ISSN 0042-321 1 ^b\ .1/4X Veliger A Quarterly published by CALIFORNIA MALACOZOOLOGICAL SOCIETY, INC. Berkeley, California R. Stohler (1901-2000), Founding Editor Volume 51 September 16, 2014 Number 4 Contents Distribution, Size, Maturity and Feeding Habits of the Squid Gonatopsis octopedatus (Cepha- lopoda: Gonatidae) in the Sea of Okhotsk and Northwest Pacific Ocean Oleg N. Katugin, Gennady A. Shevtsov and Mikhail A. Zuev 177 Anatomical Redescription of the Limpet-Like Marine Pulmonate Trimusculus Reticulatus (Sowerby, 1835) Roger Tseng and Benoit Dayrat 194 Lottia Austrodigitalis Revisited: Are Ecomorphs Incipient Species? Philip G. Murphy 208 Cretaceous and Paleogene Pteria Bivalves from the Pacific Slope of North America Richard L. Squires 216 Additions to the Genus Phyllodesmium, with a Phylogenetic Analysis and its Implications to the Evolution of Symbiosis Elizabeth Moore and Terrence Gosliner 237 Effects of Crowding on Survival and Growth of Neonatal Biomphalaria Glabmta Snails Maintained on a Nostoc sp. Diet Amanda E. Balaban and Bernard Fried 252 ( Effects of the Invasive Alga Gracilaria salicornia on Molluscan Species Abundance, Richness, and Diversity in Sheltered Shoreline Pools in East Hawai'i Robert D. Strohl and Marta J. deMaintenon 255 Contents — Continued The Veliger (ISSN 0042-3211) is published quarterly by the California Malacozoological So- ciety, Inc., % Santa Barbara Museum of Natural History, 2559 Puesta del Sol Road, Santa Barbara, CA 93105. Periodicals postage paid at Berkeley, CA and additional mailing offices. POSTMASTER: Send address changes to The Veliger, Santa Barbara Museum of Natural His- tory, 2559 Puesta del Sol Road, Santa Barbara, CA 93105. 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The Veliger 51(4):I77 193 (Scptembe 16, 2014) THE VELIGER UCMS. Inc., 2014 Distribution, Size, Maturity and Feeding Habits of the Squid Gonatopsis octopedatus (Cephalopoda: Gonatidae) in the Sea of Okhotsk and Northwest Pacific Ocean OLEG N. KATUGIN,* GENNADY A. SHEVTSOV, and MIKHAIE A. ZUEV Pacific Research Fisheries Centre (TINRO-Centre), Vladivostok 690090, Russia Abstract. The horizontal, vertical, and seasonal distribution patterns; size structure; maturity; teeding activity; and life cycle of the deep-water pelagic gonatid squid Gonatopsis octopedatus Sasaki, 1920 are described based on data collected during 25 research cruises in the Sea of Okhotsk and northwestern Pacific Ocean. This species was rather common in the Sea of Okhotsk and occurred occasionally in the Pacific Ocean. It was distributed mainly beyond the continental shelf between 0 and 1300 m depth. The largest aggregations of squid occurred in the deep southern basin in the Sea of Okhotsk. The highest abundance was observed in summer, when small young squid dominated the catches. In autumn and winter, growing squid descended down to the mesopelagic zone, where adult and maturing squid dominated the catches. Dorsal mantle lengths ranged from 28 mm (early juveniles) to 163 mm (mature adults). Analyses of seasonal changes in size structure, sexual maturity, and vertical distribution suggest that, in the Sea of Okhotsk, spawning occurred from the late summer through winter at great depths, and early life stages appear in the pelagic zone from winter through summer. Assuming that females brood their eggs in an egg mass between their arms (as do some other gonatids), and that the embryonic period lasts about a year, the estimated life cycle is about 2 yr for males and 3 yr for females. INTRODUCTION Squids of the family Gonatidae are important compo- nents in the pelagic food webs in the boreal North Pacific Ocean (Okutani et ah, 1988; Nesis, 1997), but biology of this family, particularly the distribution and life cycle patterns, is poorly documented. Individuals at advanced maturity stages (particularly prespawning, spawning, and postspawning adults) are rarely col- lected, and individuals in the early ontogenetic stages (paralarvae) are extremely hard to identify (Katugin & Shevtsov, 2006). To improve our understanding of the role of these numerous and valuable squids in subarctic marine communities, we examined the distribution patterns for the weakly studied representative of the Gonatidae, the short-tailed gonatopsis (Gonatopsis octopedatus) in the Sea of Okhotsk and the Pacific Ocean off the Kuril Islands. Gonatopsis octopedatus was originally described based on a specimen (holotype) measuring 65 mm in mantle length captured at about 792 m depth off eastern Sakhalin in the Sea of Okhotsk (Sasaki, 1920). A more detailed description of the holotype was given later (Sasaki, 1929). This species was mentioned from the southern deep basin in the Sea of Okhotsk and off northern Honshu in the northwestern Pacific Ocean * e-mail: okatugin@mail.ru; katugin@tinro.ru (Akimushkin, 1963). It was catalogued among cepha- lopods inhabiting waters around Japan (Okutani, 1967a), listed as a reference species in the discussion of species belonging to the genus Gonatopsis (Okutani, 1967b), and accepted as a valid name in a review of the gonatid systematics (Okutani, 1968). Later, a partly mutilated mature male of about 99 mm mantle length identified as G. octopedatus was captured in a deep-sea trawl towed in a trough at 810 m depth off Niigata Prefecture in the Sea of Japan and was described in detail (Okiyama, 1970). The existing published data on the occurrence of G. octopedatus suggests that this species occurs mainly in deep pelagic waters, and its geographic range is restricted to the northwestern Pacific Ocean and the adjoining seas (Okutani et ah, 1988; Nesis, 1997). This demersal sciuid is most common in the northern Sea of Japan (Shevtsov & Bessmertnaya, 1996; Shevtsov & Mokrin, 1998), some- what less common in the Sea of Okhotsk (Nesis, 1989), relatively rare in the western Bering Sea (Didenko, 1991 ), and has not been found in the eastern Bering Sea and northeastern Pacific Ocean (Jorgensen, 2009). Recently, one individual of the gonatid squid provisionally identified as G. octopedatus based on general morphology and genetic evidence was captured on the slope off the Falkland Islands (Arkhipkin et ah, 2010). Vertical, spatial, and temporal distributions and various biological features of G. octopedatus remain Page 178 The Veliger, Vol. 51, No. 4 to a greater part unknown, mostly due to data paucity anci the difficulty obtaining information about this deep-water-dwelling cephalopod. Our objectives were to analyze reliable infonnation on G. octopedatus collected during research expeditions of Tikhookean- skyi Institut Rybnogo Khosyaistva i Okeanographii (TINRO) - Centre (Pacific Research Fisheries Centre), formerly TINRO (Pacific Research Institute for Fisheries and Oceanography) in the Sea of Okhotsk and the northwestern Pacific Ocean, including spatial, vertical, and seasonal distribution; size structure; maturity; and feeding habits of this species, and to produce a possible scenario of its life cycle. MATERIALS and METHODS Data on G. octopedatus were collected in the north- western Pacific Ocean and Sea of Okhotsk during 25 expeditions on research vessels (RVs) of Russia and Republic of Korea from 1979 through 2004 (Table I ). Tows were made using pelagic (midwater) and bottom trawls at various depths from the surface layer to a maximum of 1300 m, but were usually no deeper than 1000 m. Gill nets were deployed at 700-750 m on the bottom. Tow speeds varied from 3 to 5 knots depending on the trawl type. Pelagic tows usually lasted 1 hr. and bottom trawls were towed for 30 min. Each trawl net had a cod-end liner of 10-mm mesh. The horizontal mouth openings were approximately 30-80 m of for the pelagic trawls and 16-25 m for the bottom trawls, depending on the trawl type, modifica- tion, and trawling speed. To standardize the squid catches made by different types of trawls and by different modifications of certain types of trawls, we calculated the catch per unit effort (CPUE) for each catch. Eor the trawling operations, the CPUE was calculated as the number of captured squid per unit area (sciuare kilometer) using the following formula: CPUE = n/(0.001852 X HO X TS X TT), n = number of captured squid; HO = horizontal opening (m); TS = trawling speed (knots); TT = trawling time (hours). For the gillnetting operations, the CPUE was measured as the number of sciuid per 5 km of nets; these scjuid were found in stomach contents of Greenland turbot Reiuhardtius hippoglossoides (Wal- baum, 1792) that had been captured in bottom gillnets. Gonatopsis octopedatus were sorted from the catch, and the weight, size, and number of captured squid were recorded. We analyzed (measured, weighted, and dissected) all animals in a haul when the catch did not exceed 50 individuals. When the number of individuals was larger, a subsample of 50-100 individuals was analyzed, and a total weight and number of individuals in the haul were estimated. Dorsal mantle length (DML) was used as the standard measure of the squid body size, and was measured to the nearest 1 mm. In most observations made in the field, the squid were not dissected to obtain such biological characters as sex, maturity, and stomach contents. Therefore, in order to look at the ontogenetic distribution patterns, we separated all sc]uid into two broad groups: juveniles (DME < 70 mm) and adults (DML > 70 mm). We chose DML = 70 mm to separate juveniles and adults, because at that size, it becomes easy to identify the sex of G. octopedatus. Species-level identification of gonatid squid can be difficult, especially when it comes to the earliest and latest life stages. Gonatopsis octopedatus can be distinguished by the following combination of charac- ters: small size (DML usually smaller than 140 mm), small kidney-shaped fin (fm length is about 30% of DML), absence of tentacles (in animals larger than 30 mm DML), flabby moderately wide mantle, long almost equally sized amis (arm length is about 70-85% of DML) with numerous minute suckers in their distal parts, and five teeth in a transverse row on the radula (Sasaki, 1920, 1929; Okiyama, 1970; Katugin & Shevtsov, 2006; Katugin et al., 2010). The spatial distribution patterns were obtained for juveniles and adults, and were further studied within the epipelagic (0-200 m), upper mesopelagic (200-500 m), lower mesopelagic (500-1000 m), and bathypelagic (>1000 m) layers. The vertical distribution of squid as a function of size has been studied. Capture rate was measured as a probability (%) of squid occurrence in a trawl catch within a given depth interval, and seasonal changes in vertical profiles of capture rates were analyzed. Due to the rather scanty data on the biological features of G. octopedatus, we grouped and analyzed these data by seasons and by regions (Sea of Okhotsk and northwestern Pacific Ocean). Patterns of monthly dynamics of the species size structure were separately analyzed in these two areas. Maturity was assessed using the scale described in Zuev et al. (1985). Stomach contents were roughly sorted out into three taxonomic groups: fish, crusta- ceans, and cephalopods (squid). Where possible, prey organisms were identified more precisely. Indices of stomach fullness were assessed using the following subjective scale originally developed for field studies of fish feeding activity and later adapted for squid: 0 = no food (empty stomach), 1 = few remains of food (approximately up to one-quarter-full stomach), 2 = moderate amount of food (about half-full stomach), 3 = plenty of food (approximately three-quarters-full stomach), 4 = maximum stomach fullness (Volkov & Chuchukalo 1986). The mean index of stomach fullness was calculated for females, males, and juveniles as an arithmetic mean of individual indices. The results were used to construct an electronic database for G. octopedatus including station parame- O. N. Katugin et al., 2014 Page 179 Table 1 Collections of the squid Gonatopsis octopedatus in the Sea of Okhotsk and northwestern Pacific Ocean. RV name Date (year & months) Total number of tows Number of tows with squid Number of captured squid Gear Gerakl 1979 (Jiily-September) 132 7 22 Midwater trawl Novoiilyanovsk 1984 (September- October) 109 67 1412 Midwater and Bottom Trawls Gisscir 1985 (September) 1 1 3 4 Midwater trawl Gissar 1987 (September- November) 44 2 13 Midwater trawl Darvin 1989 (June-July) 13 2 2 Bottom trawl Professor Soldatov 1989 (August- September) 116 19 156 Midwater trawl Novokotovsk 1990 (December) 64 T 31 Midwater trawl Mleclinvi Put' 1 990 (December) 1991 (January) 127 24 1324 Midwater trawl Darvin 1991 (October- December) 243 19 278 Midwater trawl Novoulvanovsk 1992 (July-August) 130 19 849 Midwater trawl Sunflower (Korea) 1992 (December) -1993 (February) 93 7 21 Bottom trawl Professor Soldatov 1993 (July-August) 131 12 76 Midwater trawl Salvia- Ho ( Korea } 1993 (January-April) 97 13 289 Bottom trawl TINRO 1994 ( August-October) 194 2 8 Midwater trawl Professor Kagano vskyi 1995 (July-August) 134 13 97 Midwater trawl Professor Levanidov 1996 (September) 132 2 5 Mid water trawl Professor Kizevetter 1996 (August) 56 6 17 Midwater trawl TINRO 2000 (February-June) 320 11 35 Mid water trawl Professor Levanidov 2000 ( September- Oclober) 212 10 13 Bottom trawl Kavrai 2001 (August) 399 1 1 Bottom trawl Dal'okean 2 2001 (August) 47 2 2 Bottom gillnets Professor Levanidov 2002 (August) 109 1 1 77 Midwater trawl Pro lessor Kagan m ’skyi 2002 (March) 316 1 1 Midwater trawl Novozlatopol’ 2003 (October- December) 191 3 5 Midwater trawl Pro lessor Levan ido v 2004 (October) 307 6 1 1 Midwater trawl Total 25 research cruises 3727 264 4749 ters (name of the RV, region, coordinates, date and time of station, station depth, trawling speed and direction, depth of place) and catch features (species name and biological characters including size, weight, sex. maturity, and stomach contents). We used the computer programs Ichthyologist (unpublished; developed by N. E. Kravchenko, TINRO-Centre), Microsoft Excel, and Microsoft Access to create the database. Data were statistically analyzed using two programs: Microsoft Excel to construct size structure patterns, and Surfer v. 8.0 to produce spatial distribu- tion patterns. RESULTS Data charts from 25 research cruises where G. octopedatus was collected were analyzed; a total of 4749 individuals of G. octopedulu.s were captured (Table 1 ). Of these, about 90% were collected in only five cruises between 1984 and 1993, when research covered a wide depth range, including meso- and bathypelagic zones. About 30% of all squid were collected in the autumn 1984 RV Novoiilyanovsk cruise, 28% in the winter 1990- 1991 RV Mleciwyi Put' cruise, 18% in the summer 1992 RV Novoiilyanovsk cruise, 6% in the winter spring 1993 RV Salvia- Ho cruise, and about 6% in the autumn winter 1991 RV Darviii cruise. The species was reported only occasionally in the other cruises. The autumn 1984 cruise of the RV Novoiilyanovsk was also characterized by the highest percentage of squid-positive hauls, 61.5%. In all 25 research cruises that were used to construct the database for G. octopedatus, squid occurred only in 7% of hauls, and a mean catch was about 18 animals per squid-positive haul. In the survey area, young (DML < 70 mm) and adult (DME > 70 mm) squid occurred beyond shelf areas. Page 180 The Veliger, Vol. 51, No. 4 primarily over deep-sea regions; catches were distributed mainly in the Sea of Okhotsk, and were sporadic in offshore oceanic areas. Young squid were captured predominantly in offshore areas, particularly in the central and southern Sea of Okhotsk, and were relatively infrequent in the Pacific Ocean (Figure 1). Adult squid were found mainly at great depths, and their distribution pattern was more discrete than that of juveniles (Figure 2). A few catches were scattered over the northwestern Pacific Ocean. Different life stages of G. octupedutus occur within a wide depth range in the pelagic zone and near the bottom (Figure 3). The smallest animals (DML 28-29 mm) were found at 300-500 m, with somewhat larger animals (DML 30-35 mm) within 0-700 m, most juveniles and immature adults (DML 36-90 mm) within 0-1300 m, larger maturing and mature adults (DML > 90 mm) predominantly at 200-1,000 m, and the largest individ- uals (DML > 140 mm) mainly below 500 m. Capture rate of G. octopedatus varied among depth layers and the seasons, and was associated with seasonal redistri- bution of growing squid to deeper layers (Figure 4). In the entire research area, the DMLs of G. octopedatus ranged from 28 to 163 mm; two peaks on size distribution curve were due to juveniles (DML mode of 40-50 mm) and adult squid (DML > 70 mm) (Figure 5). Squid size distributions were similar in the Sea of Okhotsk and northwestern Pacific Ocean. In the Sea of Okhotsk, juveniles were found all year round and adult squid, particularly those in advanced maturity stages, were observed in all seasons except spring (Table 2). In the Pacific Ocean sciuid were captured mainly in autumn, and most of them were immature (Table 3). Sex ratios differed among the seasons and regions. In the Sea of Okhotsk, the numbers of females and males were ecjual in summer, and females were captured more frequently than males in autumn and winter. Females slightly dominated in numbers in the Sea of Okhotsk catches, and were strongly outnumbered by males in the Pacific Ocean. Males mature at smaller sizes than females, and there are regional differences in size-at-maturity distributions (Figure 6). In the Sea of Okhotsk, females were larger and males somewhat smaller than in the Pacific Ocean; however, very few animals were captured in the ocean to make reliable conclusions about regional size differences. Length-weight relationships of juveniles and females were better explained by exponential curves and those of males by logarithmic curves (Figure 7). Large mature females appeared to be heavier than males of the same size, which could be attributed to the changes that happen to females in the latest ontogenetic stages. Ripe oocytes in ovaries of prespawning females were oval (maximum diameter of about 6 mm). In the Sea of Okhotsk, G. octopedatus showed one to three variously expressed peaks on size distributions. and monthly progression of modal classes (Figure 8). Early juveniles occurred in February, July-August, and December, and large adult squid were present mainly from the late summer and through winter. In the northwestern Pacific Ocean, G. octopedatus was cap- tured from August through December; however, even data collected during such a short period revealed the presence of different size groups and monthly progres- sive changes in size composition of catches (Figure 9). Stpiid preyed on a variety of microplanktonic and nektonic animals (Table 4). During all seasons, squid consumed primarily crustaceans, which accounted for 48% of all prey items in squid stomach contents in the Sea of Okhotsk and 64% in the northwestern Pacific Ocean. The most frec]uently identified crustaceans were from the orders Euphausiacea and Copepoda. Squid were the second most important prey in the Sea of Okhotsk, and fish accounted for no more than 14% of the prey in each of two areas. Ontogenetic changes in diets of G. octopedatus have been observed: smaller juvenile squid preyed mainly upon planktonic crusta- ceans, whereas in the Sea of Okhotsk, squid and in the Pacific Ocean, fish were more important than crusta- ceans in diets of larger adult squid. Seasonal shifts in diets were less evident; however, fish were predominant prey in winter and were absent in summer and autumn, and squid were not recorded as prey in winter in the Sea of Okhotsk. Percentage of empty stomachs (Table 4) and stom- ach fullness indices (Table 5) are indicators of squid feeding activity. In the Sea of Okhotsk, proportion of empty stomachs was the smallest (8.1%) in summer, twice as large (16.7%) in autumn, and the largest (25.6%) in winter. Indices of stomach fullness showed a similar seasonal trend; mean stomach fullness for all groups examined (females, males, and juveniles) was the highest in summer (2.24), somewhat lower (1.71) in autumn and the lowest (1.37) in winter. During all seasons, juvenile animals preyed more intensively than adults in the Sea of Okhotsk. Much less infonnation is available on the feeding habits of G. octopedatus in the northwestern Pacific Ocean. Our data suggested that in the ocean, squid preyed less actively than in the Sea of Okhotsk, and cephalopods were not recorded among prey items in 35 analyzed stomach contents. DISCUSSION Our data show that G. octopedatus at various ontogenetic stages (from early juveniles to mature and spawning adults) occurred beyond the shelf zone in deep-water regions almost everywhere throughout our entire research area in the Sea of Okhotsk and northwestern Pacific Ocean. However, the frequency of occurrence may vary significantly depending upon a region, depth layer, season, and size (= life stage). O. N. Katugin et al., 2014 Page 181 Northwest Pacific Ocean CPUE . - 1 - 10 • - 10-100 • - 100 - 1000 A - Gillnets Figure 1. Gonatopsis octopedatus. Spatial distribution of juvenile squid in the Sea of Okhotsk and northwestern Pacific Ocean. Page 182 The Veliger, Vol. 51, No. 4 Figure 150 160 E 2. Gonatopsis octopedatus. Spatial distribution of adult squid in the Sea of Okhotsk and northwestern Pacific Ocean. Depth (pn) , Dq)th (pi) O. N. Katugin et al., 2014 Page 183 DML (mm) Pelagic 0 T -200 ’ -400 ’ -600 ' -800 - -1000 - -1200 - -1400 -■ 0 * * #* • * 20 “T 40 # “I 1 r- 60 80 100 DML (mm) “T 1 1 120 140 160 Bottom Figure 3. Gonatopsis octopeikitus. Vertical distribution of tlie squid in the pelagic zone (Lipper panel) and near the bottom (lower panel). Wide arrow indicates ontogenetic descent to deep-water layers. There were small size differences between sexes in G. odopeclatiLs and, at advanced maturity stages, females were usually somewhat larger than males. The fact that males mature at a smaller size than females is characteristic of other gonatid species (Nesis, 1997). Our observations and published data (Nesis, 1993; Shevtsov & Bessmertnaya, 1996) suggest that. though size-at-maturity of G. octopeclatiis may vary within and among different areas, this gonatid squid matures at about the same size in the Sea of Okhotsk, Pacific Ocean, and Sea of Japan, with sexual matura- tion starting at about 90 mm DML in males and 100 mm DML in females throughout the species range (Figure 10). Page 184 The Veliger, Vol. 51, No. 4 0-200 s gt) 200-500 S 500-1,000 04 Q >1,000 0 10 20 30 40 50 60 % Figure 4. Gimatopsis octopedatus. Seasonal patterns for vertical distribution of the squid in the Sea of Okhotsk. % = capture rate. The seasonal distribution of size structure and maturity stages of G. octopedatus suggest that, in the Sea of Okhotsk and northwestern Pacific Ocean, this species spawns from the late summer through winter. The early life stages appear from winter through summer, and the largest mature animals are observed from the late summer through winter. The observed distribution of size classes could be attributable to at least two seasonal generations of squid reproducing in the Sea of Okhotsk. Having considered the presumed succession of modal classes of any selected group of G. octopedatus, we can estimate the mean group growth 0 20 40 60 80 100 120 140 160 180 DML (mm) Figure 5. Gonatopsis octopedatus. Size of the squid in the Sea of Okhotsk and northwestern Pacific Ocean. Size, sex, and maturity of the squid GonatopsLs octopedatus in different seasons in the Sea of Okhotsk. O. N, Katugin et al,, 2014 Page 185 > _0 "5 X o OJ C/!) o 00 o o sO sO 1^, «— 3 ^ — O ro r 1 tT) tr', -sC r I t/'i O — - o I — ‘T'i so r^i ri o ^ oi rf ri O + 1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 3^_0'^k0rir^ — r^ — odinr^— -'^^dor^i'/"ir<-*'sOr^ri '=^CSOOUOOOS' — 0) C — u- S p p ^ 1) OJ ^ 3 u- S bfj = < < £ j£ 'ij ' H c u' 5 X) •p 70 H U £-2=5 CZl _ c/3 ^ 3 < l; O — o a> c3 'D [±^ ■*-> 9 t2 cC o u o o 0- 'S H cr c5 ^ ^ o rj- n o — n — ’t n — — n — — rf o Os m, O , ON O O ^ 00 n — n> I ri ri n •7}- — II n n II II ri n II n, ri NO 00 ri n sd — ' n ri n — n m, ri N +1 +1 +1 +1 ri -H +i +1 +1 "i n '-toooO'— mi’^ONri — n, ri n — rt- 00 -f O — NO m, o — mi O mi O m^ O — mi n mi-^iTin ni/-, — — n 1 1 I 1 I I I I oom^omi-^pmiooo ri 00 00 n 00 00 ri S ^ £ S X) o 53 X r P ^ i B § 3 < ^ X P o 2^ QJ Q w o H standard error; min max = DML range; n = number of animals; sex ratio = n(females):n(males). Page 186 The Veliger, Vol. 51, No. 4 matiirit}^ stage females maturity stage males Figure 6. Gomitopsis ociopcdalus. Size at maturity relationships for females (upper panel) and males (lower panel) of the squid in the Sea of Okhotsk and northwestern Pacific Ocean. O. N. Katugin et al., 2014 Page 187 0 20 40 60 80 100 120 140 160 180 DML (mm) Figure 7. Gonatopsis octupcdatus. Length weight relationships tor juveniles, males, and females. BW = body weight; filled triangles and circles indicate spawning and postspawning squid. rate and produce a hypothetical scheme of the squid life cycle in the Sea of Okhotsk. In case a group with 20-30 mm DML grew to 1 10 120 mm from February through December, the mean group growth rate would be of about 10 mm per month, which suggests that G. octopedatus attains its definitive adult size in approx- imately 1 yr of free (= postembryonic) life. In that case, the period between forming of the egg mass and the appearance of hatchlings also lasts for about 1 yr. Such an extended embryonic period is reasonable if the egg size and the ambient temperature (or the temperatures at which the eggs are developed) are taken into account (Boletzky, 1994; Nesis, 1999). The following equation describing the relationship between the length of embryonic period (D) and the eggs size (d) X 10 and ambient temperature (t) for various squid and cuttlefish has been proposed, based on the analysis of incubation data ( Laptikhovskyi, 1991): D = 48.0xe-"-"‘xd'’■"‘'<■’«‘ + "-'^ This formula satisfactorily explains the time of embryonic development in a number of decapodan families, e.g., the Ommastrephidae, distributed mainly in the lower latitudes (tropical, subtropical, and temperate zones); however, it presumably underesti- mates the length of embryogenesis in high-latitude and deep-sea squid. Using this formula, Nesis (1999) calculated that, in Goiuitiis onyx Young, 1972 egg incubation should last somewhat longer than 3 months. However, other observations indicate that the embry- onic period of Gonatus onyx should last at least three times longer, about 9 months (Seibel et al., 2000). Assuming that spawning and egg-brooding of G. octopedatus occur in deep oceanic water in the Sea of Okhotsk, and this water is below 1000 1300 m and is characterized by temperatures of 1.8-2.3^C (Morosh- kin, 1966), then, according to Laptikhovskyi’s (1991) formula, the eggs will develop in approximately 4 months. However, if like in Gonatus u/;r.v, this formula yields a three-times-shorter embryonic period for cold- water and deep-water squid, then the embryonic period for the eggs of G. octopedatus could last approximately 12 months, or 1 yr. Such a duration of the embryonic period fits well into our scheme of the species life cycle, based on the analysis of size structure annual dynamics. Indirect evidence, such as swollen fiabby mantle and arms, absence c'lf suckers, and presence of traces of black tissue (most likely remnants of the egg mass) attached to the remaining hooks on the first three arm pairs in senescent spent females of G. octopedatus indicate that, having spawned out their eggs, females of this species form an egg mass between their arms and Number Page 188 The Veliger, Vol. 51, No. 4 0 10 n 5 - January February ........ 1 — 1 4 ^ . **•«. .