo, 1989; Walsh, 1992). Experimental nets like the one used for this study have been used previously in trawl efficiency studies for flatfish (Munro and Somerton, 2002), as well as for snow iChionoecetes opilio) and Tan- ner (C. bairdi) crabs (Somerton and Otto, 1999). Trawl catch data alone, however, tell little about the details involved with escapement. Therefore, we deployed a video camera on the trawl to observe crab behavior and analyzed a combination of trawl-per- 1 All references to measured crab lengths are carapace length. Weinberg et al.: Capture probablity of a survey trawl for Parahthodes camtschaticus 741 trawl codend auxiliary codend trawl footrope wrapped wire chain hangings twine hangings fishing line auxiliary footrope Figure 1 The 83/112 Eastern bottom trawl with auxiliary net used in the 2002 red king crab capture efficiency experiment (figure adapted from Munro and Somerton, 2002, with permission from Elsevierl. formance and crab-behavioral variables to help us un- derstand the escapement process. Materials and methods Description of trawl gear The 83/112 Eastern bottom trawl has been used by the AFSC in annual surveys to assess EBS crab and shelf groundfish stocks since 1982 (Armistead and Nichol, 1993). For the present experiment, an auxiliary net with an independent footrope constructed of heavy chain-link and a separate codend were attached to the bottom of the survey trawl to capture epibenthic animals passing beneath the trawl footrope (Fig. 1). Briefly, the 83/112 Eastern is a low-rise trawl that has a 25.3-m long hea- drope strung with 48 floats giving it approximately 102 kg of lift and a 34.1 m long, 5.2-cm diameter footrope constructed of 1.6-cm stranded wire rope protected with a single wrap of polypropylene line and split rubber hose. The net is constructed with nylon twine: 10.1-cm stretch mesh throughout the wing and throat sections; 8.9-cm stretch mesh in the intermediate section; a double layer of 8.9-cm stretch mesh in the codend; and a 3.1-cm stretch mesh liner in the codend. It is fished with a pair of 1.8x2.7-m steel V-doors weighing approximately 816 kg apiece. The auxiliary net attaches to the wingtips and to the bottom of the survey trawl so that the bottom panel of the trawl serves as the top panel of the auxiliary net up to the beginning of the intermediate section. At this point, the two nets part and the auxiliary net then has a top panel of 8.9-cm stretch mesh and a double layer 742 Fishery Bulletin 102(4) 61 "N- 60 N- 59 N- 58 N- 57°N- 56N- 55N- 54N- 166 W 164 = W 162 W 160 W 158 W Figure 2 The annual eastern Bering Sea survey station grid showing the number of successful tows per station block made during the 2002 red king crab capture efficiency study. Each block represents a 400-nmi2 area. codend with a 3.1-cm stretch mesh liner. The 38.2 m long auxiliary footrope constructed of heavy 16-mm-long link trawl chain was designed to drag through soft bottom and presumably captures all escaping crabs. Munro and Somerton (2002) provided detailed construction plans of this experimental gear in their appendices. Experimental design Operations were conducted from 21 to 29 July 2002, aboard the FV Arcturus, one of two commercial stern trawlers chartered by the AFSC since 1993 to carry out annual Bering Sea groundfish surveys. Trawling took place in Bristol Bay (Fig. 2) at depths from 41 to 77 m and followed standardized survey protocols that included towing during daylight hours at a 1.5 m/sec (3 knots) vessel speed and using locked winches and stan- dardized lengths of trawl warp (scope) at each towing depth. Acoustic net mensuration equipment was used to measure wing spread for each tow. Bottom contact sensors were used on the centers of both the trawl and auxiliary footropes to measure the distance (in centime- ters) between the footropes and the bottom (Somerton and Weinberg. 2001). A silicon-intensified tube (SIT) camera, which uses ambient light, was attached to the center of the trawl to view RKC interaction with the footrope. On some of our trial tows, however, a 30-W quartz halogen light was also used to increase contrast between ambient light and the sea floor. Two departures from standardized survey protocol were necessary for this experiment. First. 27.5-m long bridles were used instead of the survey standard 55- m long bridles to help offset the loss of wing spread caused by the added drag of the auxiliary net (Munro and Somerton, 2002). Second, tow length was shortened from the survey standard of 30 min to 20 min to mini- mize the decrease of path width over time due to in- creased drag from large catches in the auxiliary net. Weinberg et al.: Capture probablity of a survey trawl for Paralithodes camtschaticus 743 Towing sites were selected according to catch rates and carapace lengths obtained from the recently com- pleted 2002 EBS survey (Stevens2). Tows were made in pairs, one in a northerly direction, the other in a south- erly direction and were offset to the east or west by a minimum of 0.1 nmi; the initial direction was chosen randomly in order to mitigate any bias that the current flow might have on footrope contact with the bottom (Weinberg, 2003). Increased effort was given to sites producing favorable numbers and crab lengths by add- ing additional towing pairs. For each tow the total catch of all species from each net was first weighed before all RKC were removed from the catch, weighed, coded by sex, and measured to the nearest millimeter. Data analysis Trawl geometry Trawl geometry for standard survey nets and experimental nets was measured to confirm that the two gear types fished similarly. Average wing spreads and footrope heights off-bottom for experimental tows were compared to those from 33 standard survey gear tows taken at the same or nearby sampling loca- tions. Because the depth of sampling varied, wing spread and footrope height were linearly regressed on scope, a factor variable indicating gear type (i.e., survey or experimental), and their interaction. Two-tailed r-tests were used to test for the difference in the slopes and the intercepts between gear types. Significance of the inter- action term indicated that slopes differed between gear types. For nonsignificant interaction, significance of the intercepts indicated that wing spread or footrope height differed between gear types by a constant amount. Capture probability Capture probability for the experi- mental gear was estimated from catch data of the trawl and the auxiliary net as a function of carapace length (L) for both male and female crab. Based on the assump- tion that the auxiliary net allows no escapement, the probability of capture at the footrope was modeled as a logistic function (Munro and Somerton, 2002) by using SPLUS software (version 6.1, Insightful Corporation, Seattle, WA). Two models were considered: the first, a two-parameter model which reaches an asymptotic maximum of 1 (unity): PlLh l+e-135 mm. As a general rule, crabs could be seen in the videos 1-2 seconds prior to contact with the footrope. Assignment of codes was typically straightforward. However, in some instances, several reviews of the encounter were neces- sary in order to determine a crab's position or orientation in relation to the footrope. The probability of capture was estimated by using stepwise generalized linear modeling (GLM; Venables and Ripley, 1994) to fit a logistic model describing the probability of capture as a function of crab size, where body height, body orientation, average footrope distance from the bottom during the tow, the use of artificial light, and all possible first order interactions were con- sidered as additional potential terms. The model fitting procedure (with data from crabs for which all variables were observed) entailed a stepwise backward model se- lection process. The process began with fitting the model to all interaction terms less one, and then calculating and comparing the resulting AIC values. The interaction producing the largest decrease in AIC was subsequently eliminated. Next, the procedure was repeated with the remaining terms until no interaction term could be eliminated without increasing the AIC. Then, the above process was repeated for the main effects. For the main effects having an interaction term, both the main effects and the interaction term were eliminated together as a unit. The final model chosen contained those terms that produced the minimum AIC value. Results Effect of the auxiliary net on trawl geometry Regressions of wing spread and footrope height on scope, gear type, and their interaction were compared to determine how closely the two gear types fished. The interaction term was not significant for wing spread (P=0.08) nor for footrope height (P=0.82), indicating that the slopes did not differ between gear types. However. tests of the intercepts were significant for both wing spread and footrope height and indicated that trawl geometry differed between survey and experimental trawls (Table 1). Predicted standard survey wing spreads for the minimum (137 m), median (229 m), and maxi- mum (320 m) scopes used were 16.6, 16.9. and 17.3 m — approximately 0.8 m more than the experimental gear at the same scopes. Predicted footrope distances off the bottom were 0.8, 1.6, and 2.4 cm, at the above three scope values — approximately 0.8 cm greater than the experimental gear. Although we detected statistical differences in the trawl geometry between the two gear types, the actual difference in physical measurements was small and presumably had only a nominal effect on the results of the capture efficiency experiment. Our assumption that the auxiliary net caught all escaping crabs was reinforced by two observations: 1) the data from the bottom contact sensor on the chain footrope indicated consistent contact with the sea floor; and 2) the auxiliary net consistently had large catches of benthic organisms other than crab, such as starfish and shells, and produced enough drag on the system to reduce wing spread. The effectiveness of the auxiliary footrope at capturing escaping crab is in part due to its weight and small diameter that enable it to sweep beneath the crabs and in part due to the suspension of benthic organisms initiated by the turbulence created by the passing of the first footrope. Length-based capture probability Capture probability was estimated from length mea- surements (n=3233) collected from 43 successful experimental tows (21 north, 22 south) made within 11 standard EBS survey station blocks (Fig. 2). Male samples (n = 1667) ranged in size from 23 to 184 mm (Fig. 3). Female samples (/? = 1566) ranged in size from 51 to 162 mm. The two-parameter model (model 1) of capture prob- ability was selected over the three-parameter model (model 2) because it had a lower AIC value for both male and female RKC (Table 2). For the comparison of Weinberg et al.: Capture probabhty of a survey trawl for Paralithodes camtschaticus 745 100 i Males 80 ■ P Captures 1 n n 60 ■ ■ Escapes ' n 40- 20 - 1 1 , 1 1 1 _nJl 1 ll 1 1 1 l 1 1 1 ■ L n n. 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0) 0) LL 150 n 125 Females □ Captures 100 ■ 75- ■ Escapes n 50- 1 25 - — ill 1 1, 1 1 J 1 1 . ~. 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Carapace length (5 mm bins) Figure 3 Size frequency of red king crabs taken in the survey trawl (captures! and the auxiliary let (escapes) during the 2002 capture efficiency study. Crab have been binned into 5-mm carapace length intervals. Table 2 Estimated parameter and Akaike's and the 3-parameter logistic models strapping 1000 replicates are provi Eastern survey bottom trawl. information criterion (AIC) values for the maximum for males, females, and for sexes combined. Parameter ied for the final model used to estimate red king crab likelihood fit of the 2-parameter variance estimates based on boot- :apture probability for the 83/112 Sex Model a P 7 AIC Var a Var/3 Covariance a/3 Male 1/(1 +e-(a + W) -0.7366 0.0178 — 1725.3 0.113 1.28xl0-5 -1.14xl0-3 //(l +e-(a + ft') -0.8826 0.0209 0.9622 1727.1 — — — Female l/(l + e-(a + ») 0.3540 0.0054 — 1875.7 0.279 2.58xl0-5 -2.63xl0-3 y/(l +e-lo + b]) 0.3540 0.0054 0.9999 1877.7 — — — Combined sexes 1/(1 +e-(« + W) -0.4569 0.0143 — 3612.7 — — — selectivity curves for males and females with model 1, we found the summed AIC for separate curves to be lower than the AIC for sexes combined. Consequently, separate selectivity curves were estimated for males and females (Table 2). The fitted model predicted male capture probabil- ity to be 41.9% at 23 mm (size of the smallest male observed); 72.2% at 95 mm (size at which full vulner- ability to the survey trawl is assigned in the current management model); 80.2% at 120 mm (size assigned 746 Fishery Bulletin 102(4) in the current management model for male maturity); 84.1% at 135 mm (legal size for males); and 92.7% at 184 mm (size of the largest male crab encountered in 1.0- o Males o 0.8- ° ■■c^o 0.6- o 0.4- 0.2- O 50 100 150 Carapace length (mm) Figure 4 A 2-parameter logistic model (solid line) and 959S confi- dence bounds idashed lines) estimating male red king crab capture probability for the 83/112 Eastern survey bottom trawl. Symbols are scaled to the sample length frequency summed over all tows and binned into 5-mm carapace length intervals. Symbols range in size from the smallest circle representing a single individual to the largest circle repre- senting 138 males. 1 0n 0.8- o o. 0.6 0 0.4 0.2 o -~9 Females 0^ o ■ 06 o o 0 0 50 100 150 Carapace length (mm) Figure 5 A 2-parameter logistic model isolid line) and 959! confi- dence bounds idashed linesl estimating female red king crab capture probability for the 83/112 Eastern survey bottom trawl. Symbols are scaled to the sample length frequency summed over all tows and binned into 5-mm carapace length intervals. Symbols range in size from the smallest circle representing two individuals to the largest circle represent- ing 206 females. our experiment [Fig. 4]). The fitted model predicted female capture probability to be 65.27c at 51 mm (size of the smallest female observed); 69.8% at 90 mm (size at which both full vulnerability to the survey trawl and 50% female maturity are assigned in the cur- rent management model); 74.7% at 135 mm (same size at which males enter the fishery); and 77.4% at 162 mm (size of the largest female crab encoun- tered in our experiment [Fig. 5]). Estimated capture probability for both male and female crab was equal at 88 mm (69.9%). Model variability, as indicated by the 95% confidence bounds, was greatest at the extremes of our size ranges because of low sample frequency. This was especially true for small crabs, and the uncertainty was so large that extrapolation of the capture probability functions to either males or females below <60 mm is not recommended. Factors influencing escapement Modeling the effect of various factors on capture probability was based on observations of RKC (ra=248) from videotapes collected during 28 EBS tows. Approximately two-thirds of the counted crabs were captured. The influence of artificial lighting, body height, footrope distance from the bottom, crab size, body orientation, and the interaction of body height and body orientation were significant (Table 3). Capture probability decreased when lights were used and when the distance between the foot- rope and the bottom increased. Capture probability increased when crabs were standing up on their legs, with increased body size, and when the footrope contact was made along the body axis rather than from the side of the crab. Capture probability, based on direct observation, was predicted by the fitted logistic models to illus- trate how the various explanatory variables affect the capture outcome. We present two examples. In the first case, capture probability in natural light Table 3 Model coefficients for predicting red king crab cap- ture probability from counts obtained with a trawl- mounted video camera. Standard Value error Intercept -2.014 0.676 Light variable -0.959 0.526 Body height variable 3.789 0.624 Body orientation variable 0.207 0.613 Crab size variable 1.506 0.315 Footrope height variable -0.286 0.197 Body height and orientation -1.436 0.785 interaction Weinberg et al.: Capture probablity of a survey trawl for Paralnhodes camtschat/cus 747 conditions and when crab are oriented sideways to the oncoming footrope, was predicted as a func- tion of footrope distance off the bottom for each size class, and for both standing and crouching crab (Fig. 6). For all size groups, capture prob- ability decreased with increasing footrope height from the bottom. The importance of whether a crab was standing or crouching diminishes with decreasing crab size because the footrope is more likely to pass completely over smaller crab. In contrast, the importance of standing was higher for large crab because they were more likely to be undercut by the footrope and captured, whereas crouching crab were more susceptible to hav- ing their legs first pinned down by the footrope, which exerted a downward pressure on their carapace and allowed the footrope to pass over the crab. Capture probability of medium-size in- dividuals, which included a large proportion of egg-bearing females, was more dependent upon the body height of the crab. Footrope contact be- low the carapace typically resulted in capture; however contact above the legs often forced the crab's carapace down, causing the crab to roll forward and pass beneath the footrope. In the second case, capture probability was predicted for natural light conditions when the footrope is 1 cm off-bottom, as a function of crab size, by body orientation to the footrope, and for standing and crouching individuals (Fig. 7). Under these conditions, capture probability was greater for crab contacted along their body ax- is than for crab hit from the side. In addition, capture probability increased with crab size for both standing and crouching crab, regardless of whether the footrope first contacted the crab along their body axis or from the side. When the footrope was 1 cm off bottom, the difference in the body-orientation effect on capture probability for standing crab was greater for smaller crab than for medium and larger individuals, but rela- tively equal for crouching crab of all sizes. 1 0- Standing 08- ^~~~ . _^^ 06- Medium ~~~ — -- ________^^ 04- *■"-— _ 0.2- ^r— _____^^ 5 00- O Q- i i i i i 12 3 4 5 CD 3 CO O Crouching 0.8- Large""" — ______^ 0.6- ^^— -^__^ 0.4- ___^ ^ Medium""^" — . 02- ~~~~- — _ 0.0- Small ■ i i i i 12 3 4 5 Footrope height (cm) Figure 6 Estimated red king crab capture probability (based on direct observations! by size class as a function of footrope height above the bottom for standing and crouching individuals. For this model, it was assumed that no artificial light was used for the camera and that crab were oriented sideways to the footrope upon initial contact. Discussion Our observations confirmed that adult Bristol Bay red king crab can escape beneath the footrope of the AFSC's 83/112 Eastern survey bottom trawl under normal towing conditions. Capture probability increased with size but did not reach 100% for the largest crab caught. For the current management model used for RKC stock assessments, 100% capture probability is assumed for adult crabs and should be revised. A recruitment ogive is used in the calculation of the total spawning bio- mass for defining overfishing under the Magnuson- Stevens Fishery Conservation and Management Act (Stevens2). Revised computations of vulnerability will be required for this purpose as well. Survey trawl selectiv- ity, although similar between the two sexes at prerecruit sizes, was generally 15% higher for legal-size males than for equal-size females. This between-sex difference in capture probability may be explained by behavioral differences (for instance, egg-bearing females stand dif- ferently from large males). Unfortunately, crab, when viewed from above, mask their gender; thus sex was excluded from our modeling exercise of video data. Survey catch statistics for RKC are routinely included in the management modeling procedure to estimate the abundance of legal-size males (>135 mm), male prere- cruits (95-134 mm), the effective spawning biomass of males (>120 mm), and the spawning biomass of females (>90 mm, as determined from size at 50% maturity). We estimated capture probability for legal-size males (up to 184 mm) to range from 84% to 93%, for prerecruit males from 73% to 84%, and for the mature portion of the male spawning population (up to 184 mm) from 748 Fishery Bulletin 102(4) 80% to 93% Our estimated capture probability for the survey trawl on the female portion of the spawning RKC population ranged from 70% to 77% for crab up to 162 mm. A review of the AFSC database for EBS crab surveys showed that the largest male and female crabs taken were 200 mm and 172 mm. Corresponding capture probabilities estimated by the model for these size crabs were 94% and 78%, respectively. Two main factors affect the overall capture efficiency of epibenthic species by a bottom trawl: 1) horizontal herding, defined as movement into the path of the trawl between the wingtips in response to stimuli produced by the doors or bridles; and 2) escapement, defined as the avoidance of capture once the crab is within the path of the trawl. We believe herding is negligible because our observations of crab movement, which were consistent with those reported by Rose (1999), indicated that RKCs are slow-moving animals that can travel only slight dis- tances before being overtaken by a trawl approaching 1.0- Standing ______ 0.8- ^ ^^^^ 0.6- j^^^^^ 04- 0.2- Capture probability o o ■ i i small medium large Crouching 0.8- 0.6- /^^ 0.4- ^^^^ 0.2- ^ ad"** 0.0- I" 1 1 small medium large Crab size Figure 7 Estimated red king crab capture probability (based on direct observations) by body orientation at the time of footrope contact as a function of crab size for standing and crouching individuals. For this model, it was assumed that, no artificial light was used for the camera and that the footrope was 1 cm off bottom. at 1.5 m/sec. Our video observations of the trawl bridle revealed that RKCs consistently passed over the top of the bare cable, with one exception — where a few crabs were seen sliding along the bridle, legs entangled, to the wingtip before being cast outside the path of the trawl. Escapement is likely restricted to footrope escapement because mesh escapement is impeded by the spiny sur- face and long legs of the crab and could only occur for the smallest individuals, which we encountered in low numbers and which could not be predicted reliably by our model. We recognize from the analysis of our in situ data that capture probability is influenced not only by trawl performance but also by crab behavior. For instance, crabs standing upright, such as moving or migrating individuals, are more susceptible to capture than those with their bodies resting on the substrate. Crab density could also affect capture probability as seen for some species offish (Godo et al., 1999). The crabs we observed with our video cameras were fairly dispersed and the maximum number of crabs seen in any single video frame was two (twice observed). Crabs in relatively low abundance are likely to react di- rectly to the gear, but in areas of high abun- dance, crabs may react to each other in response to the stimuli from the approaching gear, causing them to crouch or conversely move away from perceived danger. Both of these responses would result in a different capture probability. Our estimates of capture probability apply to the conditions in which the EBS survey is conducted; that is, relatively disperse offshore populations encountered during daylight hours on sandy bottom during the summer months. There are other behavioral factors or environmental conditions that we did not consider in the present study but which could affect the efficiency of the survey trawl. These include, but are not limited to, the following: trawling where the substrate is substantially different; crabs that are either ag- gregated into pods or are buried (Dew3); and tem- peratures or tidal currents that would affect the migratory or feeding behavior, and therefore the body height of crab (Dew, 1990). Our estimates of capture probability are also based on the as- sumption that the auxiliary net is 100% efficient at capturing crab escaping beneath the footrope of the survey trawl. We have no direct evidence to believe otherwise. However, if crabs also escaped the auxiliary net, then our estimates of capture probability would be too large. In conclusion, we wish to clarify to users of our findings that, although these experimen- tally determined selectivity models indicate an upward correction in spawning biomass of red king crab may be in order, we find no reason 3 Dew. C. B. 2003. Personal commun. Alaska Fisheries Science Center, 7600 Sand Point Wav NE, Seattle, WA 98115. Weinberg et al.: Capture probablity of a survey trawl for Para/ithodes camtschaticus 749 to claim that the stock is in any better condition than the condition that was determined by the most recent assessment. The foremost utility of the AFSC annual EBS surveys is to monitor distribution and abundance trends through time. The survey accomplishes this by maintaining strict protocols and consistency in trawling methods, in computation of area-swept abundance, and in nonenvironmentally affected trawl efficiency. The survey times series is designed to detect changes in abundance, signaling advances in the population's re- building processes, regardless of whether crab are 100% or 80% vulnerable to the survey trawl. We advocate that careful consideration be given to the other factors that drive the management model, along with the results of our capture efficiency experiment, to ensure that the stock rebuilding process remains uninterrupted. Acknowledgments We are thankful to Captain Glenn Sullivan and the crew of the FV Arcturus for their professional attitudes and relentless attention to detail; to scientists Chris John- ston, Frank Shaw, and Kerim Aydin for their assistance at sea following the 2002 survey; to Craig Rose and Scott McEntire for technical support; to Dave King and Jim Smart for preparation of the experimental trawl gear; and to Gary Walters, Braxton Dew, Doug Pengilly, Jie Zheng, and our anonymous reviewers for their helpful comments during the manuscript review process. Literature cited Armistead, C. E., and D. G. Nichol. 