*•** 0 2 1 0 x=n March April May June 200 - 100 - 0 150 1 100 - 50 - 0 - 20 1 10 - 0 - 10 n 5 0 I ■ July 10 - 5 - 0 10 5 0 August September . — L- October November * I • December 10 20 30 40 SO 60 70 SO 90 100 110 120 130 140 150 160 170 DML (mm) Figure 8. Gonatopsis octopeclatus. Monthly frequency distributions for squid size at class intervals of 10 mm in the Sea of Okhotsk. O. N. Katugin et al., 2014 Page 189 DlvCL (mm) Figure 9. Gonatopsis octopeclaliis. Monthly frequency distributions for squid size at class intervals of 10 mm in the northwestern Pacific Ocean. Page 190 The Veliger, Vol. 51, No. 4 Diet of the sc]uid Gonatopsis > octopedatus in Table 4 the Sea of Okhotsk and northwestern Pacific Ocean.* Area Season Sex % E % Nonempty stomachs containing the following: Cr Fi Sq d/f n Sea of Okhotsk Summer Females 8.9 48.8 0 51.2 0 45 Males 8.7 45.2 0 52.4 2.4 46 Juveniles 6.7 78.6 0 21.4 0 45 Total 8.1 57.6 0 41.6 0.8 136 Autumn Females 31.3 27.3 0 45.5 27.3 16 Males 10.0 44.4 0 44.4 1 1.2 10 Juveniles 9.1 25.0 0 30.0 45.0 22 Total 16.7 30.0 0 37.5 32.5 48 Winter Females 31.0 27.6 58.6 0 13.8 42 Males 10.0 65.2 26.1 0 8.7 30 Juveniles 0 0 83.3 0 16.7 6 Total 25.6 39.7 48.3 0 12 78 All seasons Females 21.4 38.3 21 32.1 8.6 103 Males 9.0 51.4 8.1 35.1 5.4 86 Juveniles 6.8 55.9 7.4 22. 1 14.7 73 Total 14.9 48.0 12.6 30.0 9.4 262 Northwestern Pacific Ocean All seasons Females 40 33.3 0 0 66.7 5 Males 41.7 57.2 42.8 0 0 12 Juveniles 33.3 75 0 0 25 18 Total 37.1 63.6 13.6 0 22.7 35 * n = number of animals: % E = percentage of empty stomachs; Cr = crustaceans; Fi = fishes; Sc] = squids; d/f = digested food. perform egg-brooding behavior like some other gonatid species. Such a mode of postspawning egg care in squid was first observed in the Sea of Okhotsk near the northeastern Hokkaido coast, where large females of unidentified gonatid squid, carrying either large egg masses or remnants of egg masses attached to their arms, have been photographed (Okutani et ak, 1995). Initially, these scjuid were thought to belong to the genus Gouatopsis due to the absence of tentacles; however, later other authors suggested that these were in fact Goiuitiis sp. that had lost their tentacles upon maturation (Tsushiya et ak, 2002). Based on our data collected in the Sea of Okhotsk (where large senescent spent gonatid females with traces of black tissue attached to arm hooks and short stubs instead of broken tentacles have been observed) and taking into consideration body proportions (large size, very large fm, and long anus) of “our” animals and those photographed off Hokkaido, we suggested that all these squid were actually spent females of Gomitiis madokai Kubodera et Okutani, 1977 (Katugin & Merzlyakov, 2002; Katugin et ak, 2004). Egg- brooding has also been suggested for Gonatus fabricii (Lichtenstein, 1818) in the North Atlantic Ocean (Arkhipkin & Bjorke, 1999). In the northeastern Pacific Ocean, egg-brooding behavior has not only been documented (photographed and filmed) but also analyzed in detail for another gonatid — Gonatus onyx (Seibel et ak, 2000, 2005). It was suggested, based on molecular genetic evidence, that changes in body consistency upon maturation associated with post- spawning egg-brooding could be a synapomorphy supporting the monophyly of gonatid squid with five teeth in a transverse row on the radula (Lindgren et ak. Table 5 Mean stomach fullness indices of Gonatopsis oclopedatiis in the Sea of Okhotsk and northwestern Pacific Ocean. Sea of Okhotsk Northwestern Pacific Ocean Summer Autumn Winter Total Total Females (n) 2.24 (45) 1.63 (16) 1.19 (42) 1.72 (103) 0.60 (5) Males (n) 1.89 (46) 1.40 (10) 1.53 (30) 1.71 (86) 1.08 (12) Juveniles (n) 2.58 (45) 1.91 (22) 1.83 (6) 2.32 (73) 1.06 (18) Total (n) 2.24 (136) 1.71 (48) 1.37 (78) 1.88 (262) 1.00 (35) O. N. Katugin et al„ 2014 Page 191 Figure 10. Gomilopsis octopedatus. General view of the A, immature and B, spawned females. 2005). Therefore, G. octopcdaiii.s, being a “five-toothed” species, should have also developed an egg-brooding behavior. Like egg-brooding females of Gomitiis onyx, post- spawning females of G. octopedatus carrying their egg masses are not necessarily associated with the bottom. Most likely, they nurse their embryos at great depths in the lower mesopelagic and bathypelagic layers. The fact that no newly hatched paralarvae of this species have ever been captured in the upper epipclagic zone Page 192 The Veliger, Vol. 51, No. 4 Figure 11. Gonaiopsis octopedatiis. Schematic presentation of the species life cycle in the Sea of Okhotsk. Cross-section of the sea along approximately the 150°E is shown; DML is taken for the squid size; hatched area indicates the period of breeding and presumably 1 -yr-long postspawning egg care displayed by females. suggests that, after hatching, they reside in deep layers until they reach about 30 mm DML, and only then start ascending to the upper layers. We propose the following hypothetical scheme of the life cycle of spring-born G. octopedatus (Figure 11). Paralarvae of about 8-10 mm DML hatch from the eggs at great depths in the lower mesopelagic and bathypelagic layers or near the bottom in spring. Having grown to approximately 30 mm DML, squid ascend in the water column, where they grow to the early juvenile stage with a DML of about 40-50 mm in summer. Young squid begin extensive vertical move- ments within the epipelagic and upper mesopelagic layers. In autumn, adult squid of DML > 90 mm start descending to the lower mesopelagic and bathyal zones, where they mature at about 110-160 mm DML by winter, and spawn in spring. Males die after spawning, while females start a long-term process of brooding of an egg mass, which is formed out of the gelatinous secretion of nidamental glands, and is kept between arms I-lII attached to the arm hooks. Females stop eating, and become “floating nurseries,” as documented for some other gonatid squids with a five-toothed radula and gelatinous body. The hatch- lings appear after 1 yr of egg-brooding, and after that, the females die. Such a scheme of the individual life cycle of G. octopedatus has been corroborated by substantial evidence from patterns of the squid distribution and biology. If this scenario is correct, the life span is 2 yr for males ( 1 yr of embryonic period and 1 yr of postembryonic period) and 3 yr for females ( 1 yr of embryonic period, 1 yr of postembryonic period, and 1 yr of egg-brooding). Acknowledgments. We would like to thank John R. Bower for his useful comments on the manuscript and for editing the text. We appreciate the review of the manuscript by Richard E. Young, and useful comments on the manuscript by two anonymous reviewers. LITERATURE CITED Akimushkin, I. I. 1963. [Cephalopods of the Seas of the USSR]. USSR Academy of Sciences Press; Moscow. 236 pp. [in Russian]. Arkhipkin, a. I. & H. Bjorke. 1999. 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The duration of egg incubation in high- latitude and deep-water cephalopods: estimation and O. N. Katiigin et al., 2014 Page 193 ecological consequences. Biologiya Morya (Russian Jour- nal of Marine Biology) 25:466-474 [in Russian with English summary]. Okiyama. M. 1970. A record of the eight-anned squid, Gonatup.si.s octopeclatus Sasaki, from the Japan Sea (Cephalopoda, Oegopsida, Gonatidae). Bulletin of the Japan Sea Region- al Fisheries Research Laboratory 22:71-80. Okutani, T. 1967a. Preliminary catalogue of decapodan mollusca from Japanese waters. Bulletin of Tokai Regional Fisheries Research Laboratory 50:1 16. Okutani. T. 1967b. Preliminary note on the hitherto unknown form of the eight-armed squid, genus Gonutop- sis. from the northern North Pacific. Venus 25:65-68. Okutani, T. 1968. Review of Gonatidae (Cephalopoda) from the North Pacific. Venus 27:31-34 [in Japanese]. Okutani, T., T. Kubodera & K. Jefferts. 1988, Diversity, distribution and ecology of gonatid squids in the subarctic Pacific. A review. Bulletin of the Ocean Research Institute 26:159-192. Okutani, T., I. Nakamura & K.. Seki. 1995. An unusual egg-brooding behavior of an oceanic squid in the Okhotsk Sea. Venus 54:237-239. Sasaki, M. 1920. Report on cephalopods collected during 1906 by the U.S. Bureau of Fisheries steamer “Albatross” in the Northwest Pacific. Proceedings of the U.S. National Museum 57 (2310):163-203. Sasaki. M. 1929. A monograph of the dibranchiate cepha- lopods of Japanese and adjacent waters. Journal of Faculty of Agriculture, Hokkaido Imperial Lhiiversity 20 (Suppl. 10): 1-357. Seibel, B. a., F. G. Hochberg & D. B. Carlini. 2000. Life history of Gonatus onyx (Cephalopoda: Tcuthoidea): deep-sea spawning and post-spawning egg care. Marine Biology 137:519-126. Seibel. B. A., B. H. Robinson & S. H. D. Haddock. 2005. Post-spawning egg care by a squid. Nature 438:929. Shevtsov, G. A. & N. B. Bessmertnaya. 1996. Preliminary data on ripening and spawning of squid Gouatopsis octopcdatus (Gonatidae, Cephalopoda) in the Russian Zone of the Japan Sea. Pp. 356-359 in Okhotsk Sea and Sea Ice, 11th International Symposium: Mombetsu, Hokkaido, Japan. Shevtsov, G. A. & N. M. Mokrin. 1998. Fauna of cephalopod molluscs in the Russian zone of the Japan Sea in summer-autumn. Izvestiya TINRO (TINRO Proceedings) 123: 191-206 [in Russian with English summary]. Tslishiya, K., N. Yamamoto & H. Abe. 2002. [Cephalopods in Japanese Waters]. TBS-Britannica Co., Ltd.: Tokyo. 144 pp. [in Japanese], Volkov, A. F. & V. I. Chuchukalo. 1986. [Guide on Studies of Fish Feeding Activity.] TINRO Press: Vladivostok. 32 pp. [in Russian]. Zuev, G. V., Cii. M. Nigmatullin & V. N. Nikolskyi. 1985. [Nektonic Oceanic Squids]. Agropromizdat (Indus- trial Agriculture Press): Moscow. 224 pp. [in Russian[. The Veliger 51(4): 194-207 (September 16, 2014) THE VELIGER © CMS, Inc., 2014 Anatomical Redescription of the Limpet-Like Marine Pulmonate Trimusculus reticulatus (Sowerby, 1835) ROGER TSENG and BENOIT DAYRAT* School of Natural Sciences, University of California, 5200 North Lake Road, Merced, California 95343, USA Abstract. Trinmsculus reticulatus (Sowerby) is an air-breathing, limpet-like, pulmonate gastropod inhabiting the intertidal zone of the northeastern Pacific Ocean, from California to Washington. Although many distributional records are reported for this relatively common species, little has been published on its internal anatomy, which is also true for other Trimusculus species. The external morphology and internal anatomy of Trimusculus reticulatus are described. Scanning electron microscopy pictures of anatomical parts of Trimusculus are presented here for the first time. The anatomy of Trimusculus reticulatus is compared with other Trimusculus species. The need for a systematic revision of Trimusculus is discussed. INTRODUCTION Although Trimusculus contains less than 10 valid species, it is an important taxon in the evcTiutionary history of pulmonate gastropods. Indeed, it is one of the intriguing, phylogenetically unique, basal lineages of pulmonates, along with other taxa such as Amphi- bolidae, Siphonariidae, Ellobiidae, and Onchidiidae, which all live on the sea shore (their reproduction depends on the sea) but breath air, like land snails and slugs. Trimusculus species were previously referred to the genus Gacliitia, but Rehder (1940) demonstrated that Trimusculus Schmidt, 1818 had precedence over Gadiuia Gray, 1824. Also, Trimusculus is the only genus within the family Trimusculidae Burch, 1945. Traditionally (e.g., Thiele, 1931; Hubendick, 1946), Trimusculus species have been classified with other limpet-like pulmonates (Sipliouaria and Williamia) within Patelliformia. At present, the higher relation- ships among pulmonates are still unclear (Dayrat and Tillier, 2002, 2003), although some recent molecular analyses suggest some new, interesting possible rela- tionships. Mitochondrial and nuclear markers suggest that Trimusculus may be nested within ellobiids ( Klussmann-Kolb et ah, 2008); and mitochondrial genomes suggest that Sipliouaria might be more closely related to opisthobranchs than to pulmonates (Grande et ak, 2008). Although additional molecular data should help us understand pulmonate higher relation- ships, it is important that we continue to study the morphology of basal pulmonates because our anatom- Present address: Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA; bdayrat(g) gmail.com. ical and systematic knowledge is still remarkably poor for most basal pulmonate taxa. The objective of the present study is to redescribe the anatomy of one of the species of Trimusculus, a taxon that is still poorly known anatomically (for instance, a basic morphological structure like the radula has not been illustrated usine scanning electron microscopy [SEM]). According to the most recent revision of Trimusculus by Hubendick (1946), Trimusculus (which Hubendick refers to as Gadiuia) includes seven valid species, out of 32 nominal species: Trimusculus mammillaris (Lin- naeus, 1 758), from the Mediterranean, as well as west coast of Africa, South Africa, Mauritius, and Reunion; Trimusculus costatus (Krauss, 1848), from South Africa; Trimusculus couicus (Angas, 1867) from Aus- tralia, New Zealand, Chatham and Kerguelen islands; Trimusculus peruviauus (Sowerby, 1835) from the southeastern Pacific; Trimusculus reticulatus (Sowerby, 1835) from the northeastern Pacific, and possibly the southeastern Pacific (see below); Trimusculus odhueri (Hubenciick, 1946) from the Philippines and Polynesia (Rehder, 1980); Trimusculus goes! (Hubendick, 1946) from the Caribbean. The systematics of Trimusculus, however, needs to be revised again, because some of Hubendick's synonymies have not been accepted: for instance, Abbott (1974) regards Trimusculus stellatus (Sowerby, 1835), from the Gulf of California to Nicaragua, as a valid species. Also, the status of many generic and specific names need to be reevaluated, such as Gadiuia Gray, 1824, C/v/^ciry Scacchi, 1833, Mouretia Sowerby, 1835, and Gadiualea Iredale, 1940 (e.g.. Gray, 1840; Hubendick, 1946). Finally, some of the species distributions proposed by Hubendick are questionable; for instance, it is unclear whether T. mammillaris is R. Tseng & B. Dayrat, 2014 Page 195 present in the Mediterranean, as well as the west coast of Africa, South Africa, Mauritius, and Reunion, or if it is restricted to the Mediterranean, with other names applying to other entities, such cxs, Trinnisciilus maiiv- itiauus (Martens, 1880) for limpets from eastern Indian Ocean islands. At this stage, it is unclear how diverse Trimuscuhis really is. The anatomy of Triniuscidus has been studied in a few species: Trimiiscii/iis manwulavis (Linne, 1758) was described by Pelseneer (1901), Schumann (1911), and Dieuzeide (1935); T. peruviaiiiis, T. conicus, and T. retiailatus were described by Schumann (1911), Walsby et al. (1973), and Dali (1870), respectively; and Hodgson and Healy (1998) described the sperm morphology of T. costatus. Although some of the olcier descriptions are detailed and quite informative, they sometimes lack adequate illustrations (e.g., Dali, 1870); also, it appears that SEM, which provides us with access to many detailed anatomical features, has not been previously used. Triunisculus reticulatus is present on the western coast of North America, from Cape San Lucas, Baja California, Mexico (Dali, 1870) to Washington (God- dard et al., 1997). Dali (1870) determined for the first time that it lacked a gill and was an air-breathing pulmonate. Yonge (1958), Van Mol (1967), Walsby (1975), and Hodgson and Healy (1998) added infor- mation concerning specific parts of the body: the pulmonary cavity, cerebral ganglion and procerebrum, digestive organs involving in feeding, and spermatozoa morphology, respectively. In this study, the anatomy of T. reticulatus is entirely redescribed. SEM is used for the first time to illustrate external and internal structures (kidney, radula, stom- ach, etc.). The anatomy of T. reticulatus is compared to the anatomy of other species, based on existing literature. Characters potentially useful for alpha- taxonomy are discussed. The phylogenetic position of T. reticulatus also is discussed. MATERIALS AND METHODS Eor the present study, 39 specimens were examined, 20 of which were dissected. All specimens are held at the California Academy of Sciences, San Erancisco (CASIZ): California, Marin County, Dillon Beach, 2nd Sledge Road, 27 Eebruary 1983, 17 specimens, leg. D. Chivers, identified as Trimusculidae by unknown identifier (CASIZ 057768; 15 specimens dissected: 17/ 16 [#1], 20/20 f#2], 20/18 [#3], 20/17 [#4], 17/15 mm [#5], 17/16 [#6], 14/13 [#7], 14/13 [#8], 12/11 [#9], 17/16 f#10], 18/17 [#1 1], 18/17 mm [#12], 16/14 [#13], 14/13 [#14], 17/14 [# 15] mm);California, Santa Cruz County, Scott Creek, 6 July 2()08, three specimens, leg. R. Tseng and B. Dayrat, identified as Trimuscuhis reticulatus by R. Tseng and B. Dayrat (CASIZ 177991; 1 specimen dissected: 17/17 mm [#1] mm); California, San Luis Obispo Country, Montana de Oro, 2 January 2007, two specimens, leg. J. Goddard, identified as Trimuscuhis reticulatus by J. Goddard (CASIZ 177989); California, San Luis Obispo Country, Montana de Oro, 2 January 2007, three specimens, leg. J. Goddard, identified as Trimuscuhis reticulatus by J. Goddard (CASIZ 177990); California, Santa Cruz Country, Scott Creek, 23 October 2006, seven specimens, leg. J. Goddard and B. Dayrat, identified as Trimuscuhis reticulatus by J. Goddard and B. Dayrat (CASIZ 177988); and Washington, Clallam County, 13 July 1995, seven specimens, leg. J. Goddard, identified as Trimuscuhis reticulatus by J. Goddard (CASIZ 105924; four specimens dissected: 16/15 [#1], 8/8 [#2], 8/7 [#3], 8/8 [#4] mm). All anatomical observations were made using a dissecting microscope and drawn with a camera lucida. Parts were prepared for SEM for most specimens dissected (SEM preparations are listed in Appendix 1). Radulae were cleaned in 10% NaOH for a week, rinsed in distilled water for at least a week, briefly cleaned in an ultrasonic water-bath (less than a minute), sputter- coated with gold-palladium, and examined in an SEM (University of California Merced SEM laboratory). Soft parts were dehydrated in ethanol and critical-point dried before coating. In total, 47 SEM stubs were prepared, including 14 radulae. Because all lots included more than one specimen, each specimen was labeled, both inside the jar and on the stubs, so that future workers will be able to link unambiguously the present anatomical descriptions to particular specimens from the CASIZ collection. Abbreviations: a, anus; be, buccal commissure; bed, bursa copulatrix duct; bg, buccal ganglion; bim, buccal lateral muscle; m, buccal mass; ebe, cerebro-buccal connective; cc, cerebral commissure; eg, cerebral ganglion; epe, cerebro-pleural connective; cpec, cere- bro-pedal connective; dg, digestive gland; e, eye; es, esophagus; fo, female opening; f, foot; g, gonad; enr, circLimesophageal nerve ring; h, head; i, intestine; k, kidney; mm, mantle margin; m, mouth; mtm, mantle tissue muscle; na, anus nerve; ncapl, head nerve 1; ncap2, head nerve 2; ncap3, head nerve 3; ne, esophagus nerve; nf, foot nerve; nfo, female opening nerve; ng, genital nerve; nl, lateral nerve; nm, mantle nerve; nmo, male opening nerve; no, optic nerve; npal, parietal nerve 1; npa2, parietal nerve 2; npa3, parietal nerve 3; npa4, parietal nerve 4; npel, pedal nerve 1; npe2, pedal nerve 2; npc3, pedal nerve 3; np. posterior nerve; nr, radula nerve; ok oral lobe; orm, oral retractor muscle; ov, oviduct; p, pneumostome; pbrm, pharynxbulbus retractor muscle; pec, pedal commissure; peg, pedal ganglion; per, pericardium; pg, pleural ganglion; ppc, parapedal commissure; ppec. pleuro-pedal Page 196 The Veliger, Vol. 51, No. 4 Figure 1. Syntype of Rowetliu radiata Cooper iu Gabb, 1865 (NMNH #25027). A. Dorsal view, scale = 1 mm. B. Ventral view, scale = I mm. C. Left, lateral view, scale = I mm. connective; prm, penial retractor muscle; ps, penial sheath; re, renal pore; r, rectum; s, shell; see, subcerebral commissure; sgrm, salivary gland retractor muscle; sg, salivary gland; sm, shell muscle; spm, spermoviduct; s. stomach; vd, vas deferens; vg, visceral ganglion; vl, visceral loop. NOMENCLATURE AND TYPE MATERIAL The type species of Trimusciihis is Patella nummiillaris Linnaeus, 1758, by subsequent designation (Rehder, 1940:68). Several authors (e.g., Dali, 1870; Hubendick, 1946; Rehder, 1940) have listed synonyms for Trinnis- culits (whether they referred to it as Trinniscithis or Gadinia). The monotypic family name Trimusculidae was created by Burch (1945). Trinnisciihis reticidatus was originally described as Mouretia reticulata in a brief article by Sowerby which includes species descriptions based on the "'Shells collected by Mr. Cuming on the western coast of South America and among the Islands of the South Pacific Ocean” (Sowerby 1835:4). The original description is brief: “MoURETlA RETICULATA. Mow. testa siihde- presso-conied. suhrotundatd, superne reticulata, alhci. Hah. ad Valparaiso. Found attached to shells in deep water, from forty-five to ninety fathoms” (Sowerby 1835:6). Thus, according to Sowerby, the type locality of T. reticidatus is Valparaiso, South America, from 82 to 164 m depth. However, according to Dali (1870:18), “the species was originally described from the Gulf of California or Lower California, though by some misplace- ment of labels the habitat was published as Valparaiso, in deep water.” The type material is not at the Natural History Museum, London (Abblett, personal communi- cation) and is probably lost. It is thus not possible to verify whether the material described by Sowerby is similar to Triniusculus specimens from the northeastern Pacific. Another species name was created for Triimiscidiis from the northeastern Pacific: Gadinia (Rowellia) radiata Cooper in Gabb, 1865, based on material collected by Cooper and Rowell from Farallon Islands, Half Moon Bay, New Year’s Point, Santa Barbara, and Santa Catalina Islands. So far, authors have adopted Dali’s (1870) opinion and regarded Gadinia radiata as a synonym of T. reticidatus. The two syntypes Gadinia (Rowellia) radiata Cooper in Gabb, 1865, held at the National Museum of Natural History (#25027), two well-preserved shells from Catalina Islands, were examined for the present study; they are very similar to our non type material of T. reticidatus (Figures lA- C). When Triniusculus is revised, T. radiatus could be used as a valid name for the Trimusculus from the northeastern Pacific, if it appears that the name T. reticidatus is too problematic (because its current distribution does not match its type locality). However, this would have to be discussed within the context of a global systematic revision of Triniusculus, and the name Triniusculus reticidatus, which has always been used, is kept here so that nomenclature is not disturbed. NATURAL HISTORY Live animals are found under the roof of overhanging rocks or caves in the lower intertidal zone (the authors observed live animals on the coasts of Santa Cruz County, California; Figure 2A). Because T. reticidatus requires this very specific habitat, it is not distributed continuously along the coast but can be found easily if in the right habitat. Usually, small groups of less than 10 individuals are observed close to each other, hidden within small crevices (Figure 2B). The animals are well camoutlaged, especially with red calcareous algae covering both their shells and the surrounding rocks (Figure 2C). Barnacles can also be found attached on the shell. When the animal is detached from the rock, it immediately secretes some milky, whitish mucus from its mantle margins (near the shell margin). This mucus seems to kill the larvae of the tube worm Pliragniatopoina R. Tseng & B. Dayrat, 2014 Page 197 Figure 2. Habitat of Triiinisculiis reticiilatiis and live animals from Scott Creek, Santa Cruz County, California. A. Example of overhanging rocks and caves in the lower intertidal zone in which T. reticiilatiis can be found. B. Four individuals on the roof of an overhanging rock. C. Two live animals whose shells are covered by red algae. California and is repugnant to a number of starfish species (Rice, 1985; Manker & Faulkner, 1996). Shells of old individuals are often found fitting perfectly within the rock, suggesting that live animals are citiite inactive or that, like other limpets, they tend to return to their specific, individual position. Trinmsciiliis reticiila- tiis feeds by secreting adhesive mucus to collect diatoms and other small organisms when submerged under water (Walsby, 1975). Although T. reticiilatiis is an air- breathing animal, it is able to remain submerged for long periods of time. Haddock (1989) suggested this durability might be the reason why T. reticiilatiis is able to inhabit another unique environment: the air pockets of reef caves at 4.5 m below sea level. ANATOMICAL RE-DESCRIPTION Shell (Figures 2C, 3); The shell is patelliform, i.e., conical and slightly Hattened, and nearly circular (Figures 3A-C). Distinct dorsal ridges radiate from the protoconch toward the shell margin; their number varies from 37 to 52 in the specimens examined (Figure 3E). The shell is whitish-light grayish, but the dorsal surface of live animals is often covered with red, calcareous algae, as well as barnacles. The height varies from 2.5 to 10 mm. The apex consists of the patelliform protoconch, coiled at its very top (Figure 3D). The size of the specimens examined ranges from 8 to 20 mm in diameter. A horseshoe-shaped scar can be seen on the inner (ventral) side of the shell (Figure 3C). This scar is largely due to the trace of the shell muscle. The right anterior margin of the scar corresponds to the margin of the shell muscle and the space where the pneumo- stome opens (Figure 3F). The left anterior margin of the scar corresponds to the trace of muscular tissue attached to the pallial roof but distinct from the shell muscle (Figure 3F): see the mantle tissue muscle (mtm. Figure 5B). In some individuals, the trace of that muscular tissue is separated from the scar of the shell muscle. Page 198 The Veliger, Vol. 51, No. 4 Figure 3. Shell (CASIZ 105924, specimens #3 & 4). A. Left lateral view (#3), scale = 2 mm. B. Dorsal view (#4), scale = 2 mm. C. Ventral view (#4), scale = 2 mm. D. Protoconch (#3), scale = 100 gm. E. Shell margin, lateral view (#4), scale = 200 pm. F. Ventral view, detail of anterior muscle scar (#4), scale = 1 mm. External morphology (Figures 2C, 4, 5A— C): The foot, the two oral lobes, the mantle margin, and the pneumostome can be observed ventrally (Figure 5A). The whitish foot is slightly oval and covers most of the ventral surface; its height corresponds to approximately one-third of the total body height. Two tlattened, broad, and partially overlapping oral lobes are present anteriorly. Their ventral surface bears grooves, likely covered by chemical receptors and involved in the secretion of mucus. The right lobe (which is on the side of the male opening) bears more grooves than the left one. Anteriorly, the oral lobes also bear minute ridges on their dorsal surface. The tiny male opening (<0.5 mm in diameter) is lateral and anterior to the right lobe. The female opening, also tiny, is located at the base of the right oral lobe (Figure 5C). The head is continuous with the oral lobes, posteriorly (Figure 5C). A pair of small black eyes is present on each side. The mouth is located in between the overlapping regions of the oral lobes, and is hardly seen in live animals. The mantle covers the entire inner surface of the shell, except for the margins. The mantle located within the limits of the shell muscle, which Yonge (1958) categorized as columnar, is translucent. Be- tween the shell margin and the shell muscle is the mantle margin, or mantle skirt (Figure 5B): it is thicker, yellowish, and filled with subepithelial mucous glands (Yonge, 1958; Walsby, 1975). As pointed out by Dali (1870), the thickest area of the mantle margin surrounds the pneumostome region. The ventral surface of the mantle margin bears many microscopic pores (Figure 4A), which likely are the openings of the mucous glands. The side of the foot and the latero-dorsal region