1993. 1990 bottom trawl survey of the eastern Bering Sea continental shelf. NOAA Tech. Memo. NMFS- AFSC-7, 190 p. Burnham, K. P.. and D. R. Anderson. 1998. Model selection and inference: a practical infor- mation-theoretic approach, .353 p. Springer-Verlag, New York, NY. Dew, C, B. 1990. Behavioral ecology of podding red king crab, Paralithodes eamtsehatica. Can. J. Fish. Aquat. Sci. 47:1944-1958. Efron, B., and R. Tibshirani. 1993. An introduction to the bootstrap, 436 p. Chapman and Hall, New York, NY. Engas, A., and O. R. Godo. 1989. Escape offish under the fishing line of a Norwegian sampling trawl and its influence on survey results. J. Cons. Int. Explor. Mer 45:269-276. God0, O. R.. S. J. Walsh, and A. Engas. 1999. Investigating density-dependent catchability in bottom trawl surveys. ICES J. Mar. Sci. 56:292- 298. Millar, R. B. 1992. Estimating the size-selectivity of fishing gear by conditioning on the total catch. J. Am. Stat. Assoc. 87:962-968. Munro, R T., and D. A. Somerton. 2001. Maximum likelihood and non-parametric methods for estimating trawl footrope selectivity. ICES J. Mar. Sci. 58:220-229. 2002. Estimating net efficiency of a survey trawl for flat- fishes. Fish. Res. 55:267-279. Rose. C. R. 1999. Injury rates of red king crab, Paralithodes camts- chaticus, passing under bottom-trawl footropes. Mar. Fish. Rev. 61(21:72-76. Somerton, D. A., and R. S. Otto. 1999. Net efficiency of a survey trawl for snow crab, Chionoecetes opilio, and Tanner crab, C. bairdi. Fish. Bull. 97:617-625. Somerton, D. A., and K. L. Weinberg. 2001. The affect of speed through the water on footrope contact of a survey trawl. Fish. Res. 53:17-24. Venables, W. N, and B. D. Ripley. 1994. Modern applied statistics with S-plus, 452 p. Springer-Verlag, New York, NY. Walsh, S. J. 1992. Size-dependent selection at the footgear of a ground- fish survey trawl. N. Am. J. Fish. Manag. 12:625- 633. Weinberg, K. L. 2003. Change in the performance of a Bering Sea survey trawl due to varied trawl speed. Alaska Fish. Res. Bull. 10(11:42-49. Zheng, J., M. C. Murphy, and G. H. Kruse. 1995. A length-based population model and stock-recruit- ment relationships for red king crab, Paralithodes camts- chaticus, in Bristol Bay. Alaska. Can. J. Fish. Aquat. Sci. 52 (61:1229-1246. 750 Evidence of shark predation and scavenging on fishes equipped with pop-up satellite archival tags David W. Kerstetter School of Marine Science Virginia Institute of Marine Science College of William and Mary Gloucester Point, Virginia 23062 E-mail address: bailey@vims.edu Jeffery J. Polovina Pacific Islands Fisheries Science Center National Marine Fisheries Service Honolulu, Hawaii 96822 John E. Graves School of Marine Science Virginia Institute of Marine Science College of William and Mary Gloucester Point, Virginia 23062 Over the past few years, pop-up sat- ellite archival tags (PSATs) have been used to investigate the behav- ior, movements, thermal biology, and postrelease mortality of a wide range of large, highly migratory spe- cies including bluefin tuna (Block et al., 2001), swordfish (Sedberry and Loefer, 2001), blue marlin (Graves et al., 2002), striped marlin (Domeier and Dewar, 2003), and white sharks (Boustany et al., 2002). PSAT tag technology has improved rapidly, and current tag models are capable of collecting, processing, and stor- ing large amounts of information on light level, temperature, and pressure (depth) for a predetermined length of time before the release of these tags from animals. After release, the tags float to the surface, and transmit the stored data to passing satellites of the Argos system. A problem noted by several au- thors using early PSAT models was the occasional occurrence of tags that did not transmit data. Clearly, a tag attached to a moribund fish that would sink to a depth exceeding the pressure limit of the tag casing would be destroyed. To prevent the loss of tags due to mortality events, tag manufacturers and researchers have developed mechanisms that re- lease tags from dead or dying fish before the structural integrity of the tag is compromised at depth. These mechanisms include both mechani- cal devices that sever the monofila- ment tether that attaches the tag to the fish upon reaching a given depth and internal software subroutines that activate the normal electronic release mechanism if the tag either reaches a certain depth or maintains a constant depth for a predetermined length of time. Despite the addition of these re- lease mechanisms to PSATs, some tags still fail to transmit data. Such failure could result from any of the following events or conditions: me- chanical failure of a critical tag com- ponent; destruction by fishing crews unaware of or not participating in the present research; excessive epi- faunal growth that makes the tag negatively buoyant or prevents the tag from floating with the antenna in a vertical position; or fouling of the tag on the fish, fishing gear, or flotsam. Another cause of failure is that the tags could be lost as a re- sult of ingestion. For example, a free- swimming white marlin (Tetrapturus albidus) was observed mouthing and almost swallowing a free-floating PSAT off the Dominican Republic in May 2002 (Graves, personal observ.). Alternately, the tag could be ingested incidentally with part of the tagged fish, as described by Jolley and Irby (1979) who reported that an acoustic tag on a sailfish (Istiophorus platyp- terus) was eaten along with the fish by an undetermined species of shark. In this note, we present data from PSATs deployed on two white marlin in the western North Atlantic Ocean and on an opah (Lampris guttatus) in the central Pacific; the data from these tags indicate that the tags were consumed by sharks. Materials and methods White marlin 1 (WM1) At approximately 10:00 am local time on 1 September 2002, a white marlin was observed on pelagic longline gear set during the night near the southeastern edge of Georges Bank. The fish, which had been caught on a slightly offset, straight-shank J-style hook (size 9/0), was manu- ally guided with the leader along- side the vessel. A PTT-100 HR model PSAT (Microwave Telemetry, Inc., Columbia, MD) was attached to the dorsal musculature approximately 5 cm below the base of the dorsal fin with a large nylon anchor according to the procedure and tether design described in Kerstetter et al. (20031. The tag was activated shortly after the white marlin was first identified, although approximately one hour is required following activation for this tag model to begin collecting data. The tag was programmed to record point measurements of temperature, light, and pressure (depth I in four- minute time intervals and to detach from the animal after 10 days. After release from the fish, the positively buoyant tag was expected to float to the surface and transmit stored and real-time data. For both white marlin Manuscript submitted 27 April 2003 to the Scientific Editor's Office. Manuscript approved for publication 7 June 2004 by the Scientific Editor. Fish. Bull. 102:750-756120041. NOTE Kerstetter et al.: Shark predation and scavenging on fishes equipped with satellite archival tags 751 Table 1 Comparison of depths and temperatures recorded by three pop-up sa tellite archiva tags (PSATs) before and after the tags were ingested by an organism. Befo •e ingestion Afte r ingestion Depth Depth Temp. Temp Depth Depth Temp Temp range mean range mean range mean range mean Animal (m) (SD) CO (SD) ;; (m) (SD) CO (SD) n WM1 145.2 145.2 (±0.00) 11.6-11 11.7 (±0.07l 179 0-564.9 130.0 (±237.50) 12.1-26.5 24.1 (±0.84) 2755 WM2 0-26.9 5.9 (±4.44) 19.8-27.8 24.7 (±0.91) 207 0-699.3 131.0 (±162.61) 18.9-29.5 27.3 (±1.20) 1683 Opah 32-456 221.81 (±92.20) 8-25.6 16.68 (±4.21) 360 0-524 170.56 (±133.83) 26.2-30.6 28.64 (±0.67) 168 tags, minimum straight-line distances were calculated between the point of release and the first clearly trans- mitted location of the tag following its release (pop-off) (Argos location codes 0-3). At the time of tagging, the longline hook used to cap- ture the fish was not visible in the mouth of the white marlin. The leader was therefore cut as close as possible to the fish before the fish was released, following the standard operating procedure for the domestic pelagic longline fleet. The fish was maintained alongside the vessel for less than three minutes for the application of the PSAT and a conventional streamer tag. Although the white marlin was initially active at the side of the vessel, some light bleeding from the gills was noted. After re- lease, the fish swam away slowly under its own power. the dorsal musculature with a Wildlife Computers tita- nium anchor. The tag was programmed to record the temperature and depth occupied by the fish in binned histograms, and the minimum and maximum tempera- tures and depths for 12-hour time periods. However, these 12-hour bins encompassed both day and night periods. The tag was programmed to be released six months after deployment. In the event of a premature release, the tag was programmed to begin transmitting stored data if it remained at the surface for longer than three days. The opah was lively and quickly dived after it was released. Results White marlin 2 (WM2) At 9:05 am on 2 August 2003, a white marlin was observed on pelagic longline gear with the same configu- ration in the same approximate area of Georges Bank as WM1. The fish was caught by a circle hook (size 16/0) in the right corner of the mouth, and although the stomach was everted, the fish appeared to be in excellent physi- cal condition. A PTT-100 HR tag had been activated at 6:30 am that morning, and was therefore collecting data at the time of tagging. After the fish was brought to the side of the vessel, both the PSAT and a conventional streamer tag were attached to this fish in less than three minutes by using the same protocol as that described for WM1, and the fish swam strongly away from the vessel after release without any evident bleeding. Opah At 5:52 pm local time on 21 November 2002, a female opah was observed on pelagic longline gear set during the day east of the Island of Hawaii. The fish was brought to the side of the fishing vessel and a Wildlife Computers (Redmond. WA) PAT2 model tag was attached through WM1 Release of the PSAT was expected to occur on 10 Sep- tember 2002 and the tag was expected to begin transmit- ting data on that date, but the first transmission was not received until almost two days later. At the time of first transmission, the PSAT was 81.3 km (43.9 nmi) west-southwest of the tagging location. A total of 81.5% of the archived light level, temperature, and pressure (depth) data was recovered. The light level, temperature, and pressure (depth) readings over time are presented in Fig. 1 (A-C) and summarized in Table 1. The first light level measure- ments indicated that the fish was already in relatively dark waters within one hour following its release. Light levels continued to drop to almost zero during the next ten hours and remained at that level for the next nine days (Fig. 1A). During the next seven-day surface trans- mission period, the tag recorded real-time day and night differences in light levels, which indicated that the light sensor was functioning properly. Sea surface temperatures in the area where the gear was set and hauled back, varied from 25.2° to 26.7°C (D. Kerstetter. unpubl. data) and the first temperature 752 Fishery Bulletin 102(4) Q. CD D A WM1 flu 1 200 • i i"f f 400 • 'A i 1 !l i: 600 • i 9/1/02 9/4/02 9/7/02 9/10/02 9/13/02 9/16/02 9/19/02 B 9/1/02 9/4/02 9/7/02 9/10/02 9/13/02 9/16/02 9/19/02 c 1.0' ™ "" "T r np 08. 06- 04. 02. 1 ~ 9/1/02 9/4/02 9/7/02 9/10/02 9/13/02 9/16/02 9/19/02 WM2 8/3/03 8/6'03 8/9/03 8/12/03 8/15/03 8/18/03 8/21/03 8/24/03 E 32 28- 24- 20- 8/3/03 8/6/03 8/9/03 8/12/03 8/15/03 8/18/03 8/21/03 8/24/03 8/3/03 8/6/03 8/9/03 8 12/03 8/15/03 8/18/03 8/21/03 8 24,03 Figure 1 Graphs of data on and depth (A and Di. temperature (B and E), and light index iC and Fl for tags WM1 and WM2. Lighter lines and points are prior to programmed release date, whereas darker lines and points are "real-time" surface condition measurements transmitted by the tag in addition to the archived data. recording by the PSAT (one hour after activation) was 11°C (Fig. IB). The temperature remained fairly con- stant at 11°C for a period of approximately ten hours after which there was a rapid rise to 25°C. The temper- ature of the PSAT remained between 22.5° and 26. 5C for the next nine days (until the programmed release date), with the exception of one brief decrease to 20 C on 8 September. When the tag began transmitting on 12 September, the real-time surface temperature was 23.6°C. The pressure data (Fig. IC) indicated that the tag was at a depth of approximately 145 m at one hour following release. The PSAT remained at this depth for a little more than ten hours after which the data suggested that there was a rapid rise to the surface. For the next nine days, the tag reported considerable vertical move- ment between the surface and depths to 565 m. The tag was at the surface when it began transmitting both archived and real-time data on 12 September. WM2 The tag reported data as expected on 13 August 2003 and transmitted 57.3rr of the archived data. At the time of first transmission, the PSAT was 600.1 km (324.0 nmi) east-southeast of the tagging location. Summary depth and temperature data recorded by the PSAT are included in Table 1. From the depth and temperature data, it appears that the fish survived for approximately 24 hours af- ter release, at which point the light readings dropped to zero (see Fig, ID) and remained at that level for NOTE Kerstetter et al.: Shark predation and scavenging on fishes equipped with satellite archival tags 753 the next eight days. The depth record following this change in light level was marked by several discrete diving events, and depths (see Fig. IF) ranged between the surface and over 699 m. Recorded temperatures for this period varied between 18.9° and 29.5°C, although sea surface temperatures in the area where gear was set and hauled back varied from 20.9° to 26.0°C (Ker- stetter, unpubl. data). On 12 August, the light level returned to its maximum value and the tag remained at the surface for approximately one day until its scheduled release date (13 August) when it began transmitting data. Opah The PAT2 satellite tag was expected to pop-up 6 months after deployment, but the first trans- mission was received after only 34 days from a location about 330 km (178 nmi) northwest of the deployment site. All the archived binned light level, temperature, and pressure (depth) data from this period were recovered (see Table 1). This tag model collected eight temperature and depth samples during each 12-hour period, result- ing in 16 values per day or 528 total values for the deployment period. The two 12-hour blocks were removed from all analyses to more accu- rately represent the differences in data between specimens: 1) the 12-hour block after tagging in order to allow for the recovery of the animal, and 2) the 12-hour block during which the predation event putatively occurred in order to clarify the potentially distant depth and temperature char- acteristics of the ingesting animal. The measured sea surface temperature during the tagging of the opah was 25.9°C. The ranges of dive depths, temperature, and light based on minimum and maximum values over the 12-hour day and night periods showed two distinct pat- terns (Fig. 2). During the first period (23 days), the dive depths ranged from about 32 to 456 m (Fig. 2A). Water temperatures encountered by the tag during this period ranged from 8.0° to 25.6°C (Fig. 2B) and the light index values ranged from about 50 to 150 (Fig. 2C). During the second period (11 days), the dive depths ranged from 0 to 524 m, temperature ranged from 26.2° to 30.6°C (higher than the 24.2-24.8°C SST recorded by the tag after it was released from the fish), and the light index recorded persistently low values. Discussion WM1 Our interpretation of these data is that the PSAT on WM1 was ingested by an animal scavenging the marlin carcass. The first PSAT readings for WM1, Dive minimums and maximums 0 100 E 200 Q 300 400 500 ' 11/24/02 11/30/02 12/6/02 12/12/02 12/18/02 12/24/02 Dive minimums and maximums 28 '.•■' Ii'' I'-'-'l'l O 24- o Q. E |2 20 - 16 - 12 - R O i ' i ' 1 ' l ' I ' i 11/24/02 11/30/02 12/6/02 12/12/02 12/18/02 12/24/02 Light minimums and maximums from location data 250 200- en 0 =J CO > 150 100 _i 50- ' I I 0 , | ..,11... I I I ' 1 ' I 11/24/02 11/30/02 12/6/02 12/12/02 12/18/02 12/24/02 Date Figure 2 G raphs of depth (A), temperature (B), and light index (C) for the opah PAT tag from deployment until transmission. recorded about one hour after its release, indicated that the marlin was already dead or moribund by that time and was descending to the ocean floor. For the next ten hours, the tag and carcass remained at a constant depth of 145 m (the depth of the nearest sounding at the site of release, according to NOAA depth chart 13003 [1998], was approximately 160 m) and at a temperature of 11°C. The light level steadily decreased at approximately 4:30 pm, corresponding to changes in ambient light from the setting of the sun. At approximately 9:00 pm local time, there was a dra- matic change in conditions when temperature rapidly rose to near 26°C and depths began to vary between the surface and 600 m. 754 Fishery Bulletin 102(4) We cannot attribute these chang- es to a resuscitation of the fish for three reasons. 1) The measured light levels indicated that the tag was in complete darkness for a period often days, even though it was at the sur- face during daylight hours. A mal- functioning light sensor cannot ex- plain this observation because the tag recorded day and night differences in light levels at the surface during the seven-day transmission period af- ter it was released from the fish. 2) After a rapid increase, the tempera- ture remained relatively constant, between 23° and 26°C, even when the tag was at depths in excess of 300 m. Although dive behavior may be affected by location-specific con- ditions, previous PSAT observations of more than 20 other white marlin indicated that temperature ranges of individual dive events rarely exceed 8°C when, it is assumed, animals make foraging dives to depth (Horo- dysky et al., in press). 3) The PSAT recorded several dives in excess of 400 m, and previous observations of white marlin have revealed no dives in excess of 220 m (Horodysky et al., in press). Finally, the PSAT was scheduled to be released from WM1 after ten days on 10 September. Although archiving of light, temperature, and pressure data ceased on that date, the tag did not begin transmitting until 12 September. WM2 The shallow dive patterns reported by this fish may indicate that it survived for approximately 24 hours following its release. Between 12:45 and 3:07 pm (local time), the light level fell abruptly from the maximum light level value to zero. At 3:08 pm, the temperature was 19.8°C at 166 m depth; by 4:37 pm, the tempera- ture was above 24°C and remained above this value for the remainder of the deployment period. At 5:58 pm on 12 September, the light levels returned to maximum strength from zero — an indication that the tag had likely been egested. For the 19 hours remaining of the programmed deployment period prior to pop-off, the depth, light, and temperature data all indicated that the tag was floating at the surface. Opah Based on recovered data, our conjecture is that the tag was attached to the live opah for the first 23 days. Then, sometime during the 12-hour period from 2:00 pm 13 December to 2:00 am 14 December the tag was ingested. From our data, we cannot discern whether 1) the tag was detached prematurely from the opah and was float- ing on the surface when it was ingested, 2) an animal D CD ■o Temperature Depth Figure 3 Delayed temperature changes recorded by tag WM1 following deep dive events on the morning of 2 September 2002. Arrows indicate the lowest temperatures recorded in association with a movement of the animal to depth; note that these temperatures were often recorded while the animal was at or near the surface and therefore represent a delay between depth and temperature. attacked the opah and ingested the tag incidentally, or 3) an animal ingested the tag alone. However, it is unlikely that the opah died, sank to the ocean floor, and was scavenged because the ocean floor in the area where the opah was tagged is below 2000 m. We have observed from other tags on opahs what we believe are mortalities; these occur shortly after tagging and show that the tag reaches depths in excess of 1000 m before detaching when the emergency pressure release in the tag is triggered. We did not observe depths below 600 m at any time during this record, and therefore the pressure-induced detach- ment mechanism on the tag was not triggered. The ingestion hypothesis for the failure of these three tags to transmit data is supported by several lines of evidence. First, the light level readings were consistent with a tag residing in the complete darkness of an alimentary canal. Second, although temperature varia- tions occurred during the deployment period, the delay in temperature changes during dives to depths indicates that the tags were not directly exposed to ambient wa- ter (see Fig. 3 for an example from WM1, as well as the comparisons in Table 1) and further may indicate that the scavenger was either endothermic or of large enough size to mitigate heat loss at depth. There are several organisms that could have eaten these PSATs, whether by scavenging a carcass or at- tacking a moving fish. Clearly, each of these organisms was sufficiently large to ingest the tag without seri- ously damaging it. It is unlikely that a cetacean was responsible for any of these events because internal temperatures for odontocete whales (including killer whales, Orcinus orca) range between approximately 36° and 38°C (Whittow et al., 1974)— well above the range of temperatures recorded by the PSATs. NOTE Kerstetter et al.: Shark predation and scavenging on fishes equipped with satellite archival tags 755 The only other natural predators of large pelagic fishes are various species of sharks. Several species of lamnid sharks maintain elevated body temperatures, including the shortfin mako {Isurus oxyrinchus) and the white shark (Carcharodon carchariasl, both of which are found in the area of Georges Bank (Cramer, 2000) and the Central Pacific (Compagno, 1984). Several shortfin makos were caught by the same longline vessel during the week following each white marlin PSAT deployment (WM1: n=4, 95-189 cm FL; WM2: n=3, 94-199 cm FL) (Kerstetter, unpubl. data). The opah tag record closely resembles the relatively constant temperature noted for lamnid sharks, despite the independence of stomach temperature with ambient water for these endothermic sharks as reported by Carey et al. (1981). It is also interesting to note that although precipitous tempera- ture fluctuations were generally absent, a rapid drop in temperature from 24° to 20°C was observed with tag WM1 on 8 September at 32.3 m depth— a fluctuation that could have resulted from another feeding event that brought cool food matter into the stomach. Simi- lar reductions in stomach temperatures due to feeding have been noted for white sharks (McCosker, 1987). The range of temperatures recorded by each of the two white marlin tags appears rather broad for an endothermic shark, however, and although the temperature at depth was not measured, the delay in stomach temperature closely resembles the pattern of blue shark internal temperatures {Prionace glauca) measured in the Mid- Atlantic (Carey and Scharold, 1990). The diving behavior recorded by the three tags also corroborates ingestion of the tags by sharks. Carey et al. (1982) reported that a tagged white shark off Long Is- land, New York, made frequent dives to the bottom dur- ing a 3.5-day acoustic tracking period. White sharks are known to dive to depth while scavenging whale carcasses (Dudley et al., 2000; Carey et al., 1982). A juvenile white shark also tracked by Klimley et al. (2002) spent far more extended times at depth than either white marlin tag. Although the programming of the tag on the opah precludes such fine-scale analyses of diving behavior, the available data are not inconsistent with the mako tracks in the study of Klimley et al. (2002). However, the short duration dives with frequent returns to the surface seen with the two white marlin tags most closely resemble those of blue sharks (Carey and Scharold, 1990) and were notably missing from the tracks of three shortfin makos observed by Klimley et al. (2002). If sharks were indeed the scavenging animals, it is likely that the tags were regurgitated, rather than egested through the alimentary canal, whereupon the PSAT floated to the surface and was able to transmit the archived data. The narrow diameter of the spiral valve in the elasmobranch gastrointestinal