of the head are also rich in mucous glands (Yonge 1958; Walsby 1975). A blood sinus is embedded within the mantle margin, surrounding R. Tseng & B. Dayrat, 2014 Page 199 Figure 4. External morphology (CASIZ 057768 #7). A. Surl'ace of mantle near the pneumostome. with pores, scale = 50 pm. B. Pneumostome, scale = 200 pm. the shell muscle; it enters in the visceral cavity at the anterior gap of the shell muscle. Between the mantle margin and the foot is the ciliated pallial groove (Yonge, 1958), in which numer- ous particles and white mucus residues can be found. In live animals, tluids and small particles circulate within the pallial groove, which serves as a pathway to transport sediments about. The pneumostome is located on the edge of the mantle margin, near the right oral lobe (Figure 5B). It is distinctly U-shaped (Figure 4B). It is the distal opening of a narrow pathway that leads to the pulmonary cavity, which is located in the anterior region of the body. The shell muscle is yellowish. When all the organs of the visceral cavity (delimited by the shell muscle) are removed, the shell muscle displays a concave inner surface. Near the left anterior end of the shell muscle, a small muscle present on the mantle roof may or may not fuse with the shell muscle (Figure 5B). The posterior part of the visceral cavity is mostly filled with the digestive gland and the gonads. The gonad, located on the right side of the digestive gland, is distinguishable by its bright yellowish color and granular surface. The anus, close to the pneumostome, is shaped like a baseball glove with the pocket facing the pneumostome; the rod-shaped dark-colored feces exit from the anus to the outside through the pneumostome. Digestive system (Figures 5B-D, F, 6, 7A): The buccal mass contains the radula within the transparent radula sac. supported by the odontophore cartilage and surrounded by buccal muscles. The odontophore cartilage is shaped like a bow (front section of a boat) with an open posterior end and continuous with two lateral cartilages. Near the anterior tip of the odontophore, the radula sac opens to expose the anterior part of the radula. The radular formulae of the specimens examined range from 122 X (49-1-49) in a 8-mm-long specimen (CASIZ 105924 #4) to 157 X (82 -1-82) in a 14-mm- long specimen (CASIZ 057768 #7) and 171 X (78 1 — 78) in a 17-mm-long specimen (CASIZ 057768 #10). The size of the radulae observed ranges from 1.2 mm in length by 0.34 mm in width to 2.5 by 0.75 mm. The half rows of lateral teeth are symmetrically identical along the rachidian axis. Each half row forms an angle of approximately 45° with the rachidian axis. The rachidian teeth are small ( — 10 pm long): its base is U- shaped, with the two lateral branches pointing poste- riorly; a few median, minute denticles are present at the anterior end (Figures 6B-D). The size and shape of the lateral teeth vary, depending on their position in the half row. The innermost teeth are characterized by a long and sharp cusp and one small, inner denticle (Figure 6D). The cusp of the innermost teeth measures from 13.5 to 17.5 pm in length. The cusp of the outer lateral teeth is significantly shorter and bears up to five denticles on each side (Figure 6E). The size of the cusp of the lateral teeth decreases and the number of denticles on either side of the cusp increases from the innermost to the outermost lateral teeth (Figure 6F). The shape of radular teeth varies between individuals, especially the cusp of the innermost lateral teeth: in some individuals, the cusps are long, with a single denticle on each side (Figure 6C), whereas in other individuals the cusps are wider and shorter with one or two denticles on each side (Figure 6B). The esophagus connects dorsally to the buccal mass and passes on the left side of the visceral mass (the right side is largely filled with reproductive organs) to enter the anterior part of the stomach ventrally. In some Page 200 The Veliger, Vol. 51, No. 4 Figure 5. External morphology and internal anatomy. A. Ventral view (CASIZ 057768 #9), scale = 2 mm. B. Dorsal view, shell removed (CASIZ 057768 #15), scale = 2 mm. C. Dorsal view of the postcephalic region, illustrating the connections between the central nervous system, digestive system, and reproductive system (CASIZ 057768 #6), scale = 1 mm. D. Dorsal view of the digestive system, with digestive gland removed (CASIZ 057768 #7), scale = 1 mm. E. Dorsal view of the reproductive system, with vas deferens cut anteriorly (CASIZ 057768 #6), scale = I mm. F. Left lateral view of the buccal mass (CASIZ 1 77991 #1), scale= 1 mm. individuals, the middle part of the esophagus is larger than its anterior and posterior ends. A muscle originates from the posterior shell muscle wall and inserts onto the posterior part of the esophagus. Two unbranched salivary glands connect to the buccal mass dorsally near the origin of the esophagus. run along each side of the esophagus (up to its middle part), and finally join a muscle that attaches to the left lateral inner wall of the horseshoe shell muscle. The stomach is embedded ventrally within the digestive glands (Figure 5D). There are no gizzard plates or spines: the lining is smooth, with small R. Tseng & B. Dayrat, 2014 Page 201 Figure 6. Radular teeth. A. Complete radula (CASIZ 057768 #7). scale = 500 pm. B. Rachidian and innermost lateral teeth (CASIZ 057768 #7), scale = 10 pm. C. Rachidian and innermost lateral teeth (CASIZ 057768 #8), scale = 10 pm. I). Rachidian and innermost lateral teeth (CASIZ 105924 #2), scale = 5 pm. K. Left, outermost lateral teeth (CASIZ 057768 #8). scale = 10 pm. F. Outer to inner left, lateral teeth (CASIZ 057768 #8). scale = 20 pm. Page 202 The Veliger, Vol. 51, No. 4 Figure 7. Internal anatomy. A. Inner lining of stomach (CASIZ 057768 #7). scale = 20 |.im. B. Penial papilla (CASIZ 057768 #9), scale = 200 pm. C. Lining of penial sheet (CASIZ 057768 #9). scale = 10 pm. D. Internal surface of kidney (CASIZ 057768 #11), scale = 200 pm. E. Transversal section of mantle roof of the lung (CASIZ 057768 #7), scale = 200 pm. F. Internal surface of kidney, detail (CASIZ 057768 #11), scale = 10 pm. G. Renal pore (CASIZ 057768 #3). scale = 50 pm. grooves (Figure 7A). The intestine originates from the left, posterior region of the stomach and, after three loops, exits from the digestive gland to continue as a straight rectum up to the anus, near the pneumostome. Oral muscles (Figure 5F): Complex muscles control the movements of the buccal mass and oral lobes. Their description below follows the muscle nomenclature used by Schumann (191 1) for Trimiisciihis penivuunis. External buccal muscles include the following: two lateral muscles originating from the visceral Iloor near the pleural ganglia and inserting on the posterior part of the buccal mass; a pharynx- bulbus retractor originating from the inner wall of the posterior shell muscle with two insertion sites (the posterior radula sac and the posterior odontophore cartilage); a pharynx protractor originating from the posterior odontophore cartilage extending ventrally to the buccal mass and fusing with the dorsal interior of the oral lobes; and a series of short muscles (which can hardly be separated by dissection) connecting the oral lobes to the anterior buccal mass. The buccal mass also is rich in internal muscle fibers, which form two lateral, symmetrical masses on either side of the radula. Distinct muscles can hardly be seen by dissection. Oral lobes are moved mainly through the action of the oral retractor, which originates from the inner wall of the posterior shell muscle, runs on the floor of the visceral cavity, passes between the visceral loop and the pedal ganglia, and inserts on the ventral inner sicie of the oral lobes. It is the longest muscle of the body. Reproductive System (Figures 5C, 5E, 7B-C): The hermaphroditic gland, or ovotestis, located on the right side of the digestive gland, can be seen dorsally (after the shell is removed) and is distinguishable from it by its bright yellowish color and granular surface. Gametes exit the ovotestis through the hermaphroditic duct, which can hardly be seen by dissection, and reach the spermoviduct, which also transports both male and female gametes. The proximal part of the spermoviduct is tightly embedded within the digestive gland and the female gland mass (mucus and albumen gland). The distal part of the spermoviduct is not tightly embedded with other organs of the visceral mass and is easily seen by dissection: it is large, whitish, located on the right side (Figure 5C). Distally, it branches in two separate R. Tseng & B. Dayrat, 2014 Page 203 male and female ducts: the vas deferens and the oviduct. Proximally, the vas deferens (Figure 5C) is a thin duct that travels anteriorly and makes a first U-turn near the right eye where it becomes tiny and inconspic- uous, embedded within the tissue of the oral lobe. While traveling posteriorly, the vas deferens gradually increas- es in size and then makes a second U-turn, where the retractor muscle of the penial sheath inserts (originating from the posterior inner surface of the shell muscle). After a few loops, the vas deferens eventually enlarges and ends as a papilla (Figure 7B) in the proximal region of the penial sheath (Figure 5C). There is no distinct penis: the size of the papilla measures from 200 pm long in a 12-mm specimen (CASIZ 057768 #9) to 345 pm long in a 20-mm specimen (CASIZ 057768 #2). The straight penial sheath extends anteriorly at the male opening, on the inferior side of the right oral lobe. Its lining is ciliated (Figure 7C). From the distal region of the spermoviduct, the oviduct, larger than the vas deferens, extends anteriorly up to the female opening (Figure 5E), forming one loop. In smaller specimens, the oviduct is less convoluted than in larger specimens. A thin duct branches off the oviduct and transports exosperm up to the bursa copulatrix, a brown, spherical pouch embedded within the digestive gland (Figure 5E). Nervous system (Figures 5C, F, 8): The hypoathroid circumesophageal nerve ring consists of three pairs of left and right ganglia: two dorsal cerebral ganglia, two lateral pleural ganglia, and two ventral pedal ganglia. Those ganglia are not fused and are easily distinguished (Figure 8A). A procerebrum is present in each cerebral ganglion. The left and right cerebral ganglia are connected by a cerebral commissure and a subcerebral commissure, the latter being ventral to the former. The pharynx protractor is innervated by the subcerebral commissure. The cerebral commissure is the largest commissure of the circumesophageal nerve ring. The left and right pedal ganglia are connected by a pedal commissure and a smaller parapedal commissure, the latter being ventral to the former. The left and right pleural ganglia are connected by a visceral loop (which is not part of the circumesophageal nerve ring) with only one ganglion, the visceral ganglion, which is closer to the right pleural ganglion than to the left pleural ganglion. The visceral loop is about the same length as the cerebral commissure. On each side of the esopha- gus, the cerebral, pleural, and pedal ganglia are connected by three connectives: the cerebro-pleural, cerebro-pedal, and pleuro-pedal connectives. The cerebro-pedal connective is the longest and pleuro- pedal connective is the shortest. From the ganglia of the central nervous system, the nerves connect to all parts of the body. The nerves from the cerebral ganglia innervate mainly the cephalic region, especially the oral lobes and the eyes. The nerves from the pleural and visceral ganglia innervate the lateral sides of the body, the mantle, and the visceral organs. The nerves from the pedal ganglia mainly innervate the ventral region, especially the foot. The detailed description of the nerves provided below follows the nerve nomenclature proposed by Schumann (1911) for Triuiii.se ulus garuoti (Philippi, 1836). Four major nerves originate from each cerebral ganglion: the optic nerve, and the cap 1 , cap 2, and cap 3 nerves (Figure 8B). Only cap 3 displays some differences between the left and the right sides. Both the optic nerve and cap 1 originate from the procerebrum area of the cerebral ganglia. The optic nerve connects to the eye. The cap 1 nerve fuses temporarily with cap 2; the resulting nerve branches distally in several nerves that innervate the mouth area and the oral lobes (the points of fusion and branching vary between individuals). The nerve cap 3 is the largest cerebral nerve. It innervates the oral lobes. On the right side, cap 3 also innervates the distal end of the vas deferens and the male opening. The cerebral ganglia also connect to a pair of buccal ganglia by a pair of left and right cerebro-buccal connectives (Figure 8D). The two buccal ganglia are connected by a single buccal commissure. From each buccal ganglion, a radular nerve enters the buccal mass and an esophagus nerve runs posteriorly on the surface of the esophagus and then fuses with it; other smaller nerves innervate the buccal mass, most likely muscles. The nerves that originate from the left and right pleural ganglia are quite different (Figure 8A). From the left pleural ganglion, two nerves (pa 1 and pa 2) innervate the mantle margins near the cephalic region and the left anterior region of the shell muscle; two additional nerves (pa 3 and pa 4) innervate posteriorly the shell muscle. From the right pleural ganglion, a nerve innervates the mantle margin near the cephalic region, and two nerves innervate the female opening and the oviduct. The left and right pedal ganglia display six similar major nerves: pe I, pe 2, pe 3, par 1, par 2, and par 3 (Figure 8C): pe I innervates the anterior region of the foot; par 1 and par2 innervate the lateral region of the foot; pe 2 extends diagonally and ventrally; pe 3 innervates the posterior region of the foot; and par 3 innervates the central region of the foot. Four nerves originate from the visceral ganglion (Figure 8A). One nerve innervates the female repro- ductive organs. Two other nerves extend to the lateral and posterior shell muscle: they actually go through the muscles (which they might innervate partially) and innervate the lateral and posterior mantle margin. The fourth nerve follows the digestive tube up to the an us. Page 204 The Veliger, Vol. 51, No. 4 Figure 8. Nervous system (CASIZ 057768 #14). A. Dorsal view, scale = 1 mm. B. Dorsal view (details of the cerebral ganglia and nerves), scale = 0.5 mm. C. Ventral view (pedal ganglia), scale = 0.5 mm. D. Dorsal view (cerebral and buccal ganglia), scale = 0.5 mm. Respiratory, excretory, and circulatory systems (Figures 5B, 7D-G): The pulmonary cavity, the kidney, and the heart are all located in the mantle roof and are functionally interconnected. The respiratory system consists of the pulmonary cavity, filled with air, and the pneumostome. The pulmonary cavity is delimited dorsally by the columnar mantle, characterized in this region by two layers of epithelium (external and internal) defining a space in which blooci is carried for gas exchanges (Figure 7E). The columnar mantle, which is only 180 pm thick, connects to the kidney dorsally as well as to the floor of the visceral cavity ventrally. The kidney is flattened and translucent. On its inner surface, it bears yellowish papilla which are elongated near the shell muscle and bulb-like near the digestive gland (Figure 7D). A minute renal pore is located at the anterior tip of the kidney (Figure 7G). Posteriorly, R. Tseng & B. Day rat, 2014 Page 205 the kidney tissue becomes continuous with the pericar- dium. which delimits the pericardic cavity. The ventricle is enclosed within the pericardium. Its anterior opening is continuous with numerous thin blood vessels, which may be part of the transparent auricle, which is difficult to identify through dissection. Its posterior opening is continuous with a thin transparent tissue that surrounds the internal organs (buccal and visceral organs). DISCUSSION Previous studies have described T. reticidatiis inhabit- ing as densely populated colonies of about 30 to 50 individuals with shells in contact with each other (Dali. 1870; Yonge, 1960; Walsby, 1975; Rice, 1985). Walsby et al. (1973) observed such compact clustering in T. coiiiciis as well. However, our field observations yielded smaller groups of less than 10 individuals, with most individuals hidden within small crevices. Trinniscii/iis coniciis also shelters itself from waves within small crevices (Walsby et al., 1973). Studies have demonstrated that individuals may remain at the same position for up to 2 yr (Yonge, 1960; Haddock, 1989; Walsby, 1975). Rice (1985) suggested that this apparent absence of movements may help the air-breathing limpet to budget the oxygen consumption when submerged under water. In T. conicus, young individuals appear to be more actively moving than adults, which exhibit the same apparent lack of movement as in T. reticulatiis (Haven. 1973). However, Dali (1870) and Yonge (1958) reported T. veticidcitiis is able to move, suggesting that live animals do not stay all the time at the same position. However, the fact that individuals fit so well within the rock suggests that they may go back to their specific position after activities, whether those take place at low or high tides (Lindberg and Dwyer 1983). Due to the stationary behavior of T. rericidatiis, the animal endures long periods of time under water. The respiratory movements are mainly controlled by the pneumostome. When above water, the pneumostome opens widely to breathe in air and oxygen. When under water, an air bubble is maintained at the closed pneumostome, which opens at irregular intervals to let water in (Yonge, 1958). The exterior of the air bubble is enclosed by a mucus membrane secreted at the base of the head. Trimuscidus conicus exhibits identical pneumostome activity (Walsby et al., 1973). Both Yonge ( 1958) and Walsby et al. ( 1973) suggest that T. reticulatus may retain the ability to breathe in water as well as in air. However, Haddock (1989) found T. reticulatus colonies at 4.5 m below sea level, in a unique subtidal habitat of a trapped air pocket under rock ledges, suggesting that T. reticulatus can only survive if breathing air, indicating that it is a truly air-breathing limpet. In fact, like other marine pulmonates (such as Onchidiidae and Ellobiidae), they get out of the water if one puts them into a bucket with water (Yonge, 1958). The anatomical comparison of T. reticulatus with several past studies (Yonge, 1958, 1960; Dali, 1870; Walsby, 1975; Van Mol, 1967) reveal consistent descriptions in the shell and the external morphology. The descriptions of the internal organs in most systems are also similar, with few exceptions: a temporary fusion of the nerves cap 1 and cap 2 (from the left cerebral ganglia) was not described by Van Mol ( 1967). Dali ( 1870) erroneously referred the kidney as the lung and to the buccal ganglia as “two pear-shaped salivary glands.” Also, his problematic terminology (e.g., “ovary,” “uterus,” and “testicles”) generates some confusion in his description of the reproductive system. However, the general location of the kidney and the diaulic reproductive organs are compatible with the present observations. The anatomy of T. reticulatus can be compared with the anatomy of three other species: T. peruvianus (Schumann, 1911), T. mainnularis (Dieuzeide, 1935; Schumann, 191 1), and T. conicus (Walsby et al., 1973). The external morphology and several internal struc- tures are similar; ( I ) the pulmonary cavity and surrounding organs (pneumostome, kidney, heart, and vascularized tissue of the lung); (2) the buccal mass (ridged surface of proboscis, shape of the odontophore, and shape of the radula); (3) pattern of the intestine and rectum; (4) shape and position of the ganglia of the hypoathroid circum-esophageal nerve ring; (5) organization of the diaulic reproductive system extending from the spermoviduct; and (6) a long bundle of retractor muscles originating from the posterior end connecting to the esophagus, vas deferens, salivary glands, and buccal mass. There are a few differences. The major ganglia and innervations to the body parts are consistent between T. reticulatus and T. inaniniilaris, except for the lack of an osphradial ganglion (Schumann, 1911) and “nerf accoustique” (Dieuzeide, 1935) in T. reticulatus. Radular formulae have been reported in the literature for other species (Table 1). In T. reticulatus, the present studies demonstrate that the numbers of rows and teeth per half row vary among individuals, depending on the size of the animals (Table I). In T. conicus, half rows seem to host a smaller number of teeth, at comparable size, but nothing is known about the variation in T. conicus. Trinnisculus niainnu'laris seems to also possess fewer lateral teeth per half row we found 122 X (49 1 49) in an 8-mm-long specimen- but, again, nothing is known about the radular variation in T. inaninullaris. As for the shape of the teeth, it varies slightly between individuals. However, it is very difficult to compare SEM pictures with written descriptions of radular teeth, or even drawings, and Page 206 The Veliger, Vol. 51, No. 4 Table 1 Radular formulae of specimens dissected for the present study and from the literature Triimisciihis species Reference Specimen catalogue number Length (mm) Radular fonnula T. reticulatus Present study CASIZ 105924 #4 8 122 X (49- 1-49) T. reticulatus Present study CASIZ 057768 #7 14 157 X (82- 1-82 T. reticulatus Present study CASIZ 057768 #10 17 171 X (78- 1-78) T. peruviauus Schumann, 1911 N/A 17 175 X (60- 1-60) T. peruviauus Schumann, 1911 N/A 19 175 X (76-1-76) T. couicus Walsby et af, 1973 N/A 14 1 16 X (45- 1^5) T. luammiilari.s Dieuzeide, 1935 N/A 7 to 10 ? X (27- 1-27) T. numiuuUari.s Schumann, 1911 N/A unknown 100 X (27- 1-27) the systematic value of tooth shape in species identification is unknown. One question of interest is what kind of characters could be useful in alpha-taxonomy for distinguishing species. Given that the reproductive system is quite similar among species, and given the lack of a distinct, complex, male copulatory organ (at least in the species investigated), it might be difficult to distinguish species based on the reproductive system. Radular characters usually tend to be useful at the supraspecific level (at the generic level in particular). The use of radular characters in Trinnisculiis cannot be answered at this stage mainly because SEM pictures are only available for T. reticii/atiis (present study). Also, some species seem to have radulae with fewer teeth per half row than T. reticulatus, but infraspecific variation should first be addressed in species. The shell, of course, remains useful. However, a revision should address infraspecific variation. As for most limpets, shell shape and size vary due to environmental factors. Finally, other anatomical systems (nervous, circulatory, etc.) seem to be similar among species and will likely provide no features useful at the species level. Hodgson and Healy (1998) were able to identify two differences in the spermatozoa morphology between T. j-eticiilatiis and T. coslatus. Therefore, Trinutsculus seems to be a taxon in which species limits should probably be studied using DNA techniques in addition to morphology, as it is unclear whether morphology alone will provide enough fea- tures to separate species. Recent molecular results suggest that Trinnisculiis might be nested within ellobiids (Klussmann-Kolb et ak, 2008; Dayrat et ah, 2011; White et ah, 201 1 ). So far, no moiphological character has been identified sug- gesting such affinities (Dayrat and Tillier, 2002). However, adaptation to limpet-like shape might have affected radically the anatomy of Triniusciiliis, which could prevent us from discovering similarities. Acknowledgments. We thank Terry Gosliner (California Academy of Sciences) and Jerry Harasewych (National Museum of Natural History) for allowing us to borrow specimens. We thank Jonathan Abblett (Natural History Museum, London) for answering our questions about Sowerby’s type material. We are grateful to Jeff Goddard for sharing with us his abundant knowledge in the field, and Mike Dunlap (University of California Merced) for training Roger Tseng on SEM imaging systems. This work was performed while the first author was an undergraduate at the University of California at Merced, using funds (to BD) from National Science Foundation DEB-0933276 as well as University of California Merced start-up. LITERATURE CITED Abbott, R. T. 1974. American Seashells: The Marine Mollusca of the Atlantic and Pacific Coasts of North America. Van Nostrand Reinhold: New York. 663 pp. Angas, G. F. 1867. Descriptions of thirty-two new species of marine shells from the coast of New South Wales. 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The Veliger 51(4):208-2I5 (September 16, 2(114) THE VELIGER © CMS, Inc., 2014 Lottia austrodigitalis Revisited: Are Ecomorphs Incipient Species? PHILIP G. MURPHY Institute of Marine Sciences, University of California, 1073 7th Avenue, Santa Cruz, California 95062, USA (e-mail: murphylmpt(gymail.com) Abstract. In 1977, the marine limpet ta.xonomic concept Lottia digitalis was discovered to encompass a northern and a southern species whose geographic ranges overlapped on the California coast between Point Conception and the southern limit of Monterey Bay. In 2008, a geographic survey was performed to reestablish the species’ zone of sympatry and to confirm the partitioning of their primary microhabitats. In the three decades between surveys, the southern species. L. austrodigitalis, has advanced its northern limit to Bodega Head, and L. digitalis has kept its southern limit at Point Conception. In the northern part of their new zone of sympatry, the two species approach complete partitioning of shared microhabitats at four sites spanning two degrees latitude, with L. aiistrodigitcdis predominating on high intertidal rock faces and L. digitalis on Pollicipes polyinerus colonies. A model of allopatric speciation with a role for ecomorphs is proposed that could account for the evolution of diversity among the western North American Lottiidae. INTRODUCTION Along the coast of California, 17 intertidal species of the gastropod family Lottiidae (Lindberg 1998) share a habitat usually no more than 2 m high and a few decameters wide. Using Addicott's (1966) definition of western North American molluscan provinces, 10 of these species belong mainly to the cool temperate Oregonian, and seven to the warm temperate Califor- nian provinces (Point Conception is the boundary between these provinces), and 1 1 have geographic ranges that include 37° and 57°N latitude. There are eurytopic species that exploit multiple substrates over several intertidal zones and stenotopic species that are found only in association with single plant or animal species. By the 1970s, several authors had concluded that sympatric speciation could best explain the origin of such groups of closely related, geographically over- lapping, but ecologically distinct species (e.g.. Test, 1946; Knox, 1963; Fretter & Graham, 1963; Valen- tine, 1968; Giesel, 1970). In 1977, I began looking for consistent allozyme differences between ecomorphs of Lottia digitalis (Rathke, 1833) that might have presaged sympatric speciation. The microhabitats I compared were the high intertidal rock face and colonies of the stalked barnacle Pollicipes polymerus Sowerby 1833. On rock faces, L. digitalis specimens are usually olivaceous with prominent ribs, while Pollicipes colony specimens tend to be white, often with dark brown chevrons, blending in with, and often virtually invisible, on the barnacles’ capitula, which are covered with white plates that are rimmed with strips of dark colored mantle. In the process of that study, I discovered, on the basis of a diagnostic leucine aminopeptidase (LAP) locus, that L. digitalis, which had been thought to range from Unimak, Aleutian Islands to Cabo San Lucas, Baja California, consisted of two morphologically similar species (Murphy, 1978). These were a northern and a southern species whose ranges overlapped in central California between Point Conception and Monterey Bay. The northern species is L. digitalis, the southern is Lottia austrodigitalis, and each species has similar high-rock and Pollicipes colony ecomorphs. I noticed at every sampling site in their zone of sympatry that there were disproportionately more L. digitalis on barnacle colonies and more L. austrodigi- talis on rock faces. Considering this observation and their geographic relationship, I proposed a model of allopatric speciation with a role for ecotypes that inight produce the phenomenon of closely related but ecolog- ically distinct sympatric species that previous authors had attributed to sympatric speciation (Murphy, 1983, thesis). The present study was motivated by evidence that the current global wanning trend had begun to accelerate in the 1960s (Meehl et ah, 2004), and, therefore, the initial contact between these species might have been recently established and in fiux in 1977. Sampling at 15 sites was done to reestablish the species’ zone of geographic overlap and to ascertain if the interspecific segregation of the high rock and Pollicipes colony microhabitats had changed. MATERIALS AND METHODS Sampling was done between May and December 2008 at the following locations in California: Seal Rock, La P. G. Murphy, 2014 Page 209 Numbers of L. digitalis and L. austrodigitalis Table from rock 1 and PoUicipes mici ohabitats at 15 locations in 2008. L. digitalis L. austrodigitalis Site °N Latitude Rock PoUicipes Rock PoUicipes CTC 41.78 57 56 0 0 SHC 40.42 55 57 0 0 PTA 38.92 50 49 0 0 BOD 38.30 52 56 5 1 PFA 37.60 7 54 50 2 PIP 37.30 19 55 'il 9 SAN 36.96 5 60 59 3 PAC 36.64 7 54 50 2 MIL 36.01 18 56 39 1 CAY 35.45 10 32 47 25 JAL 34.48 2 7 55 50 GAV 34.47 0 1 57 56 MUG 34.07 0 0 71 56 CDM 33,59 0 0 35 57 LAJ 32.85 0 0 57 49 Jolla (LAJ); Corona Del Mar State Beach (CDM); Point Mugu Beach (MUG); Gaviota State Beach, Santa Barbara Co. (GAV); rocky intertidal area 6 km north of Point Conception — accessed from Jalama County Park (JAL); Cayucos State Beach (CAY); Mill Creek Picnic Ground, Los Padres National Forest (MIL); Pacific Grove (PAC); Santa Cruz (SAN); Pigeon Point, San Mateo Co. (PIP); south end of Rockaway Beach, Pacifica (PFA); Bodega Head, Bodega Bay (BOD); Point Arena Cove, Point Arena (PTA); Point Delgada, Shelter Cove (SHC); and Point George, Crescent City (CTC). Sites with °N latitude are listed in Table 1 and illustrated in Figure 1. High rock and PoUicipes are not the only microhab- itats that both L. austrocligitalis and L. digitalis occupy. 1 have chosen to focus on these microhabitats because of their clear demarcation, easily recognized eco- morphs, and ubiquity that allows sampling from both at most localities. At each site, approximately equal samples were taken from both microhabitats. On rock faces there are subtle conchological differences between L. digitalis and L. austrodigitalis (Murphy, 1978); to mitigate unconscious bias, an assemblage of limpets was located, its center of dispersal estimated, and limpets removed concentrically until the sample was attained. When one assemblage was insufficient, the procedure was repeated. Conchological differences between species on PoUicipes colonies are very subtle requiring close inspection of specimens, thereby elim- inating this type of bias. For this microhabitat, barnacle colonies were diligently searched and all limpets with the L. digitalis-aiistrodigitalis aspect over 5 mm were collected. At every site, multiple barnacle colonies needed to be searched. Live limpets, cooled in ice chests, were transported by car for up to 3 d in plastic shoe boxes containing a I -cm depth of sea water; once in the laboratory they were frozen and kept at —20°C for up to 1 mo before analysis. In this study, as in the earlier ones (Murphy, 1978, 1983), LAP allozymes were used for species assignment. Frozen limpets were removed from their shells, and the entire bodies, or pieces of hepatopancreas from larger limpets, were homogenized in 1.5 mL plastic micro- centrifuge tubes containing two drops of 2% phenoxy- ethanol in pH 7.0 gel buffer using a nylon tipped steel pestle. Tubes were centrifuged for 1 min to coalesce the contents and then frozen. Immediately before loading, the tubes were centrifuged for 5 min at 2000 g. Starch gels were 5% sucrose and 12.5% (w;v) Sigma hydrolyzed potato starch (Sigma-Aldrich Company. St. Louis, MO) in 5 mM histidine hydrochloric acid gel buffer (Brewer, 1970) adjusted to pH 8.0 (this produced a pH 7.1 gel with the starch lot used). The cooked starch was poured into the vertical molds described by Smithies ( 1959) modified by shortening to produce gels of 18.4 cm total length. Gels were cured overnight at 4°C; twenty 5.5-mm slits were cut with a thin plastic comb to accommodate 5.5 X 4.5 mm (LXH) chroma- tography paper wicks used for loading samples. Continuous electrophoresis using pH 7.1 tri-sodium citrate tray buffer was done at 4°C for 8 hr with 1 . 1 niA per cm- of gel cross section. Gel slices 1.5-mm thick were stained for LAP by incubating about 10 min in 1 mg L-leucine (I-naphthylamide and 12 mg Fast Black K Salt per 10 mL of 100 niM Tris-maleic acid staining buffer at pH 5.2. Staining was stopped as soon as the diagnostic locus could be scored to avoid interference with a slower migrating locus. The term frci/uency is used here to mean the ratio of the number of one species to the total number of both Page 210 The Veliger, Vol. 51, No. 4 Figure I. Map showing sampling sites with arrows indicating 1977 and 2008 zones of sympatry for L. austrodigitaUs and L. digindis. From the south: LAJ, CDM, MUG, GAV, JAL, CAY, MIL, PAC, SAN, PIP, PFA, BOD, PTA, SHC, and CTC. Sites sampled in both years are in bold type. L. austrodigitaUs and L. digitalis in a sample from a microhabitat at a particular site. Mean microhahitat frequency refers to the mean of a species’ frequencies in the rock face and PoIUcipes colony microhabitats at one site; this value is used as a crude estimate of a species’ overall proportion at a sampling site. RESULTS Limpets from the northernmost sites CTC, SHC, and PTA totaling 324 specimens were analyzed to deter- mine the LAP genotype frequencies for L. digitalis and total of 325 from the southernmost sites MUG, CDM, and LAJ for L. austrodigitaUs. Most of the genotypes are uniciue to one or the other species, but, for the few that occur in both species, genotypes are assigned to the species in which they occur most frequently in allopatry. This process resulted in the same genotype assignments as for the 1977 survey (Murphy, 1978). Table 1 shows the distribution of both species among sites and microhabitats for 2008. Since 1977, the species’ zone of geographic overlap, measured in “latitude, has increased 75%. L. austrodigitaUs has extended its northern range limit from PAC to, at least, BOD; while L. digitalis has maintained Point Concep- tion as its southern range limit, although in reduced numbers relative to L. austrodigitaUs (Figures 1,2). Contingency table analysis demonstrates that the distribution of species is not independent of microhab- itat for either 1977 (Table 2; yy = 34.8; df = 1; T < 0.005) or 2008 (Table 3; jy = 202; df = 1; T < 0.005) confirming the interspecific partitioning of microhab- P. G. Murphy, 2014 Page 2 1 1 Figure 2. Mean microhabitat frequencies of L. aiistroiligilulis at 1977 and 2008 sampling sites that were within the zones of sympatry. Site abbreviations are defined in the text. "0” indicates no L. austrodigituHs in sample, and "n" denotes no sample taken. itats noticed in 1977 (Murphy, 1983). L. austrodigitalis and L. digitalis have strong biases for high intertidal rock faces and Pollicipes colonies respectively. The most notable change, in this respect, over three decades is the striking segregation of species at the consecutive sites PAC, SAN, PIP, and PFA. For these four sites combined, 84% of limpets on rock faces are L. austrodigitalis and 93% of barnacle colony limpets are L. digitalis; while in 1977, PAC had the most segregated microhabitats with 79% L. austrodigitalis on rock and 67% L. digitalis on barnacles. Table 2 2x2 contingency table of pooled numbers of each species collected from two microhabitats at all sites where both species occurred in 1977 (GAV to PAC inclusive). Substrate L. austrodigitalis L. digitalis Total Rock 210 103 313 Pollicipes 151 191 242 Total 361 294 655 Table 3 2x2 contingency table of pooled numbers of each species collected from two microhabitats at all sites where both species occurred in 2008 (GAV to BOD inclusive). Substrate L. austrodigitalis L. digitalis Total Rock 339 120 459 Pollicipes 149 375 524 Total 488 495 983 DISCUSSION Species Distribution The northern range extension of L. austrodigitalis over three decades is concurrent with increases in air and water temperatures along the California coast, and poleward migration of other marine species. For instance, Nemani et al. (2001) report an increase in mean air temperature over coastal California of 1 . 14°C/ 47 yr and an increase in near-shore sea-surface temperature of 0.72°C/47 yr during the last half of the 20th century, and the resampling of an intertidal transect at Pacific Grerve, California in the 1990s, whose invertebrates had been carefully enumerated in the 1930s, showed larger populations of southern species and smaller numbers of northern species that Barry et al. ( 1995) attributed to increasing temperature. In both 1977 and 2008, there were fewer L. austrodigitalis in samples from MIL than from neighboring sites to the north and south. These results correlate with an anomalous (as compared with the general southerly increase) decrease in near-shore sea- surface temperature of about UC between Monterey Bay and Point Arguelo (25 km NW of Point Conception) reported by Sverdrup et al. (1942). Valentine (1961) attributed this temperature anomaly to upwelling and a lack of embayments along the steep western foot of the geologically young Santa Lucia Mountain Range that forms the Pacific coast south of Monterey Bay (see 100 fathoms contour map following page 658 in Morris et al. 1980). At MIL, along this mountainous stretch of coast, using the mean rock and Pollicipes colony frequency as an estimate of its overall proportion, L. austrodigitalis had a value of 0.11 in 1977, while at CAY, (he next site south, this value was 0.59, and at PAC, the next site north, it was 0.54. The cooler conditions along this part of the coast likely keeps the numbers of southern-ranging L. austrodigi- talis low, and explains why no 1977 samples were as segregated as PAC, SAN, and PFA were in 2008. For highly segregated sites, the mean microhabitat frequen- cy of a species must approach 0.5, and, as shown in Figure 2, in 1977 only samples between CAY and MIL Page 212 The Veliger, Vol. 51, No. 4 or between MIL and PAC (the range end point for L. aiistrodigitalis), where none were taken, could have had such segregated microhabitats. Recent research suggests that both the northern advance of L. aiistrodigitalis and its microhabitat partitioning with L. digitalis are the effects of a wanning climate on sibling species that have different thermal tolerances. The existence of significant ener- getic “costs of living” for intertidal organisms have been described by Somero (2002) that derive from a broad range of physiological responses to thermal perturbations. These costs are proportional to the range of temperatures that species experience and to the frequency with which their thermal limits are approached. The metabolic processes include, but are not limited to, the production of heat-shock proteins, repair and replacement of damaged enzymes, restruc- turing of cellular membranes, and shifts in gene expression. All of these thermal coping responses use adenosine triphosphate, and their employment reduces the energy available for, among others, growth, predator avoidance, feeding, and reproducticin. From their geography, L. aiistrodigitalis would be expected to be more tolerant of high temperature than L. digitalis, and this is confirmed. Their median lethal temperature (LTso) values are 40.50^I.72°C (95% confidence limits) and 39. 50-40. 72°C, respectively (Dong et ak, 2008). Therefore, with increased temper- atures along the California coast where they coexist, L. digitalis will be the species burdened with the higher “cost of living” in the high intertidal zone where the highest temperatures occur. Since L. digitalis, and presumably L. aiistrodigitalis, can live for over 6 yr and the largest and oldest limpets occupy the upper parts of their intertidal range (Nicotri, 1974; Frank, 1965), the highly disproportionate numbers of L. aiistrodigitalis in this high rock surface habitat may result from nothing more than higher attrition over the lifetimes of several energetically handicapped recruitment cohorts of L. digitalis. It is possible that even with a warmer climate L. digitalis could have continued to fill this high rock niche if not for the presence of the more heat tolerant and, therefore, faster growing and longer lived L. aiistrodigitalis. The Pollicipes colony microhabitat is more temperate than the high intertidal rock surface. First, the middle of the vertical distribution of P. polyiiieriis is about 1 m lower than the middle of the high rock limpet distribution; this reduces the time limpets on barnacles are exposed to high temperature at low tide. Then, while the body temperature of rock morph limpets is rarely more than a degree different from that of the rock (Denny & Harley, 2006), barnacle morphs spend their emergent daylight hours attached to Pollicipes whose body temperatures are 3-5°C below rock temperature due to evaporative heat loss from the peduncle (Fyhn et ak, 1972). So despite the warming climate that affords L. aiistrodigitalis its advantage on the high rock surface, temperatures in Pollicipes colonies, while higher than they have been in the past, are not high enough to affect the relative competence of L. digitalis in this microhabitat. Speciation Model with a Role for Ecomorphs Species of nearly identical ecology and morphology, from adjacent molhiscan provinces, that have estab- lished a growing zone of sympatry, and whose ecomorphs are partitioning their microhabitats suggest a model of allopatric speciation with a role for ecomorphs that is diagrammed in Figure 3. It involves five stages; ( 1 ) a parent species with ecomoiphs A and B throughout its geographic range that is (2) subdivid- ed into two populations by a physical barrier long enough for genetic incompatibility to develop between them producing (3) two species, each with ecomorphs A and B. This is followed by (4) the elimination of the physical barrier, the eventual establishment of sympat- ry, and the partitioning of microhabitats A and B between them leading eventually to (5) different monomorphic species in each of the microhabitats A and B. Huxley (1955) proposed the following general role for “morphism” in evolution: “In certain cases it may give rise to secondarily monomorphic populations, which, if isolated, may then evolve into distinct species or subspecies.” In my model, ecomorphs do not become isolated populations before speciation, nor does their microevolutionary divergence lead directly to speciation; instead, their role comes only after allopat- ric speciation when they act as pre-adaptations to facilitate ecologic divergence of the newly sympatric siblings. The existence of L. aiistrodigitalis and L. digitalis, cryptic species belonging to adjacent molluscan prov- inces, is strong, if not conclusive, evidence for the occurrence of barriers sufficiently important to result in allopatric speciation. Several authors have proposed geographic isolating mechanisms that might lead to speciation of marine species that have planktonic larvae. Buzzatti-Traverso (1960) hypothesized that larval transport by unidirectional currents to an area of different hydrographic conditions, where only rare genotypes would be adapted, could eventually lead to reproductively incompatible populations. A similar sequence of isolating events involves the mouth of the Gulf of California, which has been open for about 5 million yr. During a cooling period, larvae of a species of the cool temperate molluscan province that had contracted its range southward to the tip of Baja California could drift across the mouth of the Gulf of California and gain a foothold on the Mexican P. G. Murphy, 2014 Page 2 1 3 Figure 3. Five-stage model of allopatric speciation with a role for ecotypes. Stages are: ( 1 ) A single polytypic species with ecotypes A and B. (2) Division by a physical barrier into two populations each with ecotypes A and B. (3) Evolution of genetic incompatibility producing allopatric sibling species each with ecotypes A and B. (4) Establishment of sympatry and partitioning of habitats. (5) Two monomorphic species. mainland. With the next warming period, the maternal species could migrate northward, leaving the mainland population isolated, leading to speciation. Eventually larvae of the new species could drift north across the mouth of the gulf and become established as a member of the warm temperate molluscan province. Either of these scenarios, and probably others, could explain the allopatric origin of the species discussed here, and considering that fossil gastropods have a mean species’ duration of 10 million yr (Stanley, 1985) there seems to have been an ample number of global temperature oscillations during the Cenozoic (see Zachos et ah, 2001) for a model of speciation depending on them to have added considerable diversity to the eastern Pacific Lottiidae. When, as above, a new species originates in a climate that is wanner than its progenitor's, most evolutionary divergence in stages two and three of my model, where Page 214 The Veliger, Vol. 51, No. 4 both species still have the same breadth of habitat, should mainly involve physiological changes in the new species that will increase its tolerance to higher temperatures. After establishment of partial sympatry, in stage four, as each sibling begins to specialize in different parts of its former niche, changes in behavior and morphology in both species can be expected to follow. In time, as the species increasingly overlap geographically, they should converge, to some extent, in their thermal physiological abilities, because, even if they occupy intertidal zones with different high temperatures, they will be exposed to the same low air temperatures. Specimens of both L. aiistrodigitalis and L. digitalis are occasionally found on the rock surface microhab- itat having shell color, pattern, and architecture that indicate they are immigrants from the Pollicipes microhabitat. The shells of such specimens have a white top portion with reduced ribbing and steep inclination atop an olivaceous, strongly ribbed, and lower inclined base. Giesel (1970) confirmed the ability of L. digitalis to change shell color by breaking off pieces of shell margin and observing the regrown shell on limpets moved from one substrate to another. Evidently, then, the ecomorphs of these limpets develop plastically after individuals have chosen a microhabitat, rather than being selected by visual predators from an array of randomly dispersed shell color patterns or from variously patterned limpets searching out appro- priately colored microhabitats. This must involve regulatory mechanisms that provide stimuli to neural and hormone systems that in turn inlluence tissues to produce and appropriately deposit shell pigments. It is probable, because of habitat partitioning, that near range ends within their zone of sympatry L. aiistrodigitalis individuals will have had very few progenitors that were Pollicipes morphs and L. digitalis individuals very few progenitors that were rock morphs for many generations. This should relax selection on the regulatory mechanisms responsible for the devel- opment of each species’ obsolete ecotype and might even result in the evolution of better abilities to blend in with its remaining microhabitat (see Espinasa & Espinasa, 2005, for an explanation of the neutral mutation hypothesis for the loss of unused characters). This kind of interspecific coevolution within their zone of sympatry would tend to reduce genetic compatibility between conspecific populations within and outside of the zone of sympatry, enabling even faster stage 4 coevolution in the model above. Test ( 1946) listed several groups of stenotopic lottiids that she believed could only have been descended through sympatric divergence from still extant eury- topic species. The model presented here can be easily modified to produce one stenotopic descendant species per speciation event from a eurytopic progenitor thereby accounting for her groups, but obviating genetic isolating mechanisms that act sympatrically in species that employ broadcast gametes and have planktonic larval dispersal. This model may be applicable to other groups, and the segregation of species along environmental temperature gradients could be a generally important element in the evolution of diversity in the sea. Acknowledgments. John S. Pearse suggested this project, and I appreciate his encouragement. The criticism of an anonymous reviewer was very helpful. LITERATURE CITED Addicott, W. O. 1966. Late Pleistocene marine paleoecology and zoogeography in central California. LInited States Geological Survey, Professional Paper 483-G: Washing- ton, DC. 21 pp. Barry, J. P., C. H. Baxter, R. G. Sagarin & S. E. Gilman. 1995. Climate related, long-term faunal changes in a California rocky intertidal community. Science 267:672- 675. Brewer, G. J. 1970. An Introduction to Isozyme Techniques. Academic Press: New York, NY. 186 pp. Buzzati-Traverso, a. a. 1960. Discussion. P. 584 in A. A. Buzzati-Tra verso (ed.). Perspectives in Marine Biology. University of California Press: Berkeley, CA. Denny M. W. & C. D. G. Harley. 2006. Hot limpets: predicting body temperature in a conductance-mediated thermal system. Journal of Experimental Biology 209: 2409-2419. Dong, Y., L. P. Miller, J. G. Sanders & G. N. Somero. 2008. Heat shock protein 70 (Hsp70) expression in four limpets of the genus Lottia: interspecific variation in constitutive and inducible synthesis correlates with in situ exposure to heat stress. Biological Bulletin 215:173-181. Espinasa, L. & M. Espinasa. 2005. Why do cavefish lose their eyes? Natural History 1 14:44-49. Frank, P. W. I960. The biodemography of an intertidal snail population. Ecology 46:831-844. Fretter, V. & A. Graham. 1963. The origin of species in littoral prosobranchs. Pp. 99-107 in J. P. Harding & N. Tebble (eds.), Speciation in the Sea. The Systematics Association: London, U.K. Fyhn, H. j., j. a. Petersen & K. Johansen. 1972. Eco- physiological studies of an intertidal crustacean, Pollicipes poivnienis (Cirripedia, Lepadomorpha). Journal of Ex- perimental Biology 57:83-102. Giesel, J. T. 1970. On the maintenance of a shell pattern and behavior polymorphism in Aamiea digitalis, a limpet. Evolution 24:99-108. Huxley, J. 1955. Morphism and evolution. Heredity 9:1^2. Knox, G. A. 1963. Problem of speciation in intertidal animals with special reference to New Zealand shores. Pp. 7-29 in J. P. Harding & N. Tebble (eds.), Speciation in the Sea. The Systematics Association: London, U.K. Lindberg, D. R. 1998. Order Patellogastropoda. Pp. 639-652 in P. L. Beesley, G. J. B. Ross & A. Wells (eds.), Mollusca: The Southern Synthesis. Part B. Fauna of Australia Volume 5. CSIRO Publishing: Melbourne, Australia. Meehl, G. a., W. M. Washington, C. A. Ammann, J. M. Arblaster, T. M. L. Wigleym & C. Tebaldi. 2004. Combinations of natural and anthropogenic forcings in P. G. Murphy, 2014 Page 215 twentieth-century climate. Journal of Climate 17:3721- 3727. Morris, R. FI., D. P. Abboit & E. C. Haderly. 1980. Intertidal Invertebrates of California. Stanford University Press: Stanford, CA. Mlirphy, P. G. 1978. Co/liselhi austrodigitalis sp. nov.: a sibling species of limpet (Acmaeidae) discovered by electrophoresis. Biological Bulletin 155:193-206. Murphy, P. G. 1983. Electrophoretic discrimination of two limpet sibling species (Acmaeidae) and evolutionai-y implica- tions of their ecological, geographic, and genetic relationships. Ph.D. Thesis, University of California: Santa Cruz, CA. Nemani, R. R., M. a. White, D. R. Cayan, G. V. Jones, S. W. Running, J. C. Coughlan & D. L. Peterson. 2001. Asymmetric warming over coastal California and its impact on the premium wine industry. Climate Research 19:25-,34. Nicotri, M. E. 1974. Resource partitioning, grazing activities, and intluence on the microtlora by intertidal limpets. Ph.D. Thesis, University of Washington: Seattle, WA. Smithies, O. 1959. An improved procedure for starch-gel electrophoresis: further variations in the serum proteins of normal individuals. Biochemical Journal 71:585-587. SOMERO, G. N. 2002. Thermal physiology and vertical zonation of intertidal animals: optima, limits, and costs of living. Integrative and Comparative Biology 42:780 789. Stanley, S. M. 1985. Rates of evolution. Paleobiology I 1:13 26. Sverdrup, H. U„ M. W. .Iohnson & R. H. Fleming. 1942. The Oceans, Their Physics, Chemistry and General Biology. Prentice-Hall: New York, NY. 1087 pp. Test, A. R. G. 1946. Speciation in limpets of the genus Aamwu. Contributions from the Laboratory of Verte- brate Biology, University of Michigan, Ann Arbor No 31: 1-24. Valentine, J. W. 1961. Paleoecologic molluscan geography of Californian Pleistocene. University of California Publications in Geological Sciences 334:309 442. Valentine, J. W. 1968. Climatic regulation of species diversification and extinction. Bulletin of Geological Society of America 79:273-276. Zachos, j., M. Pagani, L. Sloan & K. Billups. 