tract would likely be too narrow to allow the undamaged passage of an object the size of a PSAT, even for a large shark. Although the available literature describing regurgita- tion abilities of pelagic sharks is rather limited, Hazin et al. (1994) reported that 35% of blue sharks brought aboard for scientific study had everted and protruding stomachs. Economakis and Lobel (1998) also stated their belief that regurgitation of ingested ultrasonic tags was the primary cause of lost tracks for grey reef sharks iCarcharhinus amblyrhynchos) on Johnston Atoll in the central Pacific Ocean. Conclusions The temperatures and dive depths recorded by the opah tag and both white marlin tags after apparent ingestion share similarities, yet also contain sufficient information to indicate the different identities of the ingesting organ- isms. The dive depths in all cases ranged from the surface to over 500 m, whereas the temperatures remained rela- tively constant at several degrees above the background SST, even during deep dive events. Temperature ranges alone strongly indicate sharks rather than odontocete whales were the ingesting organisms. However, limited literature on the internal stomach temperatures of the various pelagic sharks forces us to rely on telemetered diving behavior data for further species identification, which we used in the present study to suggest that blue sharks ingested the two white marlin tags (on account of the broad range of recorded temperatures) and that an endothermic shark ingested the opah tag. It is not possible to account for all of the factors that may result in the failure of satellite tags to transmit data, but the results from these three PSATs indicated that biological activities such as predation and scaveng- ing may play an important role. We believe that the most consistent explanation for the data transmitted by these three tags is that they were ingested by large sharks. One cannot calculate the probability that a tag could be engulfed whole without physical damage to the tag, survive for several days in the caustic en- vironment of a digestive system, and be regurgitated with sufficient battery power to transmit data to the Argos satellites, but we suspect that the probability is not very great. We expect that a far greater number of tags may have had similar fates, that is to say, they were damaged by predation or scavenging and digestion processes or were regurgitated later in the transmis- sion cycle, when the PSAT batteries had insufficient remaining power for successful data transmission. The failure of satellite tag to transmit data is frequently considered to be the result of internal tag malfunction or user error. However, these three data sets clearly indicate that the failure of PSATs to function may also be due to predation or scavenging events. Acknowledgments The authors would like to thank the Captain of the FV Sea Pearl and Captain Greg O'Neill of the FV Carol Ann, Don Hawn (University of Hawaii), who deployed the tag on the opah, Evan Howell (PIFSC) for analyses of the opah data, Andrij Horodysky (VIMS), who provided a critical review of the manuscript. Melinda Braun (Wild- 756 Fishery Bulletin 102(4) life Computers), who suggested the predation hypothesis to explain the opah data, and Lissa Werbos (Microwave Telemetry. Inc.), who independently suggested the scav- enging hypothesis for the WM1 data. This research was supported in part by the National Marine Fisheries Service, the NOAA Ocean Exploration Program, and the University of Hawaii Pelagic Fisheries Research Program (PFRP). Literature cited Block, B. A., H. Dewar, S. B. Blackwell. T. D. Williams, E. D. Prince, C. J. Farwell, A. Boustany, S. L. H. Teo, A. Seitz, A. Walli, and D. Fudge. 2001. Migratory movements, depth preferences and thermal biology of Atlantic bluefin tuna. Science 293:1310-1314. Boustany, A. M., S. F. Davis, P. Pyle, S. D. Anderson, B. J. LeBoeuf, and B. A. Block. 2002. Expanded niche for white sharks. Nature 415: 35-36. Carey, F. G., J. W. Kanwisher, O. Brazier, G. Gabrielson, J. G. Casey, and H. L. Pratt. 1982. Temperature and activities of a white shark, Car- charodon carcharias. Copeia 1982(21:254-260. Carey, F. G., and J. V. Scharold. 1990. Movements of blue sharks iPrionace glauca) in depth and course. Mar. Biol. 106:329-342. Carey, F. G., J. M. Teal, and J. W. Kanwisher. 1981. The visceral temperatures of mackerel sharks (Lamnidae). Physiol. Zool. 54(3):334-344. Compagno, L. J. V. 1984. Sharks of the world: an annotated and illustrated catalogue of shark species known to date. Part 1. Hexanchiformes to Lamniformes. U.N. FAO species synopsis 125, vol. 4, pt. I, 249 p. FAO, Rome. Cramer, J. 2000. Species reported caught in the U.S. commercial pelagic longline and gillnet fisheries from 1996 to 1998. NMFS Sustainable Fisheries Division SFD- 99/00-78:1-33. Domeier. M. L, and H. Dewar. 2003. Post-release mortality rate of striped marlin ( Tet- rapterus audax) caught with recreational tackle. Mar. Freshw. Res., 54(41:435-445. Dudley, S. F. J., M. D. Anderson-Reade, G. S. Thompson, and P. B. McMullen. 2000. Concurrent scavenging off a whale carcass by great white sharks, Carcharodon carcharias, and tiger sharks. Galeocerdo cuvier. Fish. Bull. 98:646-649. Economakis. A. E., and P. S. Lobel. 1998. Aggregation behavior of the grey reef shark, Car- charhinus amblyrhynchos, at Johnston Atoll, central Pacific Ocean. Environ. Biol. Fishes 51(21:129-139. Graves, J. E.. B. E. Luckhurst, and E. D. Prince. 2002. An evaluation of pop-up satellite tags for estimating post-release survival of blue marlin {Makaira nigricans) from a recreational fishery. Fish. Bull. 100:134-142. Hazin, F., R. Lessa, and M. Chammas. 1994. First observations on stomach contents of the blue shark, Prionace glauca, from southwestern equatorial Atlantic. Revista Brasileira de Biologia 54(21:195-198. Horodysky, A. Z„ D. W. Kerstetter, and J. E. Graves. In press. Habitat preferences and diving behavior of white marlin (Tetrapturus albidus) released from the recreational rod-and-reel and commercial pelagic longline fisheries in the western North Atlantic: implications for habitat-based stock assessment models. International Commission for the Conservation of Atlantic Tunas, Coll. Vol. Sci. Pap., SCRS 2003/033. Jolley, J. W., Jr., and E. W. Irby Jr. 1979. Survival of tagged and released Atlantic sailfish ilstwphorus platypterus: Istiophoridae) determined with acoustical telemetry. Bull. Mar. Sci. 29(21:155-169. Kerstetter, D. W., B. E. Luckhurst, E. D. Prince, and J. E. Graves. 2003. Use of pop-up satellite archival tags to demonstrate survival of blue marlin (Makaira nigricans) released from pelagic longline gear. Fish. Bull. 101(41:939-948. Klimley, A. P., S. C. Beavers, T. H. Curtis, and S .J. Jorgensen. 2002. Movements and swimming behavior of three spe- cies of sharks in La Jolla Canyon, California. Environ. Biol. Fishes 63:117-135. McCosker. J. E. 1987. The white shark, Carcharodon carcharias, has a warm stomach. Copeia 1987(11:195-197. NOAA (National Oceanographic and Atmospheric Administration). 1998. Atlantic coast chart 13003: Cape Sable to Cape Hattaras. National Ocean Service, Washington. D.C. Sedberry, G .R., and J. K. Loefer. 2001. Satellite telemetry of swordfish, Xiphias gla- dius, off of the eastern United States. Mar. Biol. 139:355-360. Whittow. G. C, I. F. G. Hampton. D. T. Matsuura, C. A. Ohata. R. M. Smith, and J. F. Allen. 1974. Body temperature of three species of whales. J. Mammol. 55(31:653-656 757 Survival rates for rays discarded by the bottom trawl squid fishery off the Falkland Islands Vladimir V. Laptikhovsky Falkland Islands Government Fisheries Department P.O. Box 598 Stanley, FIQQ 1ZZ Falkland Islands E-mail address: vlaptikhovskyigfisheries.gov fk Waters off the Falkland Islands are subject to a specialized multispecies ray fishery and were first fished by a Korean fleet in 1989. More than twenty different rajid species have been recorded from catches around the islands, and five species accounted for 87.04% of the total catch during 1993-2002. Catches peaked in 1993 at 8523 metric tons, and specific fish- ing licenses — R (second season) and F (first season) — were first introduced in 1994 and in 1995, respectively (Agnew et al. 2000; Falkland Islands Government, 2002; Wakeford et al., in press). In addition to the licensed ray fish- ery, rays are taken as bycatch in the bottom trawl fishery that targets the squid Loligo gahi and, to a lesser ex- tent, by the trawl fishery that targets finfish. A 10% bycatch of nontarget species is allowed in both these fish- eries. In 2000-2002, the reported ray bycatch of trawlers not licensed to catch rays represented between 20.2% and 31.9% of the total ray catch. However, under-reporting of elasmobranch bycatch is a common practice for trawl fisheries where sharks and rays are discarded (Ste- vens et al., 2000), and the reported chondrichthyan catch is only about half of the estimated actual global catch (Bonfil, 1994). The actual ray bycatch in Falkland waters may be much higher than reported because only large rays are processed (and therefore, reported) onboard trawl- ers. This situation makes ray fishery management in the Falkland Islands, which is already difficult because of the nature of the multispecies tar- get, even more complicated. However, good management is of primary im- portance because sharks and rays appear to be particularly vulnerable to over-exploitation because of their late attainment of sexual maturity, long life span, both low fecundity and natural mortality, and close relation- ship between recruitment and paren- tal stock (Stevens et al., 20001. In the Falkland trawl fisheries (which includes most trawlers licensed to catch rays), rays smaller than ap- proximately 30 cm disk width are discarded after spending between 5 min and 4 hours in the fish bin and passing through the factory sorting line together with other catch. Some rays that have been caught, stored, and then discarded still show signs of life. In contrast to other marine organisms whose survival after be- ing discarded has been investigated, ray survival has been studied only in Australian waters (Stobutzki et al.. 2002). The aim of this study was to investigate the survival rates of discarded rays onboard trawlers in the Falkland waters. Materials and methods The research was conducted onboard the Falkland Islands registered trawler Sil (length of 78.5 m, gross tons (GRT) of 2156 t, net tons (NT) of 647 t). The vessel used a bottom trawl with a vertical opening of 5 m, horizontal opening of 30 m, and a codend mesh size of 110 mm. Trawl- ing speed varied between 3.8 and and 4.2 kn. Fishing occurred at a depth of 80-190 m during the day and the early part of the night. The surface temperature was 8.7-9.2°C; the near bottom temperature was 6.8-7.6°C. Up to four hauls occurred daily. Each catch was released from the codend into the fish bin, which had a continu- ous supply of sea water, and the catch immediately began to be sorted on a conveyor belt. Squids and commercial fish were separated from the noncom- mercial discarded bycatch and were frozen. Of a total of 4306.2 kg of rays caught during the observed period, 67.0% were discarded and only the large rays were processed. The time taken to sort the catch was between 1 and 3 hours. A total of 66 rays that had been discarded by fishermen were sampled randomly from the conveyor belt and put into a 40-liter (44x35x26 cm) or a 60-liter (31x76x26 cm) fish box that contained running seawater. For each animal, the species and sex was identified and total length (TL) and disk width (DW) were measured within 1 cm. Their "stamina index" was assigned according to four major categories: A alive, flapping wings. I immobile, but alive, reacting to irritation, spiracles beginning to work actively after being placed in seawater. D dead; immobile, but spiracles begin to move slowly and irregularly after being placed in seawater. DD dead; paralyzed, body stiffened and wings curved but may resume breathing after being placed in seawater. Each ray (including those evident- ly dead) was kept in these boxes ei- ther until its death was evident (no breathing) or it fully recovered and began to try to swim actively. In some rays the rate of spiracle con- tractions was episodically recorded. Manuscript submitted 5 June 2003 to the Scientific Editor's Office. Manuscript approved for publication 30 June 2004 by the Scientific Editor. Fish. Bull. 102:757-759 (2004). 