2001. Trends, rhythms, and aberrations in global climate 65 Ma to present. Science 292:686-692. The Veliger 5l(4):216-236 (September 16, 2014) THE VELIGER © CMS, Inc., 2014 Cretaceous and Paleogene Pteria Bivalves from the Pacific Slope of North America RICHARD L. SQUIRES Department of Geological Sciences, California State University, Northridge, California 91330-8266, USA (e-mail: richa rd . sq ni res(@csun . ed u ) Abstract. The Pacific slope of North America's Cretaceous to late Eocene record of the pteriid bivalve Pteria Scopoli, 1 777 is studied in detail for the first time. It is mostly represented by Pteria pellacicla (Gabb, 1864) and Pteria clarki (Weaver & Palmer, 1922), which are redescribed and refined in their stratigraphic distributions. Pteria pelliiciila, known from southwestern Oregon and California, was previously reported ambiguously as being both Cretaceous and Eocene age but is actually restricted to the Eate Cretaceous (early Turonian to late Santonian). Pteria clarki ranges in age from middle to middle late Eocene. It was previously reported as occurring only in Washington State but also occurs in California. Other species are Pteria loclii, sp. nov., of late late Campanian to possibly early Maastrichtian in age, and Pteria sp., of late Paleocene age; both are found in California. The definition of Pteria is expanded based on the presence of weak radial ribs on P. lochi sp. nov. The so-called Pteria howei Nelson, 1925, from southern California and of late Paleocene age, has a hinge similar to that found in bakevelliids. Phelopteria Stephenson, 1952 is recognized for the first time as a junior synonym of Pteria. Specimens of the studied species were collected from shallow-marine, fine-grained siliciclastics that accumulated under warm-temperate conditions during the Eate Cretaceous and under subtropical conditions during the Paleogene. Specimens are rare, except at a few localities. Pteria most likely originated during the Jurassic in the Old World Tethys Sea region. It arrived in the study area during the early Turonian, which coincided with the highest sea-level stand and the warmest waters of the Eate Cretaceous. INTRODUCTION The family Pteriidae Gray, 1847, which includes the modern “pearl oysters,” consists of three genera; namely, Pteria Scopoli, 1770; Electroma Stoliczka, 1871; and Pinctada Rbding, 1798. The first two originated in the Mesozoic, and the last one originated in the Miocene (Elertlein & Cox, 1969). This present paper, which deals with the early fossil record of Pteria on the Pacific slope of North America, stemmed from the discovery by the author of a rare specimen of a late Paleocene pteriid from southern California. Identifica- tion of this specimen turned out to be less than straightforward and necessitated a complete review of the Cretaceous to Eocene fossil record of pteriids in the study area. One of the major obstacles to overcome was the confusion concerning the age of Pteria pellucida (Gabb, 1864). The name has been traditionally used ambiguously for both Cretaceous and Eocene speci- mens because Gabb did not designate a holotype or a type locality, and his type material was collected from Cretaceous and Eocene strata. In order to resolve this confusion, it was necessary to re-evaluate the paleon- tology of the Cretaceous and Eocene specimens. This involved studying the type specimens and searching for new material in museum collections. During this process, a new species of Pteria of Late Cretaceous age was detected. Its radial ribs necessitate the expansion of the definition of genus Pteria. The left- valve hinge of this new species has two small triangular resilifers, just like those found on the type species of Pteria and on the type species of Phelopteria Stephen- son, 1952, which is here synonymized with genus Pteria. The geographical and temporal distributions of the studied species are shown in Figures 1 and 2, respectively. In this present paper, remarks are given about the mode of life of Recent Pteria, the paleoecology of the study area species, and the biogeography of Pteria. MATERIALS and METHODS A total of 670 fossil specimens was studied. Material included type specimens and nontype museum speci- mens. Most of the latter was collected by Saul (1959) during the course of her thesis work. For most of the localities used in this study, it was necessary to integrate current biostratigraphic information in order to determine proper geologic stage position. A total of 53 museum specimens of Recent pteriids was studied. Knowledge of the hinge characters of pteriiform bivalves is critical because the shape that Pteria possesses has been commonly recurrent among bivalves since the Late Ordovician. Whether this recurrence is R. L. Squires, 2014 Page 2 1 7 ~ 45° - 40° 35° LATITUDINAL DISTRIBUTION OF SPECIES 1 - 2 - 3 - 4- 5 - 6- 7- 8 - 9- .10 ’ll 12 13 14 15 16 17 Northern Doty Hills, Lewis Co. Vader, Lewis Co. Phoenix area, .lackson Co. Mornbrook/Yreka area, Siskiyou East of' Redding, Shasta Co. Mill Creek, I'ehama Co. Chico Creek, Butte Co. Martinez, Contra Costa Co. Pacheco Pass/San Luis Reservoir area, Stanislaus Co. - North of Coalinga, Fresno Co. - Grapevine Creek, Kern Co. - San Marcos Pass area, Santa Barbara Co. - Pine Mountain area, Ventura Co. - Simi Valley, Ventura Co. Co. - Santa Monca Mtns., Ventura Co. - Santa Ana Mtns., Orange Co. - Carlsbad area, San Diego Co. O • ^ a. Ij Q. •-4 10 11 12 13 14 Figure 1. Index map showtng geographic locations of the studied species. related to convergence, iterative evolution, parallelism, or some combination thereof, has not been established. Without knowledge about the hinge details, placing any of these bivalves in any genus, or even family, is guesswork at best. It was necessary, therefore, to obtain information about the hinges of representative specimens used in this present study. Nearly all of the specimens were encased in well-cemented rock matrix; therefore, it was necessary, where possible, to excavate the specimens and their hinges by means of a high-speed drill and diamond-coated grinding wheels. The shell material is thin and fragile, thus careful cleaning is reciuired. The classification scheme used here follows that of Temkin (2006), which is based upon a cladistic analysis of anatomy and shell morphology. Most of the morphologic terms used in this present paper are illustrated in Figure 3. Ligament terminology generally follows that of Carter (1990) and Malchus (2004). Dentition terminology follows that of Temkin (2006), who used the terms “subumbonal” (for teeth or sockets Page 218 The Veliger, Vol. 51, No. 4 35 m.y. 40 LU 45 — LU u LU O — LU 50 — O LU _l 55 — < CL 60 U O 65 i 70 75 cn 3 80 o LU LU U st]Cfbr submarginal XiCkjta r«illfcf bPiik subunibonal tooch Figure 3. Illustrated key of morphologic terms used in describing pteriids. A. Pteria clarki. hypotype LACM I PI 3480, left valve, dorsal-margin length 13 mm, height 7 mm, X4.2. B, Pteria clarki. hypotype 13481, left-valve hinge, length 26 mm, X3. R. L. Squires, 2014 Page 219 many workers (e.g.. Boss, 1962; Carter, 1990) reported that pteriids have a single ligament (alivincular ligament), other research (e.g., Fiirsich & Werner, 1988; Malchus, 2004; and Temkin, personal communi- cation) indicates that in their early juvenile stage, most species of Previa can have more than one resilil'er and that these earlier resilifers commonly become obscured with growth. For a synopsis of the early history of the nomencla- ture of this family, see Flertlein & Cox (1969;N302), and for a synopsis of more current taxonomic studies of this family, see Wada & Temkin (2008). Genus Previa Scopoli, 1777 Avicula Klein, 1873 (nonbinominal); Bruguiere, 1792; Cuvier, 1797; Lamarck, 1799; Lamarck, 1801; (type species by subsequent designation [Kennard et ak, 1931], Myrihis hivimclo Linnaeus, 1758). Glaiiciis Poli, 1 795. Glaucoclevma Poli, 1795. Hivinuiigemis Renier, 1807 (rejected by ICZN, Opinion 427). Ammica Oken, 1815 (rejected by ICZN, Opinion 417). Prevopevna Morris & Lycett, 1853 (type species by original designation, GevviUia cosruriilu Eudes-Des- longchamps, 1 824). Ansrvoprevia Iredale, 1931 (type species by original designation, Ansrvoprevia salrara Iredale, 1931). Magmivicula Iredale, 1939 (type species by original designation, Magmivicula hewierri Iredale, 1939). Phelopievia Stephenson, 1952 (type species by original designation, PievuP. dalli Stephenson, 1936). Type Species: By original designation, Myrilus hiviiiula Linnaeus, 1758; Recent, western Europe and Mediter- ranean Sea (Abbott, 1982:30). Diagnosis: Shell medium to large, moderately thin. Posterior auricle long. Posterior sinus well developed. Byssal notch broad. Eigament multivincular in early juvenile (postlarval) stage and single in adult stage. Hinge long and usually with several noticeable subumbonal teeth. Description: Shell medium small to large size (over 200 mm in length); juveniles longer than adults relative to height (length/height approximately = 3 on juveniles and approximately = 2 on adults). Byssate, free- swinging, epifaunal. Shell thin and brittle, moderately to strongly obliquely ovate to subquadrate, with anterior and posterior auricles. Periostracum dark in color with radial lines of lighter color, dehiscent and can be scaly, matted, shaggy, wrinkled, or spiny. Outer calcareous shell smooth (rarely with weak radial ribicts) or with low commarginal lamellae coincident with growth lines. Valves laterally compressed. Umbones prominent, subcentral, backwardly obliciue. Inequilat- eral. Inequivalved, left (upper) valve more inllated (convex) that right (lower) valve, but difference can be slight. Dorsal margin long and straight with alate projections at anterior (smaller) and posterior ends. Anterior auricle on both valves variable in length, triangular to subtrigonal, narrow to wide, short to moderately short, with pointed to blunt tip, and separated from umbo by shallow radial groove leading to prominent sinus coincident with byssal notch on right valve. Byssal-notch area on right valve with sinused growth lines. Posterior auricle on both valves variable in length, triangular, narrow and very long (on juveniles) to very broad and long (on adults) with pointed to blunt tip. Posterior slope llattish relative to adjacent inflated umbonal area and coincident with parasigmoidal growth lines indicating ontogenetic growth of posterior sinus that usually creates small sinus on posterior margin of valve. Ventral margin of shell convex. Beaks low to moderately prominent, prosogyrate, weakly incurved, and located less than 25% of length of dorsal margin from anterior extremity. Early juvenile (postlarval) stage ligament multivincular, with up to po.ssibly live serial parts. Adult-stage ligament single, external, somewhat sunken, opisthodetic, long, straight, and occupying first one- third of length of hinge line between beak and extremity of posterior auricle. Ligament on left valve nearly vertical and resting on very narrow nymph; ligament on right valve resting on low-angle, platform-like nymph, widest at beak and becoming narrower posteriorward. Hinge with or without several subumbonal teeth (tooth- like processes) or crenulations just anterior to beak; commonly, left valve with subumbonal tooth (sides can be socket-like) and right valve with corresponding socket (sides can be swollen). Posterior submarginal groove on left valve accommexiating posterior submar- ginal oblitjue ridge on right valve, both occupying middle one-third of length of hinge line between beak and extremity of posterior auricle. Adult adductor scar oval-shaped and located posterior of center of valve. Pedo-byssal retractor muscle large. Byssus emerges from base of byssal groove ventral to short foot. Pallial line discontinuous to subobsolcte. Pedal-muscle scar track can extend downward and backward from anterior scar on each valve. Strong angulation on interior of left valve coincident with bounding edge of anterior auricle; strong angulation leads to bys.sal notch. Outer shell layer prismatic calcitc, inner shell layers aragonitic nacreous, imparting pearly luster on modern specimens and on well-preserved fossil ones. Blister pearls ean be pre.sent. ( De.scription based on Hertlein & Cox [1969], Davies [1971], Fiirsich & Werner [1988], Coan et al. [2()()0], Malchus |2()04], Wada & Temkin [2008], and personal observations based on speeimens borrowed from LACM). For a description of the anatomy of Page 220 The Veliger, Vol. 51, No. 4 Pteria, see Coan et al. (2000) and Wada & Temkin (2008). Discussion: According to Kennard et al. (1931), Bruguiere (1792) created the name Avicula for un- named figures. Lamarck (1799) cited Mytiliis hirimdo Linnaeus as its type species, but Lamarck (1801) unnecessarily changed the specific name to comnnuiis. Cuvier (1797) adapted and defined Avicula and cited three species. Of those species, Kennard et al. ( 1931:14) nominated A. Iiirum/o Linnaeus as the type species for genus Avicula. According to Hertlein & Cox (1969:N304), Gkiucoderwa and Glaucus are objective synonyms of Pteria in consequence of type designations by Winckworth (1930:1 16). The definition of Pteria is amended here because Pteria loclii sp. nov. has radial riblets on its left valve. Pteria, which has been defined previously as having smooth valves, is highly variable in its morphology. On individuals of the same species from the same locale, there can be variation in the angle of prosoclinality, shape of the anterior auricle, height of the posterior auricle, sinuosity of growth lines on the posterior slope, amount of indentation of the posterior sinus on the valve margin, as well as the shape, size, and inclination of the subumbonal and posterior submarginal denti- tion. Juveniles have more variability in the dentition than do the adults. According to Fursich & Werner (1988), the number, size, and shape of the teeth also vary greatly between and within species of Pteria, and some adult individuals lack teeth because they can be overgrown during ontogenetic growth. According to most workers (e.g., Stephenson, 1952; Coan et al., 2000), Pteria is characterized by having an alivinctilar ligament, but Fursich & Werner (1988) also reported that the hinges of adult specimens of Late Jurassic Pteria can have two or more roughly parallel- sided resilifers of variable width and position. Malchus (2004:pl. 1, fig. 7) illustrated the postlarval hinge of Pteria hiruudo and showecJ that it has two resilifers on its hinge; one immediately below the beak and the other within the first 2 mm posterior to the beak. Malchus’ (2004:pl. 1, fig. 7) illustration is very similar to the postlarval hinge of Pteria held sp. nov., which is discussed in detail later in this present paper. On-going research by Temkin (personal communication) has revealed that Recent Pteria can have three postlarval resilifers. In the present paper, genus Phehpteria Stephenson, 1952 is put into synonymy for the first time with Pteria because they are morphologically indistinguishable. Although Stephenson reported that Phelopteria differs from Pteria by lacking the long posterior ear and by having a shorter and less pointed anterior ear, Speden (1970) has shown in his study of Maastrichtian Phelopteria from South Dakota that these features are not of significance. Stephenson (1952) and Speden (1970) reported that Phelopteria is multivincular in its younger stages, but as discussed above, so is Pteria. Although Stephenson (1952) tentatively assigned Phe- lopteria to family Pteriidae, most workers have assigned the genus to family Bakevelliidae King, 1850 and reported that Phelopteria is the only known member of this family not having a multivincular ligamental area on adult specimens (e.g.. Carter, 1990; Cox, 1969; Temkin, 2006). The usage of the name ""Phelopteria" in the geologic literature has been mainly restricted to Cenomanian faunas in Nova Scotia, New Jersey, and northeast Texas (Stephenson, 1936, 1952) and Maastrichtian faunas in South Dakota (Speden, 1970). These workers found juvenile specimens showing multiple resilifers. Other reports are discussed below. Tamura (1976) reported Phelopteria erect a Tamura (1976:57-58, pi. 3, figs. 1-5) from Cenomanian strata of Japan. The morphology of this bivalve is identical to a Pteria. A long resilifer is present on the adult specimens, but, unfortunately, no juvenile shells were found. Kauffman ( 1977:pl. 4, figs. 1, 4) reported Phelopteria salinensis (White, 1880a:296-297, pi. 5, figs. 1, 2; I880b:l5-I6, pi. 16, figs. 2a,b; Twenhofel, 1924:82, pi. 18, fig. 1; pi. 22, fig. 4), which is known from middle upper Albian strata in Colorado, Kansas, and Okla- homa. The dorsal view of the hinge line of a paired- valved specimen (White, 1880b:pl. 16, fig. 2b) and the posterior submarginal groove of a right valve (Twen- hofel, 1924:pl. 22, fig. 4) are very similar to that found on Pteria. No juvenile specimens showing multiple resilifers were mentioned. Kauffman et al. (1977) also reported Cenomanian/ Turonian species of Phelopteria from Oklahoma, but the hinges of these species are either very poorly known or unknown. Stephenson (1952:68) suggested that another Western Interior species, Avicula (OxytomcP) gastrodes Meek (1873:491), might be a Phelopteria. Meek's species was reported by White (1879:280, pi. 10, fig. la) from Cretaceous beds in Utah and by Stanton (1893:72-74, pi. 9, figs. 7-10) from the ""Pugiiellus sandstone” in Huerfano Park, Colorado. According to Cobban & Reeside (1952:1029), the "" Pugnellus sand- stone” is of middle Turonian age and locally occupies the interval between the Codell and Fairport members of the Carlisle Shale. Although a dorsal view of a paired-valved specimen (Stanton, 1893:pl. 9, fig 10) of gastrodes shows a long resilifer that is very similar to that found on Pteria, more specimens are needed in order to determine the hinge details of this species. Avicula caudigera Zittel (1866:9, pi. 12, figs. 12a-c; Cox, I969:fig. C42, Id) from Coniacian to lowermost Campanian in Austria has been reassigned by modern workers to Phelopteria, but hinge information is R. L. Squires, 2014 Page 221 lacking. Dhondt & Dieiii (1993:176, pi. 2, fig. 7) reported Plielopferia cf. caudigeru (Zittel) from Con- iacian strata in western Europe but did not provide any hinge information. Pleria is similar to the pteriid Electronui Stoliczka, 1871. Wada & Temkin (2008) reported that E/ecrroiiia is part of a sister group to the clade containing Pteria. Electronui differs from Pteria by having a smaller and more equivalved shell, no anterior auricle, much shorter posterior auricle without a posterior sinus or no posterior auricle, shorter hinge line, smaller resilifer, more angular byssal notch (almost in-folded rather than broadly spread out), an edentulous left valve, and a much thinner and much less sturdy shell. The shell of Electronui is so thin that some lightly colored species are semitransparent, and all specimens are so fragile that they can easily break, even with very careful handling. Although Lamprell & Healy (1998) and Temkin (2006:306) reported that Electronui is edentu- lous, at least one of its species (e.g., E. georgiana Quoy & Gaimard, 1834) can have an elongate stibumbonal tooth on its right valve. Some specimens of E. georgiana differ from Pteria by having a concave postero-ventral margin just ventral of the hinge line. Pteria pellitckht (Gabb, 1864) (Figures 4-18) Aviciila pelliicUla Gabb, 1864:186 187 (in part), pi. 25, fig. 172. Pteria pellucida (Gabb). Yokes, 1939:pl. 2, fig. 1; Moore, 1983:pl. 24, fig. 3. Aviciila roguensis Anderson, 1958:106, pi. 38, fig. 7. Pteria aff P. pellucida (Gabb). Saul, 1959:73-75. Pteria cf. P. pellucida (Gabb). Bishop, 1 970:appendix. Not Pteria pellucida (Gabb). Yokes, 1939:pl. 2, figs. 4, 7, 8; Anderson & Hanna, 1925:188-189, pi. 1, fig. 1; Weaver & Kleinpell, 1963:197, pi. 29, fig. 1; Givens, 1974:43, pi. 1, fig. 10; Squires, 2001:15 16, fig. 32. Diagnosis: Small Pteria, commonly longer than high. Umbonal area well infiated, posterior slope weakly demarcated from umbonal area. Byssal notch not evident. Description: Shell medium size, average length 22 mm, height 19 mm, and paired-valved width 13.5 mm; rare specimens up to length 27 mm and height 38 mm. Shell thin, obliquely subovate to subqiiadrate, with anterior and posterior auricles present. Shell smooth, uncom- monly bearing broadly spaced commarginal ribs or lamellae on both valves of same specimen. Yalves laterally compressed. Umbones well infiated, promi- nent, subcentral, backwardly oblique. Angle of proso- clinality ranges from 47° to 73°. Inequilateral, higher than long. Inequivalved, left valve more infiated than right valve, but valves equally infiated on some specimens. Dorsal margin straight with triangular alate projections at anterior (smaller) and posterior ends. Anterior auricle on both valves short, with blunt tip, and separated from umbo by shallow, radial groove leading to weak or obscure sinus on anterior margin. Byssal notch not evident. Posterior auricle on both valves long and narrow. Posterior slope weakly demarcated from umbo. Growth lines parasigmoidal on posterior slope. Posterior sinus small to obscure. Yentral margin of shell convex. Beaks moderately prominent, prosogyrous, weakly incurved (beak on left valve commonly appears to overhang hinge line), and located approximately 25% of length of dorsal margin from anterior extremity. Ligament external, somewhat sunken, and opisthodetic. Late juvenile hinge (approx- imately 10 mm in length) unknown; adult hinge with one large resilifer extending posteriorly long distance; resilifer rhomboid shape with base (nymph) widest on right valve beneath beak. Hinge line can be slightly sinuous in vicinity of beaks. Left valve just anterior of resilifer with one tooth (elongate on juveniles). Hinge line fiat above auricle on left valve. Right valve with corresponding low socket and, and immediately anterior of it, hinge swollen. Anterior pedal-byssate retractor muscle-scar areas deeply excavated and situated high up on each valve. Anterior pedal-byssate retractor muscle scar can leave track of small blisters along anterior internal margin of umbonal ridge on both valves. Outer shell consisting of prismatic calcite; shell interior consisting of nacreous lamellar aragonite. Commarginal ribs “ripple" outer prismatic layer. Type material: Of Aviciila pellucida Gabb, 1864, lectotype UCMP 11983, designated by Yokes (1939: 51). Of Aviciila roguensis Anderson, 1958, holotype CAS 445.17. Type locality: Probably near Pacheco Pass, Stanislaus County, northern California. Geologic age: Lower Turonian to Santonian. Distribution: LOWER TLIRONIAN: Redding Forma- tion, Bellavista Sandstone Member, east of Redding, Shasta County, northern California. MIDDLE TUR- ONIAN: Redding Formation, Frazier Siltstone Member, east of Redding, Shasta County, northern California. UPPER TURONIAN: Hornbrook Formation, Osbur- ger Gulch Sandstone Member, at “Forty-nine mine” south of Phoenix, Jackson County, southwestern Oregon and at Hornbrook and Snowdon areas, Siskiyou County, northern California. UPPER TURONIAN: Ladd For- mation, Baker Canyon Member and lower Holz Shale members, Silverado and Rose canyons, Santa Ana Mountains, Orange County, southern California. UPPER CONIACIAN: Chico Formation, Ponderosa Way Member, Butte County, northern California. SANTO- NIAN: Redding Formation, Member Y, Price Hollow, Page 222 The Veliger, Vol. 51, No. 4 Figures 4 18. Specimens eoated with ammonium chloride. Pleriu peHiicida (Gabb, 1864). Figure 4. Lectotype UCMP 11983, Paclieeo Formation, Paclieeo Pass/San Luis Reservoir area, left valve (partial), dorsal-margin length 28.5 mm, height 17 mm, Xl.2. Figure 5. Holotype CAS 445.17 o\' Avicuki roguensis Anderson, 1958, Forty-nine Mine, near Phoenix, Oregon, left valve, internal mold, dorsal-margin length 13 mm, height 12 mm, X2.3. Figure 6. Hypotype LACMIP 13459, LACMIP loc. 23619, left valve, dorsal-margin length 2 1 mm, height 20 mm, X 1 .6. Figure 7. Hypotype LACMIP 1 ,3460, L ACM IP loc. 23620, lett valve, dorsal-margin R. L. Squires, 2014 Page 223 Clover Creek, and Old Cow Creek, east of Redding, Shasta County, northern California; Chico Formation, Musty Buck Member?, Mill Creek, Tehama County, northern California; Chico Member, Musty Buck Member, Chico Creek Canyon, Butte County, northern California; Panoche Formation, near top of unit II, Pacheco Pass/San Luis Reservoir area, Stanislaus County, northern California. Discussion: A total of 641 specimens of P. pellucida was e.xamined, including some Turonian and Coniacian specimens, abundant specimens of Santonian age from the lower part of Member V of the Redding F'ormation east of Redding, abundant specimens of Santonian age from the Musty Buck Member of the Chico Formation in the Chico Creek area, and the lectotype, which is a partial left valve whose ventral area is missing (Figure 4). No early postlarval shells are available for study. The smallest specimens that could be cleaned are 10 mm in length, and none shows more than one resilifer. The study area specimens of P. pelliicicia appear to be complete, but the fragile posterior auricle is broken on nearly every specimen. The shell is so thin that the breakage is not obvious. A very rare specimen (Figure 8) showing much, but not all, of its posterior auricle is evidence that the posterior auricle is long, narrow, and extends beyond the margin of the posterior side of the main disk. This phenomenon of Pteria valves missing much of their posterior auricles is also common on Recent specimens. Saul ( 1959) reported that the outline of the valves of P. pellucicla from any one locality in the Chico Creek area is variable, especially on specimens from coarse- grained, poorly sorted matrix versus those from finer grained, better sorted matrix. She also observed that the left valve is usually at least twice as inllated as its right valve, but in different individuals a left valve might be no more infiated than a right valve. The more inflated specimens are commonly higher, and the less infiated ones usually proportionately wider or more quadrate. As mentioned earlier, the ambiguous name "'Pleria pellucicla" has been traditionally used for both Creta- ceous and Eocene specimens because Gabb (1864) did not designate a holotype nor a type locality, and his type material was collected from both Cretaceous and Eocene strata. Gabb (1864) based his description of Avicula pellucicla on specimens of significantly different geologic age. He reported that this species is found in (I) Division A strata, at the Ranch of San Luis Gonzaga (now covered by the San Luis Reservoir) near Pacheco Pass, California and also in (2) Division B strata, near Martinez, California. Division A is now known to be of Cretaceous age, and Division B is now known to be of early Tertiary age (Stanton, 1896; Whitney, 1869). Merriam (1895), in his list of Gabb’s types (now stored at UCMP), placed Avicula pellucicla with the Eocene types. Stewart (1930), who reviewed all of Gabb's named species, evidently did not see this material because he did not discuss this particular species. Yokes (1939;50) stated that subsequent to Stewart’s studies, the type material o[' Avicula pellucicla was discovered in the UCMP collections. He designated a lectotype (UCMP 1 1938), which is in a weathered but hard, gritty brown very fine-grained biotitic sandstone. Prior to this present report, the age of the lectotype was uncertain, but its age is most likely Santonian, as discussed in the following paragraph. A review of the geologic and paleontologic literature concerning all the strata that were mentioned by Gabb ( 1864) as containing his Avicula pellucicla indicated that the most likely locale where he collected his type specimens of this species is the Pacheco Pass/San Luis Reservoir area on the west side of the northern San Joaquin Valley, approximately 35 km south of an area in the vicinity of Patterson, where Bishop (1970) studied the Cretaceous stratigraphy of the Panoche Eormation and the overlying Moreno Formation. These Cretaceous strata trend southward, in a contin- uous band of outcrop, directly into the Pacheco Pass area. Bishop’s (1970) geologic map of the Patterson area shows UCMP locality B-761, which is approxi- mately 100 m below the top of map unit II of the Panoche Formation. The upper part of unit II contains brown claystone and siltstone with some interbeds of length 22 mm. height 21 mm, X 1 .6. Figure 8. I lypolype L ACM IP 1 3461 , L ACM IP loe. 10787, left valve, dorsal-margin lenglli 33 mm, height 31 mm, X 1.3. Figure 9. Hypotype LACMIP 13462, LACMIP loe. 23620, left-valve interiors ol'lwo specimens nestled together, dorsal-margin length of largest specimen 19.5 mm, height 16 mm, X2.3. Figure 10. Hypotype LACMIP 13463, LACMIP loc. 24659, partial left-valve hinge, length 10,5 mm, height 3.5 mm, X.4.6. Figure I I . I lypotype LACMIP 13464, LACMIP loc. 23620, right valve, dorsal-margin length 10 mm, height II mm, X3.4. Figure 12. Hypotype LACMIP 1.3465, LACMIP loc. 23625, partial right valve, length 20 mm. height 21 mm, X2.5. Figures 13 14. 1 lypotype LACMIP 1 3466, LACMIP loc. 10801 , right valve, dorsal-margin length 24 mm. Figure 13. Dorsal view of hinge, X2.6. Figure 14. Lateral view of portion of hinge, length 16 mm, X4.5. Figure 15. I lypotype LACMIP 13467. LACMIP loc. 23620. dorsal view of paired-valved specimen, dorsal-margin length 18.6 mm, X3.6. Figure 16. Hypotype LACMIP 13468, LACMIP loc. 23620, dorsal view of paired-valved specimen, dorsal-margin length 17.3 mm. X4.4. Figure I 7. I lypotype LACMIP I 3469, LACMIP loc. 23620, dorsal view of partially complete paired-valved specimen, length 24.7 mm, X2.l. Figure 18. Hypotype LACMIP 13470, LACMIP loc. 23620. anterior view of paired-valved specimen, height 20.5 mm, X2.2. Page 224 The Veliger, Vol. 51, No. 4 fine sandstone. On page 29, based on identifications done for him by the junior author in 1967, Bishop listed Santonian-age fossils from this locality. One of the fossils is Pteria cf. P. pellucida (Gabb). Based on geographic location, rock type, geologic age, and presence of pellucida there, it is very likely that the lectotype (UCMP 1 1938) was collected from Santonian- age rocks in the Pacheco Pass area. In addition, the fact that this species is most abundant in Santonian-age rocks helps support that the lectotype is of this age. Yokes (1939) also designated a lectoparatype (UCMP 15576) of ""Pteria" pellucida, which is identi- fied here as Pteria clarki. It is in a dark-gray sandstone filled with shell fragments, among which are the Paleogene gastropods Turritella uvasami subsp. and Seapliaiider (Mirascaplia) costatus (Gabb, 1864). Yokes (1939) also mentioned a third specimen (an immature one) of rather different proportions from the rest of the type material of ""Pteria" pellucida, and he stated that it is possibly the Division A (i.e., Cretaceous) specimen mentioned by Gabb (1864). At first glance, Pteria pellucida appears to be conspecific with P. clarki, but P. pellucida differs by having a smaller average size, smaller maximum size, length commonly greater than height, more infiated umbonal areas, more variability in angle of prosoclinality, and a weaker demarcation between umbonal area and posterior slope. Pteria pellucida also has no indication of a byssal notch, whereas it is large on P. clarki. Weaver (1942, 1943) reported that pellucida has a shallower sinuosity of the growth lines on the auricles than does clarki, but both species are the same in this respect. Arnold (1910:11, pi. 1, fig. 6) illustrated a cast of a small left valve of a study area pteriid that he identified as Avicula liuguaefonuis Evans & Shumard, 1854, which is a Maastrichtian species from South Dakota and assigned to genus Phelupteria by Speden (1970). Arnold & Anderson (1910:pl. 23, fig. 6) repeated the illustration. Arnold did not provide any hinge infor- mation about his specimen, nor did he provide precise locality data. He stated that the specimen is from Upper Cretaceous strata at the head of Canoas Creek, which is west of Avenak southern part of Fresno County, California. On Stewart’s (1946) geologic map, the locale of the specimen is on the east flank of Black Mountain and plots within the undifferentiated Upper Cretaceous Panoche Formation. It seems likely that this specimen is Pteria pellucida, but more collecting in that area is needed for confirmation. Pteria loclii Squires & Saul, sp. nov. (Figures 19-28) Diagnosis: Yery small Pteria. Fine radial riblets on anterior slope. Posterior auricle short. Small flexure on posterior part of umbo of left valve. Feft-valve hinge multivincular in early juvenile (postlarval) stage, with two triangular resilifers (FI and F2?), both greatly widening distally. Description: Shell very small, average length 8 mm, height 10 mm; rare specimens up to length 11 mm and height 14 mm. Shell very thin, obliquely subquadrate, elongate. Feft valve with 7 to 12, very fine and widely spaced radial riblets on either anterior slope or on area extending from anterior slope to medial part of umbonal ridge; riblets becoming obsolete toward posterior slope; riblets accentuated by elongate minute nodes at intersections with prominent growth lines (growth checks?) on anterior slope. Right valve smooth. Yalves laterally compressed. Umbones prom- inent, backwardly oblique; posterior part of left-valve umbo with prominent sulcus extending from beak to ventral margin. Angle of prosoclinality ranges from 54° to 60°. Inequilateral, higher than long. Inequivalved, left valve lowly inflated, right valve flattish (probably crushed). Dorsal margin straight with triangular alate projections at anterior (smaller) and posterior ends. Anterior auricle on both valves short, with blunt tip, and separated from umbo by shallow, radial groove leading to weak or obscure sinus on anterior margin. Posterior auricle on both valves usually short. Posterior slope moderately well delimited from umbo. Growth lines parasigmoidal on posterior slope. Posterior sinus small. Yentral margin of shell convex. Beaks moder- ately prominent, prosogyrous?, weakly incurved (beak on left valve commonly appears to overhang hinge line), and located approximately less than 25% (adults) of length of dorsal margin from anterior extremity. Figament area opisthodetic. Feft-valve hinge with early juvenile (postlarval) very small triangular resilifer (FI) directly below beak and surrounded by small flattish to slightly convex areas on both sides; longer triangular resilifer (F2?) extending posteriorly short distance; both resilifers greatly widen distally. On left valve, posterior submarginal groove prominent. Outer shell consisting of prismatic calcite; shell interior consisting of lamellar aragonite. Interior of left valve with U-shaped (open dorsally) demarcation located medially or more dorsal than that. Holotype: F ACM IP 13471. Type locality: FACMIP 7792 (33°08'N, 117°25'W); near Carlsbad, southern California. Paratypes: FACMIP 13472-13477. Geologic age: Fate late Campanian to possibly early Maastrichtian. Distribution: Point Foma Fonuation, Carlsbad Re- search Center area near Carlsbad, northern San Diego County, southern California. R. L. Squires, 2014 Page 225 Discussion: The new species is based on 16 specimens: 15 left valves and one paired-valved specimen (the holotype). The holotype is slightly crushed, and the crushing probably imparted a flattish appearance to its right valve. Five of the 1 6 specimens show the left hinge, but only three have the beak area preserved intact. All three of these show the small subtriangular resilifer (FI) directly below the beak, but only two show the small tlattish areas adjacent to this resilifer. The holotype (Figure 27) best shows FI and F2? resilifers that resemble the two, postlarval resilifers illustrated by Malchus (2004:1547, pi. 1, fig. 7) near the antero-dorsal end of a hinge of Pteria hirundo, the type species of genus Pteria. His figure shows two resilifers, one directly below the beak and the second, longer one posterior to the beak. The resilifers of the new species are similar to the two resilifers shown by Stephenson (1952:pl. 14, fig. 10) for Phelopteria dalli (Stephenson, 1936) and to the two resilifers shown by Speden (1970):pl. 13, fig. 11) for Phelopteria lingiiaeformis (Evans & Shumard, 1854). Stephenson ( 1936, 1 952) reported that, based on P. dalli and Phelopteria timherensis Stephenson, 1952, genus Phelopteria is multivincular in the younger stages, with a series of up to four elongate-ovate resilifers that in later stages merge to form one long ligament. Speden (1970) reported that P. linguaeformis has two to five resilifers, which are initially triangular but tend to become parallel-sided with growth and generally fuse so as to reduce the number of pits to one to three. Radial sculpture is present on most of the specimens of the new species, although it can be nearly obsolete on a few. Radial sculpture has not been found before on fossil or on Recent specimens of Pteria. The radial sculpture of the new species is similar to that found on juvenile specimens of the large (up to height 105.5 mm) bakevelliid Gervillaria haradae (Yokoyama, 1890:199, pi. 25, figs. 12a-b; Hayami, 1965:269-271, pi. 35, figs. 3-6, pi. 36, fig. 1, pi. 37, fig. 2; Tashiro & Matsuda, 1986:373-374, figs. 1-5, 13) known from the late Neocomian to Albian in Japan (Hayami, 1965). In addition to being very much smaller, the new species differs from this Japanese species by having ribs that are more widely spaced and not present on the posterior wing area. The new species also lacks cancellate sculpture on its beak and early umbo, lacks a strong indentation on the interior of the valve in an area corresponding to the posterior side of the umbo, and does not have more than six subquadrangular resilifers, which are also as wide as their interspaces. The shape of Pteria lochi somewhat resembles Pseudoptera viaiia Stephenson (1952:72, pi. 15, figs. 3- 7) from Cenomanian strata of northeastern Texas. The new species differs by having a smaller and less oblique shell, rounded postero-ventral comer, rounded (rather than carinate) umbonal ridge, rounded (rather than carinate) posterior ridge, distinct posterior auricle with a sinus, and a more distinct anterior auricle. More significantly, the new species does not have three or more vei'y distinct and rectangular resilifers on the hinge, nor does it have a strong oblique cardinal tooth on the left valve. The new species differs from P. pellucida and P. darki by being much smaller, less inflated, radially ribbed, sulcate on the posterior part of the umbo, and having FI and F2? resilifers. Tlie geology of the new species’ type locality at the Carlsbad Research Center was discussed by Loch (1989). The fauna at the type locality does not represent a diminutive fauna, in spite of the assertions by Loch & Bottjer (1986) and Loch (1989) that it was. The species in this fauna are actually normal size in comparison to their size elsewhere. The preservation quality of shells at the Carlsbad Research Center is, however, one of the best for Cretaceous shells on the Pacific slope of North America. This preseiwation allowed for the very fragile valves of the new species to be preserved well enough to see the small triangular resilifer directly beneath the beak of the left valve. Etymology: Named for James Loch, who found the specimens. Pteria sp. (Figure 29) Description: Shell moderately small, length 16 mm, height 13 rnm. Shell subquadrate. Shell with numerous, closely spaced concentric riblets, becoming wavy and lamellose near ventral margin. Angle of prosoclinality 67°. Inequilateral, valve longer than high. Dorsal margin long and straight. Anterior auricle alate, subtrigonal, moder- ately short, moderately wide, with sharp tip. Anterior margin with prominent sinus coincident with broad byssal notch. Posterior auricle obscured. Posterior margin of shell unknown. Ventral margin of shell convex. Beak moder- ately low, prosogyrous, and located approximately one- third of length of dorsal margin from anterior extremity. Hinge with one long resilifer. Dentition unknown. Geologic age: Late Paleocene. Distribution: Santa Susana Lormation, upper Pulga Canyon, Santa Monica Mountains, Los Angeles County, southern California. Discussion: This species is based on a single external mold of a left valve. Its overall shape resembles a Pteria having a broken-off posterior auricle. Its concentric sculpture, however, is unlike any known pteriiform bivalve in the study area. More specimens are needed to fully characterize this species and to determine how it differs from P. pellucida and P. darki. The only other pteriiform-bivalve species known from Paleocene strata in the study area is the so-called “Ptcr/n” howei Nelson ( 1925:406^07, pi. 49, figs. 6, 7), Page 226 The Veliger, Vol. 51, No. 4 Figures 19-28. Specimens coated with ammonium chloride. Pteria lociii Sciuircs & Saul, sp. nov., LACMIP loc. 7792. Figure 19. Holotype LACMIP 13471. left valve, dorsal-margin length 12 mm. height 1 1 mm, X3.8. Figure 20. Paratype LACMIP 13472, left valve, dorsal-margin length 8.5 mm, height 8 mm, X4.7. Figure 21. Paratype LACMIP 13473, left valve, dorsal-margin length 9 mm, height 12 mm, X4.7. Figure 22. Paratype LACMIP 13474, left valve, dorsal-margin length 10 mm, height 12 inm, X4.4. Figure 23. Paratype LACMIP 13475, partial left valve, length 10 mm, height 9 mm, X4.4. Figure 24. Paratype LACMIP 13476, left-valve R. L. Squires, 2014 Page 227 Figures 29-32. Specimens coated with ammonium chloride. Figure 29. Pterin sp., hypotype LACMIP F3478, LACMIF^ loc. 17868. latex peel of external mold of left valve, dorsal-margin length 18 mm, height 13 mm, X1.4. Figures 30-31. "Pterin'" howei Nelson, 1925. Figure 30. Paratype UCMP 30516. UCMP loc. 3783, left valve, dorsal-margin length 18 mm, height 9 mm, x2. Figure 31. Holotype UCMP 30515, UCMP loc. 3767, right-valve interior, dorsal-margin length 33 mm, height 13 mm, X 1 .4. Figure 32. "Pterin" sp. of Clark and Woodford ( 1927), hypotype UCMP 31321, UCMP loc. 3 1 52, left-valve view, dorsal-margin length 23 mm, height 1 3 mm, X 1 .3. from the upper Paleocene Susana Formation of the Simi Hills, Ventura County, southern California. Although the holotype of this species shows multiple wide and rectangular resilifers. Nelson did not mention them in his description of the species, nor did he show them in his hand-drawn illustration of the hinge. Moore (1983:pl. 24, fig. 2) provided a photograph of the hinge of the interior of the holotype of "Pteria" lunvei, but it is very difficult to discern any details about the hinge. Figures 30-31 show both the exterior and interior views of the paratype and holotype, respec- tively, of this species. Based on its shell shape, size, and the presence of four very distinct and large, rectangular resilifers, "Pteria" howei is probably a bakevelliid. More work is needed to confirm its genus. "Pteria" sp. of Clark and Woodford (1927:88, pi. 14, fig. 7) is known from the uppermost Paleocene to lowermost Eocene Meganos Formation on the north side of Mount Diablo, Contra Costa County, northern California. This record is based on a single paired-valved specimen (Figure 32), which has the same shape as "Pteria" howei and is probably conspccific. Pteria clarki Weaver and Palmer, 1922 (Figures 3A-B, 33-45) Pteria clarki Weaver and Palmer, 1922:12- 13, pi. 10, figs. 5, 12, 15; Weaver, 1942, 1943:77-78, pi. 11, fig. 4; pi. 14, figs. 11, 12. Pteria pelhiciila Anderson and Hanna, 1925:188-189, pi. 1, fig. 1: Yokes, 1939:50-51, pi. 2, figs. 4, 7, 8; Weaver and Kleinpell, 1963:197, pi. 29, fig. 5; Givens, 1974:43, pi. 1, fig. 10; Squires, 2001:15-16, fig. 32. Pteria sp., cf. P. clarki Weaver and Palmer. Squires and Goedert, 1995:table 1. Diagnosis: Small medium-sized Pteria, usually higher than long. Umbonal area moderately infiated, posterior slope demarcated from umbonal area. Byssal notch well defineci. Description: Shell medium size, average length 22 mm, height 29 mm, and pairecTvalved width 12 mm; very rarely up to length 59 mm, height 57 mm, and paired- valved width 34.5 mm. Shell thin, obliquely subovate to subquadrate, with anterior and posterior auricles. Shell smooth, rarely bearing broadly spaced commarginal ribs or lamellae on both valves of same specimen. Valves laterally compressed. Umbones moderately infiated, backwardly oblique. Angle of prosoclinality ranges from 50° to 56°. Inequilateral, higher than long. Inequivalved, left valve slightly more infiated than right valve, but valves equally infiated on some specimens. Dorsal margin straight with triangular alate projections at anterior (smaller) and posterior ends. Anterior auricle on both valves short, with blunt tip, and separated from umbo by shallow, radial groove leading interior, dorsal-margin length 5.5 mm, height 7.5 mm, X7.5. Figure 25. Paratype LACMIP 13477, left-valve hinge, dorsal-margin length 5 mm, X 1 5.2. Figures 26-28. Holotype LACMIP 13471, paired-valved specimen. Figure 26. Hinge of left valve, also showing right valve, dorsal-margin length 8.5 mm, X4.4. Figure 27. Close-up of left-valve hinge, length 9 mm, X16.9; FI = first postlarval resilifer, F2? = second? postlarval resilifer. Figure 28. Anterior view, height 11 mm, X3.8. Page 228 The Veliger, Vol. 51, No. 4 Figures 33^5. Specimens coated with ammonium chloride. Pteria clarki (Weaver & Palmer, 1922). Figure 33. Flypotype UCMP 15487, UCMP loc. 460, left-valve view, dorsal-margin length 62 mm. height 59 mm, XO.7. Figure 34. Hypotype LACMIP 13479, LACMIP loc. 5654, left valve, dorsal-margin length 23 mm, height 20 mm, Xl.7. Figure 35. Flypotype UCMP 15576, near Martinez?. California, left valve, dorsal-margin length 13 mm, height 12 mm, X3.3. Figure 36. Flypotype LACMIP 13480, LACMIP loc. 15450. dorsal-margin length 13 mm, height 7 mm, X4.2. Figure 37. Hypotype LACMIP 13481, LACMIP loc. 5654, R. L. Squires, 2014 Page 229 to weak sinus coincident with byssal notch, deepest on left valve on some specimens or deepest on right valve on other specimens. Posterior auricle on both valves usually short and broad. Posterior slope moderately well demarcated from umbo. Posterior sinus small. Growth lines parasigmoidal on posterior slope. Ventral margin of shell convex. Beaks moderately prominent, prosogyrous, and located approximately 18 to 25% of length of dorsal margin from anterior extremity. Ligament external, somewhat sunken, and opisthode- tic. Late Juvenile hinge (approximately 10 mm in length) unknown; adult hinge with one large resilifer extending posteriorly long distance; resilifer rhomboid shape with base (nymph) located on right valve and widest beneath beak. Left valve with small tooth below beak at base of hinge plate. Right valve with small subumbonal indentation and immediately anterior to it, anterior auricle swollen. Outer shell consisting of prismatic calcite; shell interior consisting of nacreous lamellar aragonite. Holotype: UWBM 159 (missing). Type locality: LJWBIVI loc. 324. Other type material: Paratype UW 159b (missing); hypotype UCMP 15487 of Anderson & Hanna (1925); hypotype UCMP 15576 (originally used as lectopar- atype of Pteria pellucida [Gabb, 1864]); hypotypes UCMP 32605 and 32623 of Yokes (1939), who misidentified these three specimens as Pteria pelliicicia: and hypotypes LACMIP 13479-13484. Geologic age: Lutetian to Bartonian. Distribution: LUTETIAN: McIntosh Formation, lower part, Doty Hills area, Lewis County, southwestern Washington; questionably from the Muir Sandstone near Martinez, Contra Costa County, northern Cali- fomia; Domengine and Avenal formations, Coalinga area, Fresno County, northern California; Metralla Sandstone in Grapevine Creek and Liveoak Shale in Liveoak Canyon, Tehachapi Mountains, Kern County, southern California; Matilija Fomiation, Pine Mountain area, Ventura County, southern California; and Llajas Formation, Simi Arroyo, Simi Valley, Ventura County, southern California. BARTONIAN: Cowlitz Forma- tion, Olequa Creek member in Olequa Creek and Cowlitz River areas near Vader and Olequa Creek member near Klaber, Lewis County, southwestern Washington; “Coldwater” sandstone, west of San Marcos Pass, northwest of Santa Barbara, Santa Barbara County, southern California. Discussion: The examined material consisted of 12 specimens, with most of them from LACMIP locality 5654. Most of the material consists of paired-valved specimens. The extremities of the auricles, especially those of the posterior auricles, are usually missing. The specimens shown in Figures 36 and 39 have their anterior auricle intact. Pteria elarki was previously reported to be restricted to Washington State. This present study is the first to recognize that it also occurs in California and that Yokes’ (1939) so-called Pteria '"‘pellticida" specimens (i.e., lectoparatype UCMP 15576, questionably from the Muir Sandstone near Martinez, California [see “Discussion” of Pteria peUucida] and his hypotypes from north of Coalinga, California) are actually Pteria elarki. Pteria elarki differs from P. pelhieiila by having a larger average size, greater maximum size, height usually greater than length, commonly less inllated umbonal area, stronger demarcation between umbonal area and posterior slope, and well developed byssal notch. DISCUSSION Mode of life of Recent Pteria Approximately 24 modern species of Pteria are known (Wada & Temkin, 2()08). Most live in relatively shallow depths in subtropical and tropical seas. A few are known from temperate regions (Wada & Temkin, 2008), and rare species are found in abyssal habitats (Boss, 1982). They are most commonly found in sandy sediment, either on mud flats or in shallow-subtidal waters, no more than a few meters deep (Moore, 1983). Keen (1971) reported that Pteria sterna (Gould, 1851) can occur as clusters on mud flats, or it can occur in shallow water offshore. Most modern Pteria have a highly specialized life habit and live byssally attached to flexible alcyonarian corals (e.g., sea whips, gorgonians; left-valve hinge, dorsal-margin length 26 mm, X3. Figure 38. Hypotype UCMP 32623, UCMP loc. A-975, right-valve view, dorsal- margin length 25 mm, height 21 mm, X 1.5. Figure 39. Hypotype LACMIP 12749, LACMIP loc. 15450, right valve, internal mold, dorsal-margin length 14 mm, height 10 mm, X3. Figure 40. Hypotype LACMIP 13482, LACMIP loc. 17867, right valve, dorsal- margin length 23 mm, height 20 mm, X 1 .8. Figure 41 . Hypotype LACMIP I 3545, LACMIP loc. 5654, nearly complete right valve view, length 28 mm, height 27 mm, X1.9. Figure 42. Hypotype LACMIP 13546, LACMIP loc. 16850, antei ior end of right-valve hinge, length I I mm. height 5 mm, X5.3. Figure 43. Hypotype LACMIP 13481, LACMIP loc. 5654. dorsal view of paired-valved specimen, dorsal-margin length 26 mm, X3.2. Figure 44. Hypotype LACMIP 13545, LACMIP loc. 5654, dorsal view of paired- valved specimen, dorsal-margin length 28 mm, X2.9. Figure 45. Hypotype LACMIP 13545, LACMIP loc. 5654, anterior view of paired-valve specimen, height 27 mm, X2.1. Page 230 The Veliger, Vol. 51, No. 4 Temkin, 2006:271). Recent Pteria species are adapted for tight epifaunal anchorage by means of their byssal notch and anterior auricle complex. When attached to a tlexible coral, the full force of waves and currents is cushioned by movements of the coral’s flexible stalk, thereby, Pteria obtains stable fixation in an agitated environment. The elongate posterior auricle is used for deflection of environmental currents, thereby facilitat- ing the dispersal of the exhalant current created by the bivalve. The right (lower) valve is usually flattened somewhat in order to improve stability against disruptive-water movements (Stanley, 1972). Stanley (1970:136) reported that Pteria eolytuhus (R5ding, 1798), which lives in southern Florida and Puerto Rico, attaches itself almost exclusively to the upper portions of whip-like alcyonarians in shallow- subtidal waters, although a few individuals attach to inanimate objects. This pteriid is apparently gregarious, with as many as five or six individuals commonly inhabiting a single alcyonarian colony. Stanley (1972:189) also reported seeing P. eolywhas still attached to alcyonarians uprooted by a storm. Al- though Pteria eolynthiis attaches by a strong byssus, it can alter its posture to a limited extent by contraction of its byssal muscles. Its posterior auricles are directed upward at angles comparable to those formed by the lateral branches of the alcyonarian colony (Stanley, 1970: pi. II. fig. 3;1972:text fig. 24A). A strong exhalent current issues from the posterior sinus area (Stanley, I970:pl. 11, fig. 4; 1972:text fig. 25A). Paleoecological Implications of the Taphonomy of the Studied Fossil Pteria Only very generalized statements about the inferred taphonomy of the studied fossil species can be made because the original collectors did not use quantitative collecting techniques, nor did they provide detailed sedimentologic information. A few localities are in rocks whose depositional environments have been studied by others, and these localities are discussed below. Most of the studied Pteria specimens occur in silty fine-grained sandstone, except for P. lochi, which is found in silty mudstone. At most localities, the Pteria specimens of all the species are fewer than 30 in number and are unbroken single valves missing the tips of their auricles, especially the tip of the very fragile posterior auricle. Left valves commonly outnumber right valves, and paired valves are none to one. Nearly complete growth series are usually present, with lengths of the valves ranging from 8 to 32 mm. At every locality, the associated mollusks are indicative of normal-salinity, shallow-marine waters. The locale with the best taphonomic information about Pteria is the lower Chico Formation in Big Chico Creek, Butte County, California, because (1) Saul ( 1959) made extensive collections from several localities in a vertical stratigraphic section there and (2) Russell et al. (1986:fig. 10) plotted the ranges and abundances of Pteria pellucicla in this section and worked on the details of the depositional environments of the beds containing the localities. Russell et al. ( 1986) concluded that the Chico Fomration in this area consists of a gradational deepening-upward sequence, with the fluvial and deltaic facies of the Ponderosa Way Member overlain by the shoreface deltaic facies of the Musty Buck Member, which is, in turn, overlain by the inner shelf facies of the Ten Mile Member. Specimens of P. pellucicla are found in the lower two members but not in the deeper water and less agitated-water deposits of the Ten Mile Member. At LACMIP locality 23620 in the lower Musty Buck Member, Pteria peUiicida is very abundant. The specimens are in poorly sorted medium- to coarse- grained sandstone in association with conglomerate and wood remains. An astonishing 82 of the 94 specimens (i.e., 87%) of P. pelliieida collected from there by Saul (1959) are paired valves. A few of these paired valves have the valves agape but still attached at the hinge (i.e., “butterfiied”). This locality occurs in the part of the section that Russell et al. (1986) reported as having been deposited at the seaward edge of a deltaic complex. The only other macro-invertebrate fossils found at locality 23620 are Acila demessa and Calva tctffi. Russell et al. (1986) mentioned that the Pteria pellucicla assemblages in the lower part of this member represent a low-diversity community that probably ranged from shallow-marine to slightly brackish waters. The low diversity can be confirmed at locality 23620, but A. demessa and C. taffi are normal-marine species. Also at this locality, individual valves of P. pellucicla are nestled together in a vertically stacked manner, most likely the result of current and/or wave action. Figure 9 shows two nestled, left-valve speci- mens. Stephenson (1952:pl. 14, figs. 8-10) reported similar nestling (stacking) of two or more valves, either right or left, as well as several nestled “butterfiied” specimens, of Phelopteria Stephenson, 1952, of Ceno- manian age in northeastern Texas. In the upper Musty Buck Member at LACMIP locality 23625, 90 m stratigraphically above locality 23620, only a single specimen out of a total of 233 specimens has paired valves. The beds at 23625 consist of poorly sorted coarse- to very coarse-grained sandstone with some pebbles. This locality occurs in the part of the section that Russell et al. ( 1986) reported as having been deposited within the turbulent-water, cross-bedded shoreface part of a deltaic complex. The fossils, therefore, represent storm-accumulation lags. At locality 23625, nevertheless, most of the single- valved specimens of P. pellucicla are unbroken, and R. L. Squires, 2014 Page 231 they represent a nearly complete growth series, ranging in length from 7 to 31 mm. There are 143 left valves and 89 right valves in the LACMIP collection from this locality. A bed containing P. pelliicida at LACMIP locality 10787 in Member V of the Redding Formation, Shasta County, California, is similar to the bed at LACMIP 23625 in that a small piece of rock, 47 mm long and 20 mm high, from locality 10787 has seven valves (six left and one right) of P. pelliicida on a single bedding plane. The locality that has the most specimens of Pteria ciarki is LACMIP 5654 in the Cowlitz Formation in the vicinity of the Big Bend of the Cowlitz River just east of Vader, Lewis County, southwestern Washington. Seven specimens were collected, and six (86%) are paired valves. They range in length from 14 to 41 mm. The Cowlitz Fonnation is part of a fluvial-dominated deltaic complex consisting of several members (Hen- riksen, 1956). Using his geologic map, locality 5654 plots in the Olequa Creek member in the upper part of the formation. According to him, this member is a thick series of brackish-water, marine, and terrestrial deposits with intercalated coal beds. Many of the specimens of P. ciarki from this locality are large and paired valved. Nesbitt ( 1995:1069) reported that Pteria ciarki found in the Cowlitz Formation probably attached to the substrate and not to alcyonarian fronds, but she did not discuss any evidence. She also mentioned that unbroken, disarticulated shells of P. ciarki dominate a storm deposit in the Cowlitz Formation on Olequa Creek. It is plausible that in the study area there is a direct association between land-plant remains and presence of a significant number of paired-valved specimens of Pteria. At Saul’s LACMIP loc. 23618, in the lower part of the Musty Buck Member of the Chico Formation at Big Chico Creek, the locality consists of a single concretion surrounding a large piece of tree limb that was intricately bored by the bivalve Martesia. At this locality, four of the 35 specimens of P. pellacida are paired valves. Plant remains are also common at locality 23620, 15 m stratigraphically above 23618. Small pieces of wood (25 mm in length) are also common at LACMIP locality 30123 in the upper Turonian Baker Canyon Member of the Ladd Forma- tion, and of the seven species of Pteria pellacida at this locality, two have paired valves. Finely disseminated pieces of carbonized plant material are present at LACMIP locality 7792, the only locality where Pteria held is known, and at LACMIP locality 17868, the only locality where Pteria sp. was found. Land-plant remains are also common in the Olequa member of the Cowlitz Formation. It seems likely that P. pellacida, and possibly P. ciarki, lived attached to land-plant remains that were either sunken, submerged, or that Boated into the sites of deposition. Or, the Pteria might have been like the Cenomanian Pteria adnata (Kauffman et ah, 1977) that has been reported as having lived attached to either shell fragments, erect- algal strands, or to Boating rafts of seaweed (Kauffman et ah, 1977:51). Using plant remains or other floatable objects for attachment could account for ( I ) why the study area species of Pteria are so relatively scarce (i.e., the requirement for nearness to swamps/forests or seaweed-rich shorelines), and (2) why there is locally such a high percentage of paired valves of Pteria. Boreholes and epibionts are very rare on P pellacida. Their relative absence could be result of the effective- ness of the thick, shaggy periostracum that is present on Pteria. Naticid? boreholes are slightly more common on P. ciarki than on P. pellacida, perhaps indicating the presence of more predators in the Eocene than in the Late Cretaceous. Paleobiogeographic Comments According to Hertlein & Cox (1969), Pteria origi- nated during the Triassic in the western Tethys Sea region. Hallam (I98l:table 2) reported that Pteria was present as early as the late Middle Triassic (Ladinian). Temkin (2006) queried the Triassic occurrences because of the uncertainty arising from previous workers reporting a large number of pteriiform bivalves ascribed to Pteria without any reference to other characters. More work is needed to establish if Pteria existed during the Early .lurassic. Middle and Late Jurassic species of Pteria in western Europe and western China are confirmable because infonnation is known about their hinges (e.g., Chavan, 1952; Fursich & Werner, 1988; Yin & Fiirsich. 