758 Fishery Bulletin 102(4) Table 1 Species composition and survival of sampled rays. DW= disk width. Species n TL, cm DW. cm T ime spent in fish bin (min.) Survival rate (%) Bathyraja albomaculata 14 36-61 26-44 20-110 (mean 45) 71.4 B. brachiurops 11 15-67 9-49 31-145 (mean 72) 54.6 B. griseocauda 3 62-83 47-60 30-75 (mean 60) 0.0 B. macloviana 2 36-42 24-29 70-135 0.0 B. magellanica 5 30-44 20-30 50-125 (mean 90) 60.0 Bathyraja sp. 16 24-104 21-74 5-120 (mean 52 ) 75.0 Psammobatis sp. 15 29-47 18-33 30-200 (mean 98) 60.0 Table 2 Ray survival (S% ), mean recovery time (RT, min.), and occurrence of the four of time (T, min.) spent in the fish bin. T=time (minutes). A=alive; I=immobile; 'stamina index" categories after different D=presumed dead; DD = dead. periods T n S RT Occurrence of categories c'< l A I D DD 5-30 16 87.5 31-60 20 75.0 65-120 24 41.7 125-200 6 16.7 38.2 55.5 102.2 20' 18.75 10.0 0 0 25 18.75 30.0 40.0 20.8 50.0 16.7 83.3 37.5 20.0 29.2 0 ' Only one individual (Psammobathis sp. 1. Results The sampled rays belonged to eight species (Table 1). Of the 66 sampled rays, a total of 21 were dead at sampling, four recovered breathing but then died, and 39 survived. Two rays recorded as category DD in the "stamina index" were released before full recovery after being held for 4 to 9 hours in running water. Even though these individuals were still breathing, both were considered dead because they still had stiffened bodies and curved wings. If they had been in such a state for a long time in their natural habitat, they almost certainly would have been consumed by scavengers or caught again by another trawler. The overall survival rate was 59.1%, female survival rate was 66.7%, and male survival rate was 56.4%. All five rays assigned to the "stamina index" category A were sampled between 5 and 30 min (mean 20 min) after the catch was poured into the fish bin. All five individuals began immediately to breathe normally and recovered within 5 to 20 minutes. Of a total of 18 rays assigned to the "stamina index" category I, which were sampled between 15 and 145 min (mean 55.7 min) after haul, 88.9% (n=16) survived. The breathing of these specimens at the time of sampling was usually slow, although occasionally normal. Spira- cle contraction rates gradually increased from an initial rate of 5-15 bit/min to 25-28 bit/min for B.brachiurops specimens and to 35-38 bit/min for individuals of B. albomaculata and Bathyraja sp. Upon attaining normal breathing, they remained immobile, but fully recovered between 15 minutes and 3 hours. The survival rate of 28 rays that were assigned to the "stamina index" category D was 39.3% («=11). Of the remaining individuals, two rays died after 15 and 45 minutes after being placed in running seawater and 15 rays were dead at the time of sampling. The skates were sampled between 30 and 200 min (mean 84.2 min) after the haul. Those that survived took 5-80 minutes to recover normal breathing and between 15 and 315 minutes to attain full recovery. A total of 15 rays were assigned the "stamina index" category DD. However seven of them (46.7%) survived. These individuals were sampled between 20 and 115 minutes (mean 63.9 min.) after the haul and fully re- covered within 40 to 150 minutes. Survival rate varied substantially among the eight species sampled (Table 1). In general, ray survival dras- tically decreased and recovery time increased with the time spent in the fish bin (Table 2). The critical dura- tion in the fish bin appeared to be between one and two hours; only one Psammobathis sp. survived more than two hours in the fish bin and exhibited a surprisingly fast recovery. NOTE Laptikhovsky: Survival rates for rays by the bottom trawl squid fishery 759 Discussion Acknowledgments The survival of discarded rays during trawling opera- tions in the Falkland waters is quite important. Although 65.2% of the individuals were initially assigned as dead, the actual mortality was 40. 99c, although it took some rays up to six hours to recover. Survival of shallow-water shelf species such as Psammobatis sp., in particular, but also B. brachiurops and B. magellaniea, was somewhat higher than relatively deep-water species such as B. albomaculata, B. griseocauda, and Bathyraja sp., which inhabit the shelf edge and upper part of the slope. This survival rate was most likely related to the greater resilience to environmental changes for shallow-water species, whose habitat is more changeable both season- ally and spatially. Male survival was lower, which is in accordance with data for rays and skates obtained in northern Australian waters (Stobutzky et al., 2002). Recent data from a tropical prawn fishery off northern Australia showed that on average 449c of individuals of a number of ray and shark species survived a trawl- ing event (Stobutzky et al., 2002). The Falkland ray survival rate was higher. This difference may be due either to the higher metabolic rates of tropical ray spe- cies (and therefore a higher vulnerability to asphyxia), or to an overestimation of their mortality, which was assessed immediately after individuals where landed on deck (unlike the recovery time allowed in the present study). The latter factor is more probable because in the present study 41.9% of rays initially recorded as dead (D and DD) eventually recovered. Despite the demonstrated ability of skates to survive after being caught and stored in fish bins, their contin- ued survival is not guaranteed once they are discarded. They may fall prey to the hundreds of albatrosses and other scavenging birds that are associated with trawl- ers (author's pers. obs.). The consumption of differ- ent discarded fish species and squids from trawlers in Falkland waters by seabirds, primarily by black-browed albatrosses, has been studied (Thompson, 1992), but it is not known whether rays are also taken by sea birds and to what extent. Despite the great abundance of seabirds around vessels in the Southwest Atlantic, it is likely that they consume a minor part of discards as found in Australia (Hill and Wassenberg, 2000). Most of the discarded fish probably fall to the sea floor and attract and are consumed by bottom scavengers and bottom dwellers (Laptikhovsky and Fetisov, 1999; Lap- tikhovsky and Arkhipkin, 2003). Consequently, even after recovering and successfully avoiding the seabirds, the discarded skates may be consumed or mortally in- jured by these bottom scavengers during the recovery time, which appears to be about 0.5-1.5 hours. I would like to thank the crew of FV Sil for their valu- able help during sampling procedures and their hospital- ity onboard; the Director of Fisheries, John Barton, for supporting this work; A. I. Arkhipkin and an anonimous reviewer for valuable comments; and Helen Otley (FIFD) for language editing. Literature cited Agnew, D. J., C. P. Nolan, J. R. Beddington. and R. Baranovski. 2000. Approaches to the assessment and management of multispecies skate and ray fisheries using the Falkland Islands fishery as an example. Can. J. Fish. Aquat. Sci. 57:429-440. Bonn], R. 1994. Overview of world elasmobranch fisheries. FAO Fish. Tech. Pap. 341. 119 p. Falkland Islands Government. 2002. Fisheries Department fisheries statistics, 7, 1993-2002. Falkland Islands Government Fisheries Department, Stanley, Falkland Islands. Hill, B. J., and T. J. Wassenberg. 2000. The probably fate of discards from prawn trawl- ers fishing near coral reefs. A study in northern Great Barrier Reef, Australia. Fish. Res. 48:277-286. Laptikhovsky, V.. and A. Arkhipkin . 2003. An impact of seasonal squid migrations and fishery on the feeding spectra of subantarctic notothenioids Patagonotothen ramsayi and Cottoperca gobio around the Falkland Islands. J. Appl. Ichthyol. 19:35-39. Laptikhovsky, V., and A. Fetisov 1999. Scavenging by fish of discards from the Patagonian squid fishery. Fish. Res. 41:93-97. Stevens, J. D., R. Bonfil, N. K. Dulvy, and P. A. Walker. 2000. The effects of fishing on sharks, rays and chimae- ras (chondrichthyans). and the implication for marine ecosystems. ICES J. Mar. Sci. 57:476-494. Stobutzki, I. C, M. J. Miller, D. S. Heales, and D. T. Brewer. 2002. Sustainability of elasmobranchs caught as bycatch in a tropical prawn (shrimp) trawl fishery. Fish. Bull. 100:800-821. Thompson K. R. 1992. Quantitative analysis of the use of discards from squid trawlers by black-browed albatrosses Diomedea melanophris in the vicinity of the Falkland Islands. Ibis 134:11-21. Wakeford, R .C, D. A. J. Middleton, D. J. Agnew, J. H. W. Pompert, and V. V. Laptikhovsky. In press. Management of the Falkland Islands mul- tispecies ray fishery: addressing sustainability and diversity. Can. J. Fish. Aquat. Sci. 760 Acknowledgment of reviewers The editorial staff of Fishery Bulletin would like to acknowledge the scientists who reviewed articles published in 2003-2004. Their contributions have helped ensure the publication of quality science. Dr. G.R. Abbe Dr. Pere Abello Mr. Douglas H. Adams Dr. Vera N. Agostini Dr. Juergen Alheit Dr. Robert J. Allman Mr. Michael D. Arendt Dr. Alexander I. Arkhipkin Dr. David A. Armstrong Dr. Colin Attwood Ms. Larisa Avens Mr. M. Scott Baker Jr. Dr. Donald M. Baltz Dr. A. Banner Dr. Jay Barlow Dr. Steve Berkeley Dr. Eric R Bjorkstedt Dr. James A. Bohnsack Ms. Genevieve Briand Dr. Richard W. Brill Dr. Alejandro M. Brockmann Dr. Fiona M. Brook Dr. Elizabeth Brooks Dr. Nancy Brown-Peterson Dr. Jay Burnett Mr. Michael Burton Dr. Morgan S. Busby Dr. Michael Canino Dr. John K. Carlson Dr. Milani Y. Chaloupka Dr. David M. Checkley Jr. Dr. Susan J. Chivers Dr. Phillip J. Clapham Dr. William Coles Mr. L. Alan Collins Dr. Craig Dahlgren Dr. Marilyn E. Dahlheim Dr. Louis B. Daniel III Dr. Jana L.D. Davis Dr. Earl G. Dawe Dr. Edward E. DeMartini Dr. Heidi Dewar Dr. Robert W. Elner Dr. Tomo Eguchi Dr. Charles E. Epifanio Dr. Sheryan P. Epperly Dr. S. Frantini Dr. Gregory L. Fulling Ms. Moira Galbraith Dr. Francisco J. Garcia-Rodriguez Mr. Bert Geary Dr. Harry J. Grier Dr. Churchill B. Grimes Dr. Donald R. Gunderson Dr. Chris Habicht Dr. Lewis J. Haldorson Dr. J. Mark Hanson Dr. E. Brian Hartwick Dr. James T. Harvey Dr. Jonathan Heifetz Dr. Kevin T. Hill Dr. Simeon Hill Dr. David B. Holts Dr. J. Jeffrey Isely Dr. George D. Jackson Ms. Nadine Johnston Dr. Lindsay Joll Dr. Michel J. Kaiser Mr. Craig R. Kastelle Dr. Izhar A. Khan Dr. J. King Dr. A. Peter Klimley Dr. Suzanne Kohin Dr. Thomas E. Laidig Dr. Richard W. Langton Ms. Amy Lapolla Dr. Robert N. Lea Dr. Christopher M. Legault Dr. Steven T. Lindley Dr. Romuald Lipcius Dr. Kwang Ming Liu Dr. Kai Lorenzen Dr. Milton S. Love Mr. Mark S. Lowry Dr. Mark Luckenbach Dr. R. Bruce MacFarlane Dr. William K. Macy Dr. Richard McBride Dr. Susanne McDermott Ms. Kitty Mecklenburg Dr. David A. Milton Dr. T.J. Minello Mr. Karl W. Mueller Dr. Ashley Mullen Dr. Keith D. Mullin Dr. Michael D. Murphy Dr. Kate Myers Dr. Wallace J. Nichols Dr. Peter F. Olesiuk Dr. Ernst Peebles Dr. Karl M. Polivka Dr. Kenneth H. Pollock Dr. Allyn B. Powell Dr. Hans-Joachim Raetz Dr. Stephen Ralston Mrs. Tone Rasmussen Dr. Sherrylynn Rowe Dr. Peter Rubec Mr. D.E. Ruzzante Dr. Yvonne Sadovy Dr. Bernard Sainte-Marie Dr. Kurt M. Schaefer Dr. George R. Sedberry Dr. Jeffrey Seminoff Mr. Lawrence Settle Dr. James B. Shaklee Dr. Alan Sinclair Dr. Oscar Sosa-Nishizaki Dr. Gretchen Steiger Dr. David L. Stein Dr. Allan W. Stoner Dr. D.P Swain Dr. Yonat Swimmer Dr. Yuji Tanaka Dr. Sven Thatje Dr. A.M. Tokranov Dr. M.J. Tremblay Dr. Marc Trudel Dr. Fred M. Utter Dr. Peter Van Tamelen Dr. Michael Vecchione Dr. Claire M. Waluda Mr. William Watson Dr. George Watters Dr. Elizabeth L. Wenner Mr. A.J. Winship Dr. Sabine Petra Wintner Dr. Bernd Wursig Dr. Orio Yamamura Dr. Richard E. Young 761 Fishery Bulletin Index Volume 102(1-4), 2004 List ot titles 102(1) 142 Growth, mortality, and hatchdate distributions of larval and juvenile spotted seatrout (Cynoscion jiebulosus) in Florida Bay, Everglades National Park, by Allyn B. Powell, Robin T. Chesire, Elisabeth H. Laban, James Colvocoresses, Patrick O'Donnell, and Marie Davidian 1 The effects of size-selective fisheries on the stock dynamics of and sperm limitation in sex-changing fish, by Suzanne H. Alonzo and Marc Mangel 14 An environmentally based growth model that uses finite difference calculus with maximum likeli- hood method: its application to the brackish water bivalve Corbicula japonica in Lake Abashiri, Japan, by Katsuhisa Baba, Toshifumi Kawajiri, Yasuhuro Kuwahara, and Shigeru Nakao 25 Juvenile salmonid distribution, growth, condition, origin, and environmental and species associations in the Northern California Current, by Rick D. Brodeur, Joseph P. Fisher, David J. Teel, Robert L. Emmett, Edmundo Casillas, and Todd W. Miller 47 Spatial and temporal variation in the diet of the California sea lion (Zalophus californianus) in the Gulf of California, Mexico, by Francisco J. Garcia- Rodriguez and David Auarioles-Gamboa. 