1991). Pteria occurrences are undoubtedly more common than earlier reported because, according to Fursich & Werner ( 1988), the pteriid genus Pteroperna Morris & Lycett, 1853, in which mcist Jurassic pteriids have been assigned to in the past, is a junior synonym of Pteria. On the Pacific slope of North America, three pre- Albian species have been assigned to genus Pteria, but none is actually Pteria (= Avicala). Avicala macroaata Gabb (1864:30, pi. 5, fig. 27) from Triassic strata, Plumas County, northern California has fine radial ribs and a very narrow alation of the posterior hinge. It is probably an aviculopectinid. Avicala (O.xytoma) wla'te- ave.si Stanton (1895:38, pi. 4, fig. 1) and Pteria {O.xytoma) califondca Anderson (1945:963. pi. 15, figs. 5, 6), both from Lower Cretaceous strata in northern California, have costate radial ribs and belong to genus O.xytoma Meek, 1864. On the Pacific slope of North America, the only Albian species that has been assigned to genus Avicala is Avicala gaia.saaa Anderson (1958:106, pi. 1. fig. 7) from northern California. Its Page 232 The Veliger, Vol. 51, No. 4 shell is very elongate and does not possess an anterior auricle. Its hinge is unknown. This species most likely belongs in another genus. The geologic record of Pteria is bolstered somewhat by the recognition here that Phelopteria is a junior synonym of Pteria. A few Cenomanian occurrences in eastern North America (Stephenson, 1936, 1952) and a Maastrichtian occurrence is South Dakota (Speden, 1970) are important because of the excellent preserva- tion of the juvenile hinges of these fossil Pteria. The arrival of Pteria into the Pacific slope of North America corresponds to the appearance of Pteria pellucida in the early Turonian. Its arrival coincided with what has been reported (Saul, 1986; Frakes, 1999) as the warmest interval of the Late Cretaceous. Concurrently, there was a high-sea stand during the Turonian, and the seaway across southern Mexico was apparently at its widest (Imlay, 1944; Alencaster, 1984). These conditions would have facilitated migration of Pteria larvae into California via westward-flowing, warm-equatorial currents originating from possibly the western part of the Tethys Sea region. There are nearly continuous records of Pteria from lower Turonian through Santonian strata in the study area. Its occurrence in the Coniacian and Santonian, which were slightly cooler times relative to the Turonian (Saul, 1986), attests to its adaptation to warm- temperate conditions. The geologic range of Pteria pellucida corresponds to approximately 10 million yr. This duration, however, is less than some other “archaic” (i.e., not well derived) bivalves from the study area. For example, the pinnid Pinna calamitoides ranges for approximately 22 million yr, from the Turonian to late Campanian (Packard & Jones, 1965); and the nuculid Acila (Truncacila) deme.s.sa ranges for approximately 20 million yr, from late Turonian to latest Campanian or to possibly early Maastrichtian (Squires & Saul, 2005). There are major gaps in the study area’s record of Pteria for most of the Campanian and Maastrichtian, as well as for the Paleocene and early Eocene. These gaps are puzzling for a thermophilic genus like Pteria because the surface-water ocean temperature during the Campanian and Maastrichtian in the study area gradually became warmer relative to the Santonian (see Saul, 1986), and the late Paleocene and early Eocene coincided was the warmest time of the Cenozoic (i.e., the PETM = the Paleocene- Eocene thermal maximum event; Gradstein et al., 2004:402, fig. 20.5). The last- appearance datum for P. clarki is at the end of the Bartonian, just prior to a late Eocene global-cooling event that initiated a faunal turnover in the molluscan fauna of the Pacific slope of North America (see Squires, 2003). The study area’s late Oligocene and Miocene record of Pteria consists of a few species from the central to southern California area, and there are no Pliocene and Pleistocene records (Moore, 1983). Although no species lives today off the coast of the study area, Pteria sterna lives in Baja California through the Gulf of California, Mexico and south to Peru, in shallow-water offshore. In particularly warm years (e.g., El Nino years), P. sterna has occasionally been reported as a temporary species as far north as the warm-temperate waters off southern California (Coan et al., 2000). The paleobiogeographic distribution pattern of Pteria is like that observed (Squires et al., 2001) for the thermophilic neritid and cypraeoidean gastropods, which also retreated south- wardly in the study area after the late Eocene. Acknowledgments. LouElla Saul (LACMIP) shared her considerable knowledge about Cretaceous bivalves. Lindsey T. Groves (LACM) helped in obtaining literature and provided very useful loans of Recent pteriids. 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Denkschriften Akademie der Wissenschaften Wien. Mathematihisch-Naturwis- senschaftliche Klasse, 24, 105-179; v. 25: 77 198. APPENDIX LOCALITIES CITED LACMIP 5654. Section 28, T. 11 N, R. 2 W, north bank Cowlitz River, 1.5 mi E of Vader, Castle Rock Quadrangle (7.5 min, 1953), Lewis County, southwest- ern Washington. Cowlitz Fomiation. Age; Late middle Eocene (“Tejon Stage”). Collector: J. L. Goedert. August 29, 2003. LACMIP 7792. North of Palomar Airport, near some clay pits south of Letterbox Canyon, 5 m above base of temporary cut bank exposing I 7 m of section, N side of Faraday Ave, E of intersection with Rutherford Road, Carlsbad Research Center, ap- proximately 1 mi E of city limits of Carlsbad. San Luis Rey Quadrangle (7.5 min, 1975), southern California. Point Loma Formation. Age: Latest Campanian or possibly early Maastrichtian. Collec- tor: .1. Loch, January I, 1984. LACMIP 10787. Near crest of N slope of divide between Clover Creek and Basin Hollow, near NE corner of NW 1/4 of section 33 and not more than 122 m S of section line, T. 32 N, R. 2 W, Millville Quadrangle (15 min, 1953), Shasta County, northern California. Redding Formation, Member V, lower part. Age: Early Santonian. Collectors: W. P. Popenoe & D. W. Scharf, August 8. 1931. LACMIP 15450 (= LACMIP 25837 = CSUN 548). At elevation of 288 m on S side of Simi Arroyo just above the streambed, 335 m N and 533 m W of SE corner of Page 236 The Veliger, Vol. 51, No. 4 section 12, T. 2 N, R. 18 W, Santa Susana Quadrangle (7.5 min, 1951, photorevised 1969), Ventura County, southern California. Llajas Formation. Age: Middle Eocene (“Domengine Stage”). Collector: G. Slak, 1997 1998. (Note: locality plotted by Squires [2001 :fig. 1]). LACMIP 16850 (= CSUN 1570). Thin interval of sedimentary rocks interbedded with a basalt unit in quarry at E end of bluff overlooking W side of Garrard Creek. 1 50 ft N and 1 700 ft W of SE corner of section 21, T. 15 N, R. 5 W, Cedarville Quadrangle (7.5 min, 1986), extreme NW corner of Lewis County, Washington. Transition zone between upper Crescent Formation and lower member of McIntosh Forma- tion. Age: Middle Eocene. Collectors: J. L. & G. H. Goedert. 1993-1994. LACMIP 17867. NEl/4 of section 36, T. 13 N, R. 4 W on the South Fork of the Chehalis River. 0.8 km (0.5 mi) N of Klaber, Lewis County, southwestern Washington. Cowlitz Eormation, Olequa Creek member. Age: Late middle Eocene. Collector: J. L. Goedert, 1998. (Loc. = 10° dip on plate 1 of Henriksen [1956]). LACMIP 17868. On small ridge immediately E of new large water tank, 18,000 ft W and 14,700 ft W of NE corner of Topanga Quadrangle (7.5 min, 1952, photorevised 1981), upper Pulga Canyon, The Enclave. Palisades Elighlands, east-central Santa Monica Mountains, Los Angeles County, southern California. Upper part of Santa Susana Formation. Age: Late Paleocene (Thanetian). Collector: R. L. Squires, March 17, 2007. LACMIP 23619. First ravine to S of bridge below Mickey’s House on E side of Chico Creek and approximately 1/6 mi upstream from Chico Creek country road, 1300 ft S, 2050 ft W of NE corner of section 12, T. 23 N, R. 2 E, Paradise Quadrangle (1953), Butte County, northern California. Chico Formation. Musty Buck Member. Age: Early Santo- nian. Collectors: L. R. & R. B. Saul, August 1 1, 1952. LACMIP 23620. Fossils from blue-giuy concretion, much plant remains. First stream to S of bridge below Mickey’s House on E side of Chico Creek, 1200 ft W and 1750 ft W of NE corner of section 12, T. 23 N, R. 2 E, Paradise Quadrangle (1953), Butte County, northern California. Chico Fomration, Musty Buck Member. Age: Early Santonian. Collectors: L. R. & R. B. Saul. August 1 1. 1952. LACMIP 23625. East bank of Chico Creek, 300 ft N of right-angle bend in Chico Creek, 2000 ft north, 1000 ft E of SW corner of section 12, T. 23 N, R. 2 E, Paradise Quadrangle (1953). Chico Formation, Musty Buck Member. Age: Late Santonian. Collec- tors: L. R. & R. B. Saul, August 23, 1952. LACMIP 24659. Buff greywacke with concretionary fossiliferoLis lenses cropping out in small canyon north of dirt road heading downstream about 0.5 mi W of Black Rock, approximately 45 ft S, 1800 ft E of NW corner of section 23, T. 27 N, R. 2 E, Mill Creek Canyon, Butte Meadows Quadrangle, Tehama County, California. Chico Eormation, Musty Buck Member (might be Ten Mile Member). Age: Early Santonian. UCMP 460. Four miles S, 10° W of 1085 ft bench mark near pumping station on highway (this would be in or near Tecuya Creek), Tejon Quadrangle. Tejon Group, near top. Age: Eocene. Collector: R. E. Dickerson. UCMP B-761. 2400 ft S and 950 ft E of NW corner of section 12, T. 6 S, R. 6 E, approximately 13 km SW of Patterson, Patterson Quadrangle (7.5 min, 1971, photorevised), Stanislaus County, northern Califor- nia. Panoche Fomiation, near top of unit II. Age: Santonian. Collector: C. C. Bishop, 1960s. (Locality is plotted on geologic map of Bishop [1970]). UCMP A-975. Second “reeE’ above base of Domen- gine Formation in draw across ridge to S of Big Tar Canyon, T. 23 S, R. 17 E, Garza Peak Quadrangle (7.5 min). Kings County, central California. Domen- gine Eormation. Age: Middle Eocene. Collector: H. E. Yokes, circa 1939. UCMP 2291. Elevation 1200 ft, NE corner of SE 1/4 of SW 1/4 of section 29, T. 18 S, R. 15 E, Coalinga Quadrangle, Fresno Co, central California. Domen- gine Fomiation. Age: Middle Eocene. Collector: B. L. Clark, date unknown. UCMP 3152. Northwest 1/4 of section 20, T. 1 N, R. 2 E, Mt. Diablo Quadrangle, Deer Valley, Contra Costa County, northern California. Meganos Formation. Age: Latest Paleocene to early Eocene. Collectors: B. L. Clark & A. O. Woodford, circa 1927. UCMP 3767. Elevation 1 300 ft, on ridge, NW 1/4 of NW 1/4 of section 24, T. 2 N, R. 18 W, 9700 ft N14°E of 2150-ft hill in Simi Hills. Calabasas Quadrangle, Ventm'a County, southern California. Santa Susana Fomiation. Age: Late Paleocene. Collector: R. N. Nelson, circa 1925. UCMP 3783. Elevation 1 150 ft, a little SE of center of section 23, T. 2 N, R. 18 W, 7000 ft N5°W of 2150-ft hill in Simi Hills, Calabasas Quadrangle, Ventura County, southern California. Santa Susana Forma- tion. Age: Late Paleocene. Collector: R. N. Nelson, circa 1925. UWBM 324. Qn Olequa Creek about 1/8 mi N of Vader, in section 29, T. 1 1 N, R. 2 W, Castle Rock Quadrangle (15 min, 1953), Lewis County, south- western Washington. Cowlitz Formation. Age: Late middle Eocene. Collectors: C. E. Weaver & K. V. W. Palmer, circa 1922. The Veliger 5I(4):237-251 (September 16. 2014) THE VELIGER ' CMS, Inc., 2014 Additions to the Genus Phyllodesmium, with a Phylogenetic Analysis and its Implications to the Evolution of Symbiosis ELIZABETH MOORE AND TERRENCE GOSLINER Department of Invertebrate Zoology, California Academy of Sciences, 55 Music Concourse Drive, San Francisco, California 94118, USA (e-mail: tgosliner@calacademy.org) Abstract. The facelinid genus Pliyl/cn/esmiiini (Ehrenberg, 1831) consists of approximately 24 described species that prey upon soft-bodied cchrals. At least five additional species have yet to be described, making it an interesting genus for testing phylogenetic hypotheses. The genus is extremely morphologically diverse, with many species adapting specifically to a specific host coral. One of the most interesting adaptations found in this genus is the widespread participation in a symbiotic relationship with photosynthetic dinofiagellates in the genus SymhioiUniwn. Two new species, Phyllodeswiwu acaiit/iorliiinini n. sp. and Pliyllodesiuiwu iindulatiini n. sp., from the Philippine Islands and Japan are described, and a morphological phylogeny is created to include the two new species, as well as three undescribed species. An examination of the Phyllodesmiinn phylogeny suggests that species with digestive gland branching and zooxanthellae are more derived. Confidence and robustness in this analysis are lacking, however, and further studies using molecular data could add confidence to this conclusion. INTRODUCTION The facelinid genus Pliyllodesiniuni (Ehrenberg, 1831) consists of approximately 24 described species that prey upon soft-bodied corals. At least five species have yet to be described, making it an interesting genus for taxonomic and phylogenetic studies (Burghardt and Gosliner, 2006; Burghardt et ak, 2008a; Moored Gosliner, 2009). While this paper was in press, eight additional species were described (Burghardt et ak, 2008a). Each newly described species leads to exciting discoveries about the adaptive history of this group, and adds another piece to the evolutionary puzzle. One of the most interesting adaptations found in this genus is the widespread participation in a symbiotic relationship with photosynthetic dinofiagellates (zooxanthellae) in the genus Synihiodinium. Many species of Phyllodesmium are able to retain zooxanthellae, which they obtain from their alcyonarian food source (Kempf, 1984; Rudman, 1981, 1991). This is a unique relationship compared to other Syndiiodbuinn symbioses because the nudibranchs are not the primary host of the zooxanthellae (Rudman, 1981). Instead, the nudibranch consumes and digests the coral, the primary host of the symbiont, while selectively preserving and translocating intact zooxan- thellae into the cells of the digestive gland. Symbiotic Pliyllodesniinm species have branched digestive glands that often ramify into the cerata and dorsal surface of the body, where exposure to sunlight is at its highest. Once harvested, the zooxanthellae are passed into these branches and the nudibranch receives nutrients produced by the photosynthesizing algae. Histological studies have shown a positive correlation between the extent of digestive gland branching and the zocixanthellae retention abilities of these nudibranchs. Highly specialized species are able to accumulate vast amounts of zooxanthellae in extensively branched digestive tissue, maximizing the photosynthetic product they receive from their stolen symbionts. Based on this observation, Rudman (1991) suggested that animals with minimal or no branching, and thus few or no zooxan- thellae, are more primitive species, whereas animals with vastly branched digestive tissue have further evolved to accommodate algal symbionts. Although correlative evidence supports this idea, phylogenetic study to support this hypothesis has not been completed, and long-term starvation experiments have just recently begun (Bur- ghardt et ak, 2005, 20()8b; Burghardt & Gosliner, 2006; Burghardt & Wagele, 2006). In addition, phytogenies based on genetic characters (Moored Gosliner, 201 1 ) and have been published. Another interesting aspect of the symbiotic relation- ship in the genus Pliyllodesniinm is the variation in ability to retain zooxanthellae. Some species, such as Phyllodesmium opalescens R udman, 1991, are complete- ly aposymbiotic and digest the algae with the rest of their prey, or sometimes pass the cells unharmed. Some, such as Phyllodesmium hyuUuum Ehrenberg, 1831, can retain the algae for a shcirt time, and others, like Phyllodesmium longicirrum (Bergh. 1905), and Phyllo- desmium hviareum (Bergh, 1896) have extremely ad- vanced mutualistic relationships that allow them to survive significant periods of time in the absence of a food source (Rudman, 1981. 1 991; Kempf, 1991; Burghardt et ak. Page 238 The Veliger, Vol. 51, No. 4 Table 1 Literature references used to create the data matrix for phylogenetic analysis. Taxon Reference Favorinus japonicus Baba. 1949; Riidman. 1980 Godiva qnadricolor Willan, 1987 P. horridimi Rudman. 1981. 1991 P. seiralnin Rudman. 1991; Baba, 1991a P. opalescens Rudman 1991 P. poindimiei Rudman, 1981, 1991 P. hriareimi Rudman, 1991; Gosliner et al 1., 1996 P. coleniani Rudman, 1991 P. nuiginim Rudman 1991 P. parangatum Ortiz & Gosliner, 2003 P. hvaliinmi Rudman, 1981, 1991; Baba, 1991b P. crypticum Rudman, 1981, 1991 P. niacphersouae Rudman, 1981, 1991; Baba 1 991b P. longicirrum Rudman, 1981, 1991 P. guamensis Avila et al., 1998 P. pecteu Rudman, 1981 P. iriomotense Baba, 199 lb. This study P. kahiranwn Baba. 1991b P. jakohsenae Burghardt & Wagele, 2004 P. nulmani Burghardt & Gosliner, 2006 P. acani/wrin'nnm n. sp. This study P. undulatwn n. sp. This study P. luherciilutwu Moore & Gosliner. 2009 P. pinnatiun Moore & Gosliner, 2009 P. karenae Moore & Gosliner, 2009 2005, 2008b). Phyllodesmiiiw longicivvwn is especially notable, housing thriving communities of algae. This species may even cultivate populations of zooxanthellae by regularly digesting a fraction of the symbionts (Rudman, 1981; Kempf, 1991). The range of intimacy between the nudibranch and zooxanthellae that exists within species of Phyllodesmiiim makes it an ideal place to study the progression of symbiosis as it evolved in these animals (Wiigele, 2004). In this study, two new species of Phyllodesmium are described using anatomical dissections and scanning electron micrographs. An updated phylogeny that includes the new species was then generated based on morphological characters. METHODS Drawings of anatomical structures were completed using a Nikon SMZ-U binocular microscope with drawing tube. Buccal mass structures were coated with gold/ palladium using a Denton Desk II vacuum sputter coater, and scanning electron micrographs were produced by a LEO 1450 VP scanning electron microscope. Specimens were deposited at the California Academy of Sciences in the Invertebrate Zoology Department collection (CASIZ). Using anatomical characters, a morphological phy- logeny was created to infer placement of the new species and to map symbiosis within the genus. Using the character matrix constructed by Ortiz & Gosliner (2008) as a guide, characters and character states were reevaluated by careful examination of the literature (Table 1). When necessary and possible, dissection of specimens was used to decipher character states that were not made clear by descriptions in the literature. The reevaluated characters were then entered into MacClade 4 software (Maddison & Maddison, 2005) to generate an updated matrix (Table 2). As was done by Ortiz & Gosliner (2008), Godiva ciiiadricolor (Barn- hard, 1927) and Favorinus japonicus Baba, 1949, were retained as outgroups due to their likely relationship with species of Pliyllode.smiuiii. Other characters and character states, however, were modified from the previous study. Parsimony analysis was conducted using a heuristic search with 100 replicates of starting trees using random stepwise additions in PAUP 4.0 (Swofford 2002). All uninfomiative characters were excluded from the analysis, as well as character number 2. This character was difficult to quantify and was removed to avoid confounding the data set. A permutation tail probability test was conducted using PALIP to determine if the resulting phylogenetic tree is significantly different from randomness. Decay analysis was performed using a heuristic search in PAUP for all trees greater than or equal to the shortest trees obtained. RESULTS Phyllodesmium acauthorliimim n. sp. (Figures lA; 2 A; 4A, B; 5 A, B; 8 A) Phyllodesmium spec. Wagele et. al, 2006: 38, figure 51. Phyllodesmium spec. 6 Gosliner et. al, 2008: 389, bottom photo. Material examined Holotype: California Academy of Sciences, CASIZ 099093, 3-A m depth, near Onna Village, Horseshoe Cliffs, Okinawa, Ryukyu Islands, Japan, 1 July 1994, R. F. Bolland. Paratypes: Three specimens, two dissected, CASIZ 104702, m depth, near Onna Village, Horseshoe Cliffs, Okinawa, Ryukyu Islands, Japan, 8 July 1994, R. F. Bolland. Geographic range This species is known from the Horseshoe Cliffs, Okinawa, Ryukyu Islands, Japan (this study) and E. Moore & T. Gosliner, 2014 Page 239 Character states present in species Table of Phyllodesmiiim 2 and outgroups G. (jiiadricoloi and F. /upon i CHS. 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Fuvorimis japouicus 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 1 1 7 1 ? 0 7 7 0 1 1 1 0 Godivu cjiiadricolor 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 1 1 1 1 0 1 0 P. hon idiun 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 0 0 0 1 1 1 0 0 1 0 1 1 0 9 P. sermtum 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 1 1 1 0 1 0 0 9 9 9 1 P. opalesceus 0 0 0 0 0 0 0 0 f) 1 0 0 0 1 0 0 1 1 0 0 0 0 0 1 0 0 1 0 1 1 0 1 P. poinduuiei 1 0 1 0 0 1 1 0 0 0 1 1 1 1 0 1 1 2 7 0 0 0 0 0 0 0 1 0 1 1 1 3 P. hriareiDu 0 1 2 1 0 0 0 0 0 0 1 1 1 1 0 0 1 2 7 0 0 1 1 1 0 0 0 0 1 1 1 2 P. colommi 0 1 2 1 0 1 0 0 0 0 1 1 1 1 0 1 1 2 7 1 1 1 1 1 0 0 0 0 1 1 0 4 P. magnum 1 0 2 1 0 1 1 0 0 0 1 1 1 1 0 0 1 2 7 1 1 1 1 0 0 0 1 0 1 0 1 3 P. parangatum 1 0 2 1 0 1 1 1 0 0 1 1 1 1 0 0 1 1 1 0 1 0 0 1 0 1 1 0 1 0 1 1 P. hyalinum 0 0 2 1 1 1 1 0 0 0 0 0 0 1 1 1 1 1 1 0 0 1 1 1 0 1 0 0 1 1 1 3 P. crypticum 1 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 1 1 0 7 0 0 1 1 1 3 P. macphersoiuie 0 1 2 1 0 1 1 0 0 0 1 1 1 1 0 1 1 2 7 0 0 0 0 0 0 7 0 0 0 1 1 1 P. /ongicirriim 1 0 2 1 0 1 1 0 0 0 1 1 1 0 0 0 1 2 7 1 1 1 1 1 0 7 1 0 1 0 1 3 P. guamensis 1 0 2 1 0 1 1 1 0 0 1 1 1 1 0 0 1 2 7 0 1 0 1 1 1 0 0 0 1 1 0 3 P. pecten 0 0 2 7 0 1 0 0 0 0 0 1 1 1 0 0 1 1 1 0 1 1 0 1 0 1 1 0 1 1 0 3 P. in'omotense 0 1 1 0 0 1 0 0 0 0 0 1 1 0 0 1 1 1 1 0 0 0 1 1 0 9 7 0 1 0 0 9 P. kahiranum 0 0 2 1 I 1 1 0 0 0 0 0 0 1 0 0 1 1 1 0 0 0 1 1 0 1 1 0 1 1 1 3 P. jakohsenae 0 0 2 1 0 1 1 7 0 0 7 1 0 0 1 1 1 0 0 1 1 1 0 1 0 0 1 1 0 3 P. riichnani 1 0 2 1 0 0 0 1 0 0 1 1 1 0 0 1 1 1 1 0 0 1 0 1 0 1 1 0 1 1 0 3 P. ucanthorhiniim n. sp. 