63 Recruitment and spawning-stock biomass distribu- tion of bay anchovy {Anchoa mitchilli ) in Chesapeake Bay. by Sukgeun Jung and Edward D. Houde 78 Coupling ecology and economy: modeling optimal release scenarios for summer flounder (Paralichthys dentatus) stock enhancement, by Todd G. Kellison and David B. Eggleston 94 Sex-specific growth and mortality, spawning season, and female maturation of the stripey bass (Lutjanus carponotatus) on the Great Barrrier Reef, by Jacob T. Krtizer 156 Age determination and growth of the night shark (Carcharhinus signatus) off the northeastern Brazil- ian coast, by Francisco M. Santana and Rosangela Lessa 168 Distribution and biology of prowfish (Zaprora sile- nus) in the northeast Pacific, by Keith R. Smith, David A. Somerton, Mei-Sun Yang, and Daniel G. Nichol 179 Fish lost at sea: the effect of soak time on pelagic longline catches, by Peter Ward, Ransom A. Myers, and Wade Blanchard 196 Effects of density-dependence and sea surface tem- perature on interannual variation in length-at-age of chub mackerel (Scomber japonicus ) in the Kuroshio- Oyashio area during 1970-1997, by Chikako Wata- nabe and Akihiko Yatsu 207 Latitudinal and seasonal egg-size variation of the anchoveta (Engraulis ringens) off the Chilean coast, by Llanos-Rivera, Alejandra, and Leonard R. Castro 213 Molecular methods for the genetic identification of salmonid prey from Pacific harbor seal iPhoca vitulina richardsi) scat, by Maureen Purcell, Greg Mackey. Eric LaHood, Harriet Huber, and Linda Park 221 Diel vertical migration of the bigeye thresher shark (Alopias superciliosus), a species possessing orbital retia mirabilia, by Kevin C. Weng and Barbara A. Block 108 Examination of the foraging habits of Pacific harbor seal (Phoca vitulina richardsi) to describe their use of the Umpqua River, Oregon, and their predation on salmonids, by Anthony J. Orr, Adria S. Banks, Steve Mellman, Harriet R. Huber, Robert L. DeLong, and Robin F. Brown 118 Larval development of the sidestriped shrimp (Pan- dalopsis dispar Rathbun) (Crustacea, Decapoda, Pandalidae) reared in the laboratory, by Wongyu Park, R. Ian Perry, and Sung Yun Hong 127 Sources of age determination errors for sablefish (Anoplopoma fimbria) by Donald E. Pearson and Franklin R. Shaw 102(2) 233 Annual estimates of the unobserved incidental kill of pantropical spotted dolphin (Stenella attenuata attenuata) calves in the tuna purse-seine fishery of the eastern tropical Pacific, by Frederick Archer, Tim Gerrodette, Susan Chivers, and Alan Jackson 245 A remarkable new species of Psednos (Teleostei: Liparidae ) from the western North Atlantic Ocean, by Natalia V. Chernova and David L. Stein 251 Age and growth of sailfish (Istiophorus platypterus) in waters off eastern Taiwan, by Wei-Chuan Chiang, Chi-Lu Sun, Su-Zan Yeh, and Wei-Cheng Su 762 Fishery Bulletin 102(4) 264 A habitat-use model to determine essential fish habitat for juvenile brown shrimp {Farfantepenaeus aztecus) in Galveston Bay, Texas, by Randall D. Clark, John D. Christensen, Mark E. Monaco, Philip A. Caldwell, Geoffrey A. Matthews, and Thomas J. Minello 102(3) 407 Testicular development in migrant and spawning bluefin tuna (Thunnus thynnus (L.>) from the east- ern Atlantic and Mediterranean, by Francisco J. Abascal. Cesar Megina, and Antonio Medina 278 Translocation as a strategy to rehabilitate the queen conch {Strombus gigas) population in the Florida Keys, by Gabriel A. Delgado, Claudine T Bartels, Robert A. Glazer, Nancy J. Brown-Peterson, and Kevin J. McCarthy 289 Genetic differentiation among Atlantic cod (Gadus morhua) from Browns Bank, Georges Bank, and Nantucket Shoals, by Christopher Lage, Kristen Kuhn, and Irv Kornfield 298 Conserving oyster reef habitat by switching from dredging and tonging to diver harvesting, by Hunter S. Lenihan and Charles H. Peterson 306 Fecundity, egg deposition, and mortality of market squid iLolilgo opalescens), by Beverly J. Macewicz, John R. Hunter, Nancy C. H. Lo, and Erin L. LaCasella 418 Maturity, ovarian cycle, fecundity, and age-specific parturition of black rockfish (Sebastes melanops), by Stephen J. Bobko and Steven A. Berkeley 430 Maori octopus ( Octopus maorum ) bycatch and south- ern rock lobster (Jasus edwardsii) mortality in the South Australian lobster fishery, by Daniel J. Brock and Timothy M. Ward 441 Small-boat surveys for coastal dolphins: line-tran- sect surveys of Hector's dolphins (Cephalorhymhus hectori), by Stephen Dawson, Elisabeth Slooten, Sam DuFresne, Paul Wade, and Deanna Clement 452 Description and growth of larval and pelagic juvenile pygmy rockfish (Sebastes wilsoni) (family Sebasti- dae), by Thomas E. Laidig, Keith M. Sakuma, and Jason A. Stannard 328 The dusky rockfishes (Teleostei: Scorpaeniformes I of the North Pacific Ocean: resurrection of Sebastes variabilis (Pallas, 1814) and a redescription of Sebastes ciliatus iTilesius, 1813), by James Wilder Orr and James E. Blackburn 349 Recruitment as a evolving random process of aggre- gation and mortality, by Joseph E. Powers 366 Diet shifts of juvenile red snapper (Lutjanus campechanus) with changes in habitat and fish size, by Stephen T Szedlmayer and Jason D. Lee 464 Estimating the emigration rate of fish stocks from marine sanctuaries using tag-recovery data, by Richard McGarvey 473 Reproductive dynamics of female spotted seatrout (Cynoscion nebulosus) in South Carolina, by William A. Roumillat and Myra C. Brouwer 488 Estimating Dungeness crab (Cancer magistvr^ abundance: crab pots and dive transects compared, by S. James Taggart, Charles E. O'Clair, Thomas C. Shirley, and Jennifer Mondragon 376 Individual growth rates and movement of juvenile white shrimp (Litopenaeus setiferus) in a tidal marsh nursery, by Stacey Webb and Ronald T. Kneib 389 Does the California market squid (Loligo opalescens) spawn naturally during the day or at night? A note on the successful use of ROVs to obtain basic fisher- ies biology data, by John Forsythe, Nuutti Kangas, and Roger T. Hanlon 393 Incidental capture of loggerhead (Caretta caretta) and leatherback (Dermochelys coriacea) sea turtles by the pelagic longline fishery off southern Brazil, by Jorge E. Kotas, Silvio dos Santos, Venancio G. de Azevedo, Berenice M. G. Gallo. and Paulo C. R. Barata 400 Diet changes of Pacific cod {Gadus macrocephalus) in Pavlof Bay associated with climate changes in the Gulf of Alaska between 1980 and 1995, by Mei-Sun Yang 498 A method to improve size estimates of walleye pol- lock (Theragra chalcogramma) and Atka mackerel (Pleurogrammus monopterygius) consumed by pin- nipeds: digestion correction factors applied to bones and otoliths recovered in scats, by Dominic J. Tollit. Susan G. Heaslip, Tonya K. Zeppelin, Ruth Joy, Katherine A. Call, and Andrew W. Trites 509 Sizes of walleye pollock (Theragra chalcogramma) and Atka mackerel (Pleurogrammus monopter- ygius) consumed by the western stock of Steller sea lions (Eumetopias jubatus) in Alaska from 1999 to 2000, by Tonya K Zeppelin, Dominic J. Tollit, Katherine A Call, Trevor J. Orchard, and Carolyn J. Gudmundson 522 Sizes of walleye pollock (Theragra chalcogramma) consumed by the eastern stock of Steller sea lions {Eumetopias jubatus) in Southeast Alaska from 1994 to 1999, by Dominic J. Tollit, Susan G. Heaslip, and Andrew Trites List of titles 763 533 Multidirectional movements of sportfish species between an estuarine no-take zone and surrounding waters of the Indian River Lagoon, Florida, by Derek M. Tremain, Christopher W. Harnden, and Douglas H. Adams 545 Distribution, age, and growth of young-of-the year greater amberj ack (Seriola dumerili ) associated with pelagic Sargassum, by R. J. David Wells and Jay R. Rooker 555 Identification of formalin-preserved eggs of red sea bream iPagrus major) (Pisces: Sparidae) using monoclonal antibodies, by Shingo Hiroishi, Yas- utaka Yuki, Eriko Yuruzume, Yosuke Onishi, Tomoji Ikeda, Hironobu Komaki, and Muneo Okiyama 102(4) 563 Distribution and abundance of humpback whales (Megaptera novaeangliae) and other marine mam- mals off the northern Washington coast, by John Calambokidis, Gretchen H. Steiger, David K. Ellifrit, Barry L. Troutman, and C. Edward Bowlby 581 Reproductive biology of male franciscanas (Ponto- poria blainvillei) (Mammalia: Cetacea) from Rio Grande do Sul, southern Brazil, by Daniel Danile- wicz, Juan A. Claver, Alejo L. Perez Carrera, Edu- ardo R. Secchi, and Nelson F. Fontoura 593 Red snapper (Lutjanus campechanus) demographic structure in the northern Gulf of Mexico based on spatial patterns in growth rates and morphomet- ries, by Andrew J. Fischer, M. Scott Baker Jr., and Charles A. Wilson 604 Elemental signatures in otoliths of larval walleye pollock (Theragra chalcogramma) from the north- east Pacific Ocean, by Jennifer L. FitzGerald. Simon R. Thorrold. Kevin M. Bailey, Annette L. Brown, and Kenneth P. Severin 617 A sudden collapse in distribution of Pacific sar- dine (Sardinops saga.x} off southwestern Australia enables an objective re-assessment of biomass estimates, by Daniel J. Gaughan, Timothy I. Leary, Ronald W. Mitchell, and Ian W. Wright 634 Fish recolonization in temperate Australian rock- pools: a quantitative experimental approach, by Shane P. Griffiths, Ron J. West, Andy R. Davis, and Ken G. Russell 661 Fishery dynamics of the California market squid (Loligo opalescens), as measured by satellite remote sensing, by Michael R. Maxwell, Annette Henry, Christopher D. Elvidge. Jeffrey Safran, Vinita R. Hobson. Ingrid Nelson, Benjamin T. Tuttle, John B. Dietz. and John R. Hunter 671 Magnitude and distribution of sea turtle bycatch in the sea scallop (Placopecten magellanicus) dredge fishery in two areas of the northwestern Atlantic Ocean, 2001-2002, by Kimberly T. Murray 682 Validation and interpretation of annual skeletal marks in loggerhead (Caretta caretta) and Kemps ridley (Lepidochelys kempii) sea turtles, by Melissa L. Snover and Aleta A. Hohn. 693 The Hudson-Raritan Estuary as a crossroads for dis- tribution of blue iCallinectes sapidus), lady (Ovali- pes ocellatus), and Atlantic rock (Cancer irroratus) crabs, by Linda L. Stehlik. Robert A. Pikanowski, and Donald G. McMillan 711 Radiometric validation of age, growth, and longevity for the blackgill rockfish (Sebastes melanostomus), by Melissa M. Stevens, Allen H. Andrews, Gregor M. Cailliet. Kenneth H. Coale. and Craig C. Lundstrom 723 Descriptions of larval, prejuvenile, and juvenile finescale menhaden (Brevoortia gunteri) (family Clupeidae), and comparisons to gulf menhaden (B. patronus), by James M. Tolan and David A. Newstead 740 Capture probability of a survey trawl for red king crab (Paralithodes camtschaticus), by Kenneth L. Weinberg, Robert S. Otto, and David A. Somerton 733 Diet of the minimal armhook squid {Berryteuthis anonychus) (Cephalopoda: Gonatidae) in the north- east Pacific during spring, by Kazuhisa Uchikawa, John R. Bower, Yasuko Sato, and Yasunori Sakurai 750 Evidence of shark predation and scavenging of fishes equipped with pop-up satellite archival tags, by David W. Kerstetter, Jeffery J. Polovina, and John E. Graves 757 Survival rates of rays discarded by the bottom trawl squid fishery off the Falkland slands, by Vladimir V. Laptikhovsky 648 Factors influencing the timing and frequency of spawning and fecundity of the goldlined seabream iRhabdosargus sarba) (Sparidae) in the lower reaches of an estuary, by S. Alexander Hesp, Ian C. Potter, and Sonja R. M. Schubert 764 Fishery Bulletin 102(4) Fishery Bulletin Index Volume 102(1-4), 2004 List ot authors Abascal, Francisco J. 407 Adams, Douglas H. 533 Alonzo, Suzanne H. 1 Andrews, Allen H. 711 Archer, Frederick 233 Auarioles-Gamboa, David 47 Azevedo, Venancio G. de 393 Baba, Katsuhisa 14 Bailey, Kevin M. 604 Baker Jr., M. Scott 593 Banks, Adria S. 108 Barata, Paulo C. R. 393 Bartels, Claudine T. 278 Berkeley, Steven A. 418 Blackburn, James E. 328 Blanchard, Wade 179 Block, Barbara A. 221 Bobko, Stephen J. 418 Bower, John R. 733 Bowlby, C. Edward 563 Brock, Daniel J. 430 Brodeur, Rick D. 25 Brouwer, Myra C. 473 Brown, Annette L. 604 Brown, Robin F. 108 Brown-Peterson, Nancy J. 278 Cailliet, Gregor M. 711 Caldwell, Philip A. 264 Call, Katherine A. 498, 509 Calambokidis, John 563 Casillas, Edmundo 25 Castro, Leonard R. 207 Chernova, Natalia V. 245 Cheshire, Robin T. 142 Chiang, Wei-Chuan 251 Chivers, Susan 233 Christensen, John D. 264 Clark, Randall D. 264 Claver, Juan A. 581 Clement, Deanna 441 Coale, Kenneth H. 711 Colvocoresses, James 142 Danilewicz, Daniel 581 Davidian, Marie 142 Davis, Andy R. 634 Dawson, Stephen 441 Delgado, Gabriel A. 278 DeLong, Robert L. 108 Dietz, JohnB. 661 DuFresne, Sam 441 Eggleston, David B. 78 Ellifnt, David K 563 Elvidge, Christopher D. 661 Emmett, Robert L. 25 Fischer, Andrew J. 593 Fisher, Joseph P. 25 FitzGerald, Jennifer L. 604 Fontoura, Nelson F 581 Forsythe, John 389 Gallo, Berenice M. G. 393 Garcia-Rodriguez, Francisco J. 47 Gaughan, Daniel J. 617 Gerrodette, Tim 233 Glazer, Robert A. 278 Graves, John E. 750 Griffiths, Shane P. 634 Gudmundson, Carolyn J. 509 Hanlon, Roger T, 389 Harnden, Christopher W. 533 Heaslip, Susan G. 498, 522 Henry, Annette 661 Hesp, S. Alexander 648 Hiroishi, Shingo 555 Hobson, Vinita R. 661 Hohn,AletaA. 682 Hong, Sung Yun 118 Houde, Edward D. 63 Huber, Harriet R. 108,213 Hunter, John R. 306, 661 Ikeda, Tomoji 555 Jackson, Alan 233 Joy, Ruth 498 Jung, Sukgeun 63 Kangas, Nuutti 389 Kawajiri, Toshifumi 14 Kellison, Todd G. 78 Kerstetter, David W. 750 Kneib, Ronald T 376 Komaki, Hironobu 555 Kornfield, Irv 289 Kotas, Jorge E. 393 Kritzer, Jacob P. 94 Kuhn, Kristen 289 Kuwahara, Yasuhiro 14 Laban, Elisabeth H. 142 LaCasella, Erin L. 306 Lage, Christopher 289 LaHood, Eric 213 Laidig, Thomas E. 452 Laptikhovsky, Vladimir V. 757 Leary, Timothy I. 617 Lee, Jason D. 366 Lenihan, Hunter S. 298 Lessa, Rosangela 156 Llanos-Rivera, Alejandra 207 Lo, Nancy C. H. 306 Lundstrom, Craig C. 711 Macewicz, Beverly J. 306 Mackey, Greg 213 Mangel, Marc 1 Matthews, Geoffrey A. 264 Maxwell, Michael R. 661 McCarthy, Kevin J. 278 McGarvey, Richard 464 McMillan, Donald G. 693 Medina, Antonio 407 Megina, Cesar 407 Mellman, Steve 108 Miller, Todd W. 25 Minello, Thomas J. 264 Mitchell, Ronald W. 617 Monaco, Mark E. 264 Mondragon, Jennifer 488 Murray, Kimberly T 671 Myers, Ransom A. 179 Nakao, Shigeru 14 Nelson, Ingrid 661 Newstead, David A. 723 Nichol, Daniel G. 168 O'Clair, Charles E. 488 O'Donnell, Patrick 142 Okiyama, Muneo 555 Onishi, Yosuke 555 Orchard, Trevor J. 509 Orr, Anthony J. 108 Orr, James W. 328 Otto, Robert S. 740 Park, Linda 213 Park, Wongyu R. 118 Pearson, Donald E. 127 Perez Carrera, Alejo L. 581 Perry, R. Ian 118 Peterson, Charles H. 298 Pikanowski, Robert A. 693 Polovina, Jeffery J. 750 List of authors 765 Potter, Ian C. 648 Powell, Allyn B. 142 Powers, Joseph E. 349 Purcell, Maureen 213 Rooker, Jay R. 545 Roumillat, William A. 473 Russell, Ken G. 634 Safran, Jeffrey 661 Sakuma, Keith M. 452 Sakurai, Yasunori 733 Santana, Francisco M. 156 Santos, Silvio dos 393 Sato,Yasuko 733 Secchi, Eduardo R. 581 Severin, Kenneth P. 604 Shaw, Franklin R. 127 Shirley, Thomas C. 488 Schubert, Sonja R. M. 648 Slooten, Elisabeth 441 Smith, Keith R. 168 Snover, Melissa L. 682 Somerton, David A. 168, 740 Stannard, Jason A. 452 Stehlik, Linda L. 693 Steiger, Gretchen H. 563 Stein, David L. 245 Stevens, Melissa M. 711 Su, Wei-Cheng 251 Sun, Chi-Lu 251 Szedlmayer, Stephen T. 366 Taggart, S. James 488 Teel, David J. 25 Tolan, James M. 723 Tollit, Dominic J. 498, 509, 522 Thorrold, Simon R. 604 Tremain, Derek M. 533 Trites, Andrew W 498, 522 Troutman, Barry L. 563 Tuttle, Benjamin T. 661 Uchikawa, Kazuhisa 733 Wade, Paul 441 Ward, Peter 179 Ward. Timothy M. 430 Watanabe, Chikako 196 Webb, Stacey 376 Wells, R. J. David 545 Weng, Kevin C. 221 Weinberg, Kenneth L. 740 West, Ron J. 634 Wilson, Charles A. 593 Wright, Ian W. 617 Yang, Mei-Sun 168, 400 Yatsu, Akihiko 196 Yeh, Su-Zan 251 Yuki, Yasutaka 555 Yuruzume, Eriko 555 Zeppelin, Tonya K. 498, 509 766 Fishery Bulletin 102(4) Fishery Bulletin Index Volume 102(1-4), 2004 List ot subjects Abundance Crab, Dungeness 488 dolphin. Hector's 441 estimates (pelagic longline) 179 harbor seal. Pacific 108 whales 563 Age and growth amberjack, greater 533 rockfish, blackgill 711 sailfish 251 shark, night 156 determination problems with 127 sablefish 127 validation 711 Aggregation 349 Alabama 366 Alaska 488, 498, 509, 522 Aleutian Islands 168 Alopias superciliosus - see shark, bigeye thresher Alternative fishing practices 298 Amberjack, greater 545 Anchoa mitchilli - see anchovy, bay Anchoveta 207 Anchovy, bay 63 Anoplopoma fimbria - see sablefish Antibodies, monoclonal 555 Assemblages 634 Atlantic 245,407,671 Australia 430, 464, 634, 581, 617. 634 Bass, stripey 94 Batch fecundity 473 Bering Sea 509 Berryteuthis anonychus - see squid, minimal armhook Biomass distribution 63 estimation (sardine) 617 spawning stock 63 Bones 498 Bottom trawl 168, 757, 740 Brazil 156,393,581 Brevoortia gunteri - see menhaden, finescale patronus -see menhaden, gulf Browns Bank 289 Bycatch in longline fishery 393 in lobster fishery 430 in sea scallop fishery 671 in squid fishery 757 in tuna purse-seine fishery 233 of dolphins 233 of octopus 430 of sea turtles 393,671 California 25, 306, 389, 453 Callineetes sapidus - see crab, blue Cancer irroratus - see crab, Atlantic rock magister - see crab, Dungeness Capture incidental (sea turtles) 393 probability 740 -recapture 563 Carcharhinus signatus - see shark, night Caretta caretta - see sea turtles, loggerhead Catch rate 430 Catchability 740 Cephalorhynchus hectori - see dolphin. Hector's Cetaceans 661 Chesapeake Bay 63 Chile 207 Climate change 400 Clupeidae 723 Cod Atlantic 289 Pacific 400 Conch, queen 278 Controlled access areas 563 Corbicula japonica 14 CPUE 489 Crab Atlantic rock 693 blue 693 Dungeness 489 lady 693 red king 740 Crassostrea virgin ica -see oyster, American Crustacea 118,489 Cynoscion nebulosus - see seatrout, spotted Daytime spawning 389 Demographic structure 593 Density (population) 179 Dermochelys coriacea - see sea turtle, leatherback Descriptions (taxonomic) Brevoortia gunteri 723 patronus 723 Psednos 245 Sebastes ciliatus 328 variabilis 328 wilsoni 452 Diet cod. Pacific 400 prey volume 366 prowfish 168 sea lion California 47 Steller 498, 509, 522 seal, harbor 108 snapper, red 366 squid 733 Digestion correction factor 498 Dissolved oxygen 63 Distribution amberjack, greater 533 crab 693 hatchdate ( seatrout ) 142 prowfish 168 salmon, juvenile 25 whale 563 Diurnal cycle 389 Dive transects 489 Dissolved oxygen (DO) 63 Dolphin Hector's 441 Spotted, pantropical 233 Dorsal-fin spine 251 Egg deposition 306 identification (seabream) 555 -size variation 207 Elasmobranch 156, 221, 757, 750 Emigration rate 464 Engraulis ringens - see anchoveta Escapement 740 Estuaries 376,533,648 Eumetopias jubatus -see sea lion. Steller Everglades 142 Falkland Islands 757 Farfantepenaeus aztecus - see shrimp, brown Fecundity 473, 648 batch 648 relative 473 rockfish, black 418 seabream 648 squid, market 306 List of sublets 767 Finite difference calculus 14 Fish size (red snapper) 366 Fisheries interaction 509 management 581 size-selective 1 tuna 233 Fishery dynamics (squid) 661 Fishery reserve 533 Florida Bay 142 eastern coast 533 Keys 278 Flounder, summer 78 Footrope 740 Formalin 555 Foraging (harbor seal) 108 Franciscana 581 Gadus macroeephalus - see cod, Pacific morhua - see cod, Atlantic Galveston Bay 264 Generalized linear model 563 Generalized additive model 563 Genetic differentiation (among cod) 289 identification 108, 213 Georges Bank 289 GIS 264 Glacier Bay 489 GLOBEC 25 Goldlined seabream 648 Gonadosomatic index 94 Great Barrier Reef 94 Growth amberjack, greater 545 curve 156 prowfish 168 rockfish452, 711 seatrout, spotted 142 sex-specific 94 shark, night 156 shrimp, white 376 snapper, red 593 Gulf of Alaska 168,400,509 Gulf of California 47 Gulf of Mexico 593 Habitat conservation 298 use of 264 Hand harvesting 298 Harvesting techniques 298 Hatching date seatrout, spotted 545 snapper, red 366 histology 407 Hudson-Raritan Estuary 693 Humerus 682 Hypergeometric likelihood 464 Identification (fish eggs) 555 Incidental kill 233 Indeterminate 648 Indian River Lagoon 533 Intertidal 634 Istiophorus platypterus - see sailfish Japan 14, 196 Jasus edwardsii - see lobster, southern rock Juvenile studies 25, 142, 264 menhaden 723 mortality 233 pollock 604 rockfish 453 shrimp 264, 376 snapper 366 Kuroshio-Oyashio 179 Lake 14 Larval description 723 development 118, 723 rockfish 453 Lead210 711 Length at age, chub mackerel 179 frequency, shark 156 Life history model 1 seatrout. spotted 142 Line-transect survey 441, 563 Liparidae 245 Litopenaeus setiferus -see shrimp, white Lobster, southern rock 430 Loligo opalescens - see squid, market Longevity ( rockfish ) 711 Longline fishery 179,393 Lutjanus campechanus - see snapper, red carponotatus - see bass, stripey Mackerel Atka 498, 509 chub 179, 196 Mammals 47,108, 213, 581, 563 Marginal increment analysis 156 Mark-recapture 78, 376 Marlin, white 750 Marine sanctuaries 464 Maturity bass, stripey 94 rockfish, black 418 Maximum likelihood method 14 Mediterranean 407 Megaptera novaeangliae -see whale, humpback Menhaden finescale 723 gulf 723 Mexico 47, 593 Microwire tags 376 Mid-water trawl survey 63 Migration diel 221 from marine reserves 533 shark 221 vertical 221 Models growth 14 habitat use 264 life history 1 optimal release 78 recruitment 349 Mortality 349 amberjack, greater 545 dolphin, pantropical spotted 233 lobster 430 longline fishery 179 seatrout, spotted 142 squid, market 306 Movement shrimp 376 sportfish 533 MtDNA rockfish 453 salmonids 213 Multidimensional scaling 36 Nantucket Shoals 289 National marine sanctuary 563 New South Wales 634 New species 245 New Zealand 441 North Carolina (flounder) 78 Northern California Current 25 Nursery habitat 366, 376 Octopus, maori 430 Octopus maorum - see octopus, maori Oncorhynchus kisutch - see salmon, coho 108 Opah 750 Orbital retia mirabilia 221 Orcinus orca - see whale, killer Oregon 108,418 Otoliths amberjack, greater 545 elemental signature in 604 in fecal samples 47, 108, 498, 522 rockfish 453 Ovalipes ocellatus - see crab, lady Ovarian cycle 418 Oxytetracycline 127 Oyster, American 298 768 Fishery Bulletin 102(4) Pacific Ocean 168, 233, 328, 733, 604, 733 Pagrus major - see sea bream, red Pandalopsis clispar - see shrimp, sidestriped Paralichthys dentatus -see flounder, summer Paralithodes eamtschaticus -see crab, red king Parturition (rockfish) 418 PavlofBay 400 Penaeidae 376 Phoca vitulina richardsi - see seal, harbor Photo-identification (marine mammals) 563 Pinnipeds 498, 509, 522 Phocoenoides dalli - see porpoise, Dall's Placopecten magellanicus - see sea scallop Pleurogrammus monopterygius - see mackerel, Atka Poikilotherm 682 Pollock, walleye 498, 509, 522, 604 Pontoporia blainvillei -see franciscana Pop-up satellite archival tags 221, 750 Population dynamics 661 structure 604 Porgy, red 1 Porpoise, Dall's 563 Postovulatory follicle 473 Prey-size selectivity 509, 522 Protogynous 1 Prowfish 168 Psednos rossi - see snailfish Purse-seine fishery 233 Radiometric age 711 Radium226 711 Rays 757 Recolonization (fish in rockpools) 634 Recovery 278 Recruitment anchovy, bay 63 processes of 349 Reef habitat 298 Remote sensing 661 Remotely operated vehicle 389 Reproduction bass, stripey 94 behavior 389 franciscana 581 prowfish 168 rockfish, black 418 seatrout, spotted 473 squid 389 tuna, bluefin 407 Residence time 376 Restoration conch 278, oyster 298 Rhabdosargus sarba — see seabream, goldlined Rio Grande do Sul 581 Rockfish black 418 blackgill 711 dusky 328 pygmy 452 rockpools 634 ROV (remotely operated vehicle) 389 Sablefish 127 Sailfish 251 Salmon coho 25 decline in Onchorynchus spp. 108 juvenile 25 predation on 108 prey of 213 Salmonids 25, 213 juvenile 25 Sampling bias 488 Sardine, Pacific 581, 617 Sardinops sagax - see sardine. Pacific Sargassum 545 Satellite archival tags 750 remote sensing 661 Scale-free networks 349 Scat 47, 108, 213, 498, 522 Scomber japonicus - see mackerel, chub Scorpaenidae 711 SCUBA 366 Sea bream goldined 648 red 555 Sea lion California 47 Steller 498, 509, 522 Sea scallop 671 Sea surface temperature 179 Seal, harbor 108, 213 Seatrout, spotted 142, 473 Sebastes ciliatus - see rockfish, dusky melanops -see rockfish, black melanostomus - see rockfish, blackgill variabilis - see rockfish, dusky uilsoni - see rockfish, pygmy Sea turtle Kemp's ridley 682 leatherback 393 loggerhead 393,682 Seriola dumerili - see amberjack, greater Sex change 1 ratio 693 Sexual maturity 418 Sharks thresher, bigeye 221 night 156 predation 750 Shrimp brown 264 sidestriped 118 white 376 Size-selective fisheries 1 Skeletochronology (sea turtles) 682 Snailfish 245 Snapper red 366, 593 Spanish flag 93 Soak time (gear) 179 South Carolina 473 Sparidae 555,648 Spawning anchovy, bay 63 bass, stripey 94 diel 389 frequency 473 seabream 648 season 418, 648 squid 306 stock biomass 63 tuna, bluefin 407 Species protogynous 1 Squid 73 fishery 757 market 306,389,661 minimal armhook 733 SteneUa attenuata attenuata -see dolphin, spotted, pantropical Stock enhancement 78 Strombus gigas - see conch, queen Surveys dolphin 441,489 with fish traps 127 Survival rates 179, 757 Tagging 464, 750, 604 marine reserves 464, 533 natural 604 shark 221, 750 Taiwan 251 Temperate estuaries 693 Temperature Effect on fish size I mackerel) 196 List of subjects 769 Testicular development 407 Tetrapturus albidus - see marlin, white Theragra chalcogramma - see pollock, walleye Thunnus thynnus - see tuna, bluefin Translocation (for rehabilitation) 278 Traps 430 Trawl efficiency 740 Trophic niche breadth 693 Tuna, bluefin 407 Turtle - see sea turtle Umpqua River 108, 213 Underwater video 740 Vertebral sections 156 Washington 563 Western North Atlantic 245 Whale humpback 563 killer 563 Young-of-the-year (amberjack) 545 Zalophus californianus - see sea lion, California Zaprora silenus -see prowfish 770 Fishery Bulletin 102(4) Superintendent of Documents Publications Order Form *5178 I I YJl/O, please send me the following publications: Subscriptions to Fishery Bulletin for S55.00 per year ($68.75 foreign) The total cost of mv order is $ . 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