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 0 1 1 0 1 P. uiuhilatiim n. sp. 0 1 0 0 0 0 1 0 0 1 0 0 0 0 0 1 1 2 7 0 1 0 0 1 0 0 0 0 1 1 1 9 P. tuhercidatium 0 0 2 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 1 0 0 1 0 1 0 0 1 P. pinnatum 0 0 2 1 1 1 1 0 “> 0 0 0 0 1 0 1 1 1 1 0 0 0 0 1 0 0 1 0 1 0 1 1 P. kurenae 0 0 1 1 0 0 0 0 0 0 1 1 1 1 0 1 1 1 1 0 0 0 1 1 0 1 0 0 1 0 1 1 Lizard Island, Great Barrier Reef, Australia (Wagele et al„ 2006). Etymology This species was named after the spiny appearance of the papillae on the surface of the rhinophores. Natural history Specimens were all found in shallow water on vertical walls with miscellaneous red algae. The wall where specimen 099093 was found was near a bottom covered with a live, stony coral reef, whereas the others were found in a rocky cut near shore. The diet of this species is still unknown. Description Color and external morphology: The living animals are elongate, with edges of the foot extending laterally just beyond the mantle. They range in length from 1 7 to 28 mm for living animals studied. Preserved specimens examined (CASIZ 104702 include three specimens, two of which were dissected and are later referred to as 104702a and 104702b) were 14 mm (CASIZ 104702a), 8 mm (CASIZ 104702b), and 8 mm (CASIZ 099093). The anterior portion of the foot margin is broad with short, blunt angular foot corners while the posterior end is tapered to a point. The body of the living animal, including the rhinophores, oral tentacles, and foot is predominantly transparent with opaque white markings on the dorsum. These markings vary and can appear as white specks or as distinct white lines creating a network along the dorsum and head (Figure lA). The anterior margin of the foot is white in some specimens, extending to the angular foot corners. The viscera and gonad are readily visible through the mantle tissue. The cerata are elongate and cylindrical, with larger cerata near the medial region of the dorsum. The digestive gland is undulating and undivided within the cerata and is cream or yellow colored near the body leading to red, and then yellow at the apex of each ceras. The cerata are primarily transparent, with slight blue coloration near the tips and have a cnidosac without nematocysts. The ceratal arrangement consists of arches and rows, with arches forming in the anterior ceratal groups (Figure 8A). The precardiac cerata are grouped into one arch on each side of the body containing 6-7 cerata. The genital aperture is located between the arms of the precardiac arch on the right side of the animal. The renal opening is situated in the Page 240 The Veliger, Vol. 51, No. 4 Figure 1. A, Phylhnle.snihim acanthorhiuum n. sp.. photo: Robert Bolland; B, Phyllodcsuiium iimhilatum n. sp., photo: T. Gostmer. interhepatic space, slightly toward the posterior be- tween the precardiac arch and the first postcardiac arch on the right side. The postcardiac cerata are grouped on both sides into arches containing 5-6 cerata in the first two arches, and 4-5 cerata in the third arch. The anal papilla is located within the first postcardiac arch on the right side. The fourth postcardiac group appears as a partial arch in some animals and as a row in others containing 3^ cerata. One or two additional postcardiac ceratal groups appear as rows containing 2-3 cerata. The rhinophores are conical in shape and are roughly half as long as the oral tentacles. In addition. E. Moore & T. Gosliner, 2014 Page 241 1 nim Figure 2. A, Reproductive system of Pliy/loclesmium acanlliorhimim n. sp.; B, Reproductive system of Phyllodes- iniiini umhdation ii. sp. Abbreviations: «/h, ampulla; vs, receptaculum seminis; ml, oviduct; rd, vas deferens; fynt, female gland mass; p, penial papilla; pv, prostate; alh, albumen gland; mu, mucous gland; me, membrane gland. there are numerous, yellow-cream-colored tubercles on the entire surface of the rhinophores that lead to a yellow-cream, pointed tip. The oral tentacles are smooth, and taper from the anterior edge of the head to pointed apices. They are transparent cir slightly bluish basally, leading to white or yellow-cream tips. Reproductive wystem (Pigure 2A): The large gonad occupies the posterior portion of all specimens. As in most mature animals evf this group, the female gland mass is large and consists predominantly of the mucous gland with smaller albumen and membrane glands. The large, looped ampulla branches to the oviduct and the prostatic portion of the vas deferens. The oviduct connects to the S-shapcd receptaculum seminis. A second branch extends from near the base of the receptaculum and joins the female gland mass near the Figure 3. Reproductive system of Phydodcsmium irionio- teii.se. Abbreviations: am, ampulla; vs, receptaculum seminis; od, oviduct; vd, vas deferens; p, penial papilla; pr, prostate; alh, albumen gland; mu, mucous gland; me, membrane gland. albumen gland. The second branch of the ampulla connects to the vas deferens. The proximal portion is prostatic with the prostate being highly convoluted and prominent, with a short, conical-shaped penial bulb. Buccal armature: The jaws are thin and coriaceous. There are four to seven knobby denticles situated along the masticatory border of each jaw (Figure 4A, B). The radulae have a formula of 33 X 0.1.0 for specimen 104702b and 34 X 0.1.0 for specimen 099093 (specimen l()4702a had a radular formula of 19 X 0. 1 .0; however, this may be an incomplete radula). The teeth are triangular in shape leading to a pointed and slightly curved primary cusp. The rib on the ventral side of each tooth extends from the posterior of the tooth to the apex of the cusp in some specimens and in others stops slightly short of the apex. Denticulation extends along the margin from the base of the tooth nearly to the apex. The number of denticles varies between specimens. Specimen I04702a has 19 28 denticles (Figure 5A). 104702b has 19-22 denticles (Figure 5B), and 099093 has 25 28 denticles per radular tooth. The denticles vary in appearance and can be triangular and broad, with well-separated points or slightly elongated with closely spaced denticles that reach slightly under the edge of the tooth (Figure 5 A, B). The denticles at the base of the tooth are generally more defined, where denticles near the apex of the cusp are fused together in some specimens. Remarks: Of the previously described species of Pliyl/o(/e.S'niiiini, there are only two, Pliyllode.sniiiim liorricliini (Macnae, 1954), and Pltylhnle.snuinu opale.s- cen.s Rudman. 1991, that have undivided digestive tissue within the cerata. Rudman (1981, 1991) described P. Page 242 The Veliger, Vol. 51, No. 4 Figure 4. A and B, Jaw plate and masticatory denticles of Phyllodesmiuni accmthorhiiwm n. sp. (CASIZ 099093); C and D, Jaw plate and masticatory border o( Phyllodesiniimi imdulutum n. sp. (CASIZ 105746). Iion h/um as pale translucent, which is notably different from the predominantly transparent mantle of P. acanthovhimmh The digestive gland undulates notice- ably, without nodulation, in cerata of P. acauthorhiinmi, compared to the straight and sometimes nodulose digestive gland in cerata of P. horridwu. In addition, P. honiduw and P. opcdescens have smooth rhinophores, unlike the rhinophores of P. acaiitliorliiuum, which are covered in papillae. The body and cerata of P. opcdescens are superficially similar to P. aeautlwrliimmi in coloration and transparency, but the digestive gland within the cerata of P. opcdescens is generally straight, with little or no undulation. Also, the opaque white markings along the dorsum of P. opcdescens are individual diamond- or teardrop-shaped spots running the length of the dorsum. Phyllodesinimn cicantliorliiniini has many white flecks, or thin lines creating a network along the dorsum. The reproductive system of P. ciccintlwrin'nuni also differs from the drawings of P. opcdescens and P. Iiorridnni shown by Rudman (1981, 1991). The prostate is highly convoluted compared to that of P. Iwnichi)}!, and the receptaculum seminis is notably S-shaped in contrast to the teardrop-shaped structure in P. opcdescens. Also worth noting in these papers is the incorrect labeling of the receptaculum seminis as a bursa copulatrix. The structure is found a good distance from the genital opening and is connected to the female gland by the oviduct in these animals, indicating it should be described as a receptaculum seminis. Pliylloclesiniiini iindulatum n. sp. (Figures IB; 2B; 4C, D; 5C, D; 8B) Phyllodesmiuni sp. 4 Gosliner et al, 2008: 389, top three photos. Material examined Holotype: California Academy of Sciences, CASIZ 177171, not dissected, 14 m depth. Waterfall Bay, E. Moore & T. Gosliner, 2014 Page 243 Figure 5. A and B, Radular cusps and denticles of Phylh)des)iiiuiii acauthorhimiin n. sp. (CASIZ 104702a and CASIZ 104702b, respectively; note the variation between specimens); C and 13, Radular cusps and denticles of Phyllodesmiwti umlulcitiim n. sp. (CASIZ 105746 and CASIZ 1 15810, respectively; note wear on denticles in image D). Pulau Tioman, Malaysia, 4 October 2007, T. M. Gosliner. Paratypes: CASIZ 105746, 0-17 m depth, Sepok, Maricaban Island, Batangas Province, Luzon, Philip- pines, 24 February 1995, T.M. Gosliner. CASIZ 115810, 12 m depth, beneath Tengan pier 14 km west of Ikei-shima, Okinawa, Ryukyu Islands, Japan, 25 June 1995, R. F. Holland. CASIZ 176717, 14 in depth. Waterfall Bay, Pulau Tioman, Malaysia, 4 October 2007, T. M. Gosliner. Geographic range Known from Sepok, on Maricaban Island off southern Luzon, Philippines, near Ikei-shima in the Ryukyu Islands, Japan, and Waterfall Bay, Pulau Tioman, Malaysia (present study). There is one photograph of this species from Manado, Indonesia, taken by Pauline Fiene in 1991, but there are no specimens available from this region. Etymology This species is named in recognition of the exten- sively undulating digestive duct within the cerata. Natural history Specimens are often found crawling on a red gorgonian octocoral in the genus Acahuria. This is likely the prey of this species, but actual feeding has not been observed. Description Color and external morphology: Living animals are very elongate with the mantle extending laterally just beyond the narrow foot. Preserved specimens are 15 mm (CASIZ 105746), 18 mm (CASIZ 115810), and 45 mm (CASIZ 176717) in length. The length of specimen 1 15801 when living was 46 mm. The anterior Page 244 The Veliger, Vol. 51, No. 4 portion of the foot margin is broad with moderately tentacular foot corners. The body of the living animal is predominantly transparent, with viscera and gonads visible through the mantle and foot. A single thin, opaque white line follows the entire length of the animal midctorsally between the rhinophores and along the dorsum. The anterior foot corners have a cream-yellow line extend- ing from the lateral sides of the head to the tips of the tentacular processes (Figure IB). The cerata are generally transparent with the exception of a slight blue color near the tips followed by yellow at the temrinus of each ceras. They are cylincirical and contain extensively undulating and undivided digestive tissue. The digestive gland within each ceras is a cream-yellow color near the dorsum, turns slightly pink just before the blue portion of the ceras, and turns cream-yellow again at the tip of the ceras. The cerata contain distinctive but nonfunctional cnidosacs at the distal ends. The longest and widest cerata are near the median region of the dorsum, with newly developing cerata at the edge of the mantle. The ceratal arrangement consists of arches and rows, with anteriormost groups forming arches and posteriormost groups fonning rows (Figure 8B). One specimen (CASIZ 115810) was missing all cerata, but the raised basal groupings appear consistent with the other specimens. The precardiac and first postcardiac arches both contain 7-11 cerata, with the largest animal (CASIZ 176717) having ceratal groups containing the most cerata, and the smallest animal (CASIZ 105746) with the least. The genital aperture is located just anterior to the precardiac arch on the right side. The renal pore is in the interhepatic space between the precardiac and first postcardiac arches on the right side, slightly closer to the postcardiac group of cerata. The anal papilla is directly under the first postcardiac arch on the right side. The second postcardiac ceratal arch contains 6-11 cerata, followed by 5-10 cerata in the third postcardiac arch. The fourth postcardiac ceratal group contains 5-7 cerata and is an incomplete arch, while the fifth and sixth postcardiac groups each contain 2-3 cerata in rows (specimen number 105746 had only one ceras in the fourth, fifth, and sixth postcardiac ceratal groups, but this was suspected to be due to prior injury). Specimen number 176717, being the largest specimen, had a seventh postcardiac ceratal row containing three cerata. The oral tentacles and rhinophores are smooth, and taper to pointed apices. The rhinophores are extended in length, as long as the oral tentacles in preserved specimens, and both are generally transparent with variable blue and/or yellow bands. Two specimens (CASIZ 105746 and 176717) have only yellow on the rhinophores, whereas the other has a hint of blue midway up each rhinophore. The oral tentacles and rhinophores terminate in broadly yellow tips for all specimens. Reproductive system (Figure 2B): All three dissected specimens were mature, with well-developed female glands consisting mostly of mucous gland. The albumen and membrane glands were completely developed but smaller in comparison. A large, looped ampulla branches to the oviduct and the prostatic portion of the vas deferens. The oviduct joins a nodulose receptaculum seminis, and a second branch extends from the base of the receptaculum to join the mucous gland near the albumen gland. The second branch of the ampulla connects to the vas deferens with the proximal portion being prostatic. The moderately sized prostate is slightly convoluted in two specimens (CASIZ 115810 and 176717) and totally straight in the other (CASIZ 105746), leading to a small, conical-shaped penial bulb in both specimens. The genital aperture has two openings, one each for the male and female genital systems (Figure 2B). Buccal armature: The jaws are thin and coriaceous with undeveloped, possibly vestigial, bumps on the masticato- ry border. These are not denticulate, but noticeable in both specimens (Figure 4C, D). The radulae have fonmilae 17 X 0.1.0 for specimen 105746 and 22 X O. 1.0 for specimen 115810. The radular teeth are triangular in shape leading to long, slightly curved, primary cusps in one specimen (105746; Figure 5C) and blunt, short primary cusps in the other (115810; Figure 5D). The latter specimen appears to have an abnomiality in the length of the primary cusp, or this may have been caused by severe wear on the teeth. The rib on the ventral side of each tooth extends from the posterior of the tooth to the point of curvature on the primary cusp in specimen 105746. In the specimen with the blunt, short primary cusp, the ventral rib extends nearly to the apex. Denticulation extends along the margin of each tooth, starting at the base of the tooth and ending at the curvature of the primary cusp in one specimen, and nearly to the apex of the cusp in the other. The denticles numbered 12-14 in specimen 105746 and 10-14 in specimen 11581 0. The denticles are elongate and pointed, reaching slightly underneath the ventral portion of the tooth in specimen 105746, and appear shorter and more blunt in specimen 115810. This further indicates increased wear on the teeth in the latter specimen. Remarks: Of the previously described species of Phylloclesjiiiwn. only P. opalesceus, P. Iiovvidunu and P. acanthorhiuum have undivided digestive tissue within the cerata. Flowever, in 1991, Rudman noted the presence of nodules or buds on the digestive gland in P. honidiim, which are not seen in P. opalesceiis or in the new species, P. iiiuiiilatiim and P. acaiitliorliiniuu. In addition, the predominantly transparent mantle and cerata of P. imchilatiini varies noticeably from the translucent and pale appearance of the mantle in P. E. Moore & T. Gosliner, 2014 Page 245 horriduni. When compared to P. opalescens, P. im- c/ulatiim differs noticeably in a number of ways. Externally, the digestive tissue within the cerata of P. iincliilatiini undulates unmistakably whereas in P. opatescens the tissue is relatively straight. Also, the opac]ue white line down the dorsum of P. umlulatiini is different from the distinctive white diamonds or teardrop-shaped patches down the dorsum of P. opalescens. The jaw structure of P. opalescens includes large, blunt denticles (Rudman, 1991), whereas the jaw of P. uiululatuiu has no obvious denticles. The reproductive system of P. opatescens and P. iiinliilatwn also differ, as P. iimlulatuni has a nodulose rcceptacu- lum seminis and a moderate, predominantly straight prostate. Phyllodesniium opcdescens has a smooth receptaculum seminis and a prominent, highly convo- luted prostate. When externally comparing P. iindida- tiun and P. acanthorhiniim, they can be easily distin- guished by the papillate rhinophores in P. acaiuhortunwn compared with the smooth rhinophores in P. iindiilatimu and by the single white line down the dorsum of P. iindiilatuni and the network or decked pattern on the dorsum of P. aeantliorhiniini. Addition- ally, P. iindidafuin has elongate angular foot corners, whereas those of T. aeauthorhiniiin are short and blunt. Their reproductive systems also vary. Phvl/odesmiuin iindidaliini has a nodulose receptaculum seminis and a straight, moderately sized prostate, whereas P. acamlwiinniiin has an S-shaped receptaculum seminis and prominent, convoluted prostate. The jaw morphol- ogy is definitive based on the absence of masticatory denticles in P. wuhdatwn and 4-7 knobby denticles in P. acanthorhiniun. Phy/lodesmiuni iriomoteiise Baba, 1991 (Figure 3) Material examined One specimen, CASIZ 84878, 59 m depth, Seragaki Beach 1.3 km ENE of Maeki-zaki. Okinawa, Japan, 16 October 1991, R. F. Bolland. Geographic range This species is known only from its type locality Okinawa, Japan (Baba, 1991b). Further description Although Baba created very detailed drawings of P. iriomoteiise, he did not include any data about the reproductive system of this species (Baba, 1991b: figs. 6,7). Description Reproductive system (Figure 3): The specimen was mature, with well-developed female glands consisting mostly of mucous gland. The albumen and membrane glands were completely developed but smaller in comparison. A large, hook-shaped ampulla branches to the oviduct and the prostatic portion of the vas deferens. The oviduct joins a small, nodulose recepta- culum seminis, and a second duct extends from the base of the receptaculum to join the albumen gland. The second branch of the ampulla connects to the vas deferens with the proximal portion being prostatic. The prostate is large and slightly convoluted in the distal- most portion, leading to a large, conical-shaped penial bulb. The genital aperture has only one opening for the male and female genital systems. PHYLOGENETIC ANAEYSIS In the present study, 23 species of Pliy/lodesiiiiiiiii are examined. This includes P. acaiithoiiiiinini and P. iindidatuiii. The two new additions were included in the final matrix to produce an updated phylogeny (Figure 6). Characters and their states were as follows: 1. Body size: Refers to the overall length of the animal. A distinct gap appears to be evident between species that are smaller than 40 mm and those that are usually 50 100 mm in length. 0 = Eess than 40 mm 1 = 40 mm or greater 2. Foot width: Refers to the width of the foot in relation to the mantle and body. 0 = wider than mantle 1 = the same width as, or narrower than the mantle 3. Digestive gland: Refers to the degree of ramifica- tion in the duct of the cerata. The unbranched state is suspected to be the plesiomorphic state (Rudman. 1991). 0 = unbranched 1 = branched 2 = secondarily branched or further ramified 4. Zooxanthellae: Refers to the presence or absence of zooxanthellae in digestive diverticula. The absence of zooxanthellae is suspected to be the plesiomorphic state (Rudman. 1991). 0 = absent 1 = present 5. Ceratal texture: Refers to the texture of the external ceratal surface. 0 = smooth 1 = nodulose, with distinct tubercles or papillae 6. Ceratal shape: Refers to the shape of an individual ceras. Page 246 The Veliger, Vol. 51, No. 4 I 1 P, pecten P. rudmani P. parangatum P. magnum P. longicirrum P. guamensis 1 1 1 1 1 1 1 1 1 1 P. poindimiei P. briareum P. colemeni P. iriomotense P. karenae P. macphet^onae P. hyaknum P. jakobsenae P. crypticum P. kabiranum P. tuberculatum p. pinnatum X 1 1 P. horridum P. serratum P. opaiescens P. acanthoitiinum n. sp. P. ur\dulatum n. sp. Favorinus faponicus Godiva quadricoior Figure 6. Strict consensus of two most parsimonious trees. L = 1 1 1. Cl = 0.306. RI = 0.601. Numbers above branches are decay analysis values. 0 = cylindrical 1 = tlattened 7. Ceratal apex: Refers to the shape of the tips of the cerata. Based on comparisons of many photos of living animals in varied states of activity. Species listed as having curved or curled ceratal apices consistently exhibit this arrangement. 0 = straight 1 = curved or curled 8. Cnidosac: Refers to the presence or absence of a functional cnidosac. Presence of a functional cnidosac is suspected to be the plesiomorphic state (Rudman, 1991). 0 = present and nonfunctional 1 = present and functional 9. Shape of digestive gland duct: Refers to the presence or absence of undulation in the digestive diverticula of the cerata. 0 = straight 1 = undulating 10. Precardiac cerata: Refers to the shape of the anteriormost ceratal grouping. Arch-shaped cer- atal groups are suspected to be the plesiomorphic state (Rudman, 1991). 0 = arch-shaped 1 = not arch-shaped 1 1. Postcardiac cerata: Refers to the shape of the first postcardiac ceratal grouping. Arch-shaped ceratal groups are suspected to be the plesiomorphic state (Rudman, 1991). 0 = arch-shaped 1 = not arch-shaped 12. Second group of cerata: Refers to the shape of the second postcardiac ceratal grouping. Arch-shaped E. Moore & T. Gosliner, 2014 Page 247 OJOJClClOJCL ClCLcl ClClCL ClCLCLCL CLCL OJClClCl CLU-W Figure 7. Consensus tree with map of characters. Numbers correspond to character numbers listed in the phylogenetic analysis. Numbers in parentheses are reversals. In cases where changes followed an equivocal node, the changes were treated as regular changes, not reversals. ceratal groups are suspected to be the plesio- morphic state (Rudman, 1991). 0 = arch-shaped 1 = not arch-shaped 13. Third group of cerata: Refers to the shape of the third postcardiac ceratal grouping. Arch-shaped ceratal groups are suspected to be the plesio- morphic state (Rudman, 1991). 0 = arch-shaped 1 = not arch-shaped 14. Anterior foot corners: Refers to the shape of the anterior foot corners. 0 = moderately tentacular or tentacular 1 = not tentacular 15. Anus position: Refers to the location of the anus. (This character was uninformative as it is autapo- morphic for P. hyciliuuni. ) 0 = within ceratal grouping 1 = dorsal to ceratal grouping 16. Rhinophore size: Refers to the length of the rhinophores. Species that have rhinophores that are as long as or longer than the oral tentacles are considered to be greatly extended. 0 = moderately long 1 = greatly extended 17. Rhinophore surface: Refers to the texture on the surface of the rhinophores. 0 = ornamented 1 = smooth 18. Masticatory border: Refers to the number of rows of denticles on the jaw plate. 0 = many rows of denticles 1 = one row of denticles 2 = smooth (no rows) 19. Jaw denticles: Refers to the overall appearance of the jaw denticles. 0 = uniform size, or evenly graded 1 = obvious size gradient Page 248 The Veliger, Vol. 51, No. 4 A 14nini B 45mm Figure 8. Lateral view of preserved specimens showing ceralal arrangement. A, Phyllodesiniiim acanthorhimiiu n. sp. (CASIZ 104702a); B, Phyllodesiuiiiiii imdidatimi n. sp. (CASIZ 176717). Scale bars represent preserved specimen length. 20. Radular denticles; Refers to the overall appear- ance of the radular denticles. 0 = well developed 1 = reduced or absent 21. Cusp of teeth: Refers to the location of denticles on the radular cusp. 0 = denticulate nearly to apex 1 = denticulate only well below apex 22. Arrangement of radular denticles: Refers to the spacing between radular denticles. 0 = well separated 1 = congested 23. Length of radular denticles: Refers to the length of the radular denticles. 0 = elongate, longer than wide 1 = short, width equal to or wider than length 24. Tip of radular denticles: Refers to the shape of the tips of the radular denticles. 0 = blunt 1 = pointed 25. Rows of radular denticles: Refers to the number of rows of radular denticles. (This character was uninformative as it is autapomorphic for Phyllo- desniium gmimensis Avila et ah, 1998.) E. Moore & T. Gosliner, 2014 Page 249 0 = one 1 = two 26. Base shape of teeth: Refers to the shape of the base of the radular cusps. 0 = triangular 1 = horseshoe-shaped 27. Base limb length of teeth: Refers to the length of the base of the radular cusps. 0 = longer than cuspidate portion bearing denticles 1 = shorter than cuspidate portion bearing denticles 28. Penial spine: Refers to the presence or absence of a penial spine. (This character was uninformative as it is autapomorphic for Godivci qiuidricolor. ) 0 = absent 1 = present 29. Female gland shape: Refers to the overall shape of the female gland mass (This character was uninformative as it is autapomorphic for P/iyl/o- desmiiim nuicphersoiuie Burn, 1962.) 0 = elongate 1 = bulbous 30. Penial papilla: Refers to the size of the penial papilla. 0 = large, much wider than the vas deferens 1 = small, ec]ual in width to vas deferens or narrower 31. Prostate length: Refers to the length of the prostate. 0 = long, generally consisting of two or more deep convolutions 1 = short, with one or no shallow convolutions 32. Prey: Refers to the kind of prey consumed. 0 = other 1 = octocorals generally 2 = only gorgonians 3 = only soft corals 4 = only stoloniferans Parsimony analysis revealed two most parsimonious trees with 1 1 1 steps. The permutation tail probability test revealed a P = 0.01 chance that the tree could be reproduced with random data. The character trace tree is shown in Figure 7. The consistency index was 0.306 and the retention index was 0.601. Decay analysis results are shown in Figure 6. DISCUSSION Based on the phylogeny presented in this study, the genus Phyllodesmiwn is a monophyletic group (Fig- ure 6) with P. uiuliilatiim as the most basal species in the phylogeny. This is expected due to the undivided digestive duct within cerata of this species, and the lack of zooxanthellae found within nudibranch tissues. Phyllodesiuiuni ucanthorhinum is in a clade along with P. ojudesceiis. P. Iiorridum, and Phyllodesiuiuni serra- tuni (Baba, 1949), which are all basal in comparison to species with branched digestive tissue and symbiosis with zooxanthellae. The permutation tail probability test indicates that the dataset is significantly different from randomness, and is thus informative. Generally, this phylogeny is in accordance with the strict consensus tree produced by Ortiz & Gosliner (2()08) with the exception of a few differences. In the present tree, the relationship between Phyllodesniium paraugatiim Ortiz & Gosliner, 2003, Phyllodesiuiuni jukohsenae Burghardt & Wiigele, 2004, and Phyllodes- iiiiuui |■udulaui Burghardt & Gosliner, 2006 is fully resolved. However, the placement of these species is different, with P. jakohsenae clustering within a clade of species that mimic Xenia corals, and P. nidniaiii clustering with Phyllodesiuiuni peeten Rudman, 1981 and P. parangatuin. In addition, the relationship between Phyllodesniiuni poiiidiiniei (Risbec, 1928). P. hricireiiin, and P. niaephersonae is also fully resolved in a clade that also includes P. sp. 3. As was seen in Ortiz & Gosliner (2008), Phyllodesniiuni appears to be monophyletic with a decay value of 2, supported by characters 16, 27, and 33 (Figure 7). As was suggested by Rudman (1991), species with the highest degree of digestive gland ramification and dense populations of zooxanthellae appear to be the most highly derived. This is supported by characters 4, 5, 7. and 18 at the node dividing the nonsymbiotic species from symbiotic, and also has a higher decay value than most of the surrounding nodes. Phyllodes- niiuni longieirniin and Phyllodesiniiiin inagnuni Rud- mani. 1991 also form a moderately supported clade within a trichotomy that includes the most highly derived species. This is not surprising due to the Battened cerata and highly ramified digestive divertic- ula present in these species. These attributes allow the animals to provide maximum sunlight to large popu- lations of symbiotic zooxanthellae, likely maximizing their photosynthetic output. The large body size of P. longiciiTuin and P. inagnuni may also suggest that the additional nutrients provided by the symbionts allow the animal to achieve greater size, further indicating a highly advanced relationship with the alga. In addition, P. hyalinuin, P. jakohsenae, PhyUodesiniiiin eryptieiun Rudman, 1981, and Phyllodesniiuni kahiranuni Baba, 1991. which all have cerata that mimic Xenia or Heteroxenia coral polyps, are grouped into an exclu- sive, but moderately derived clade. Phyllodesniiuni eryptieiun and P. hyalinuin are considered moderately advanced in their symbiont retention abilities (Kempf, 1991), which is in acccndance with their placement in this phylogeny. However, Bughardt & Wiigele (2004) suggest that P. Jakohsenae is further advanced in its symbiosis with zooxanthellae than P. eryptieiun or I\ hyalinuin, indicating it may be more derived than Page 250 The Veliger, Vol. 51, No. 4 presently shown. Phyllodesmiuw poimUmiei and P. iriomoteiixe, which do not harbor zooxantliellae but have moderately ramified digestive tissue, appear in clades with species that have moderate to advanced symbioses with zooxantliellae. The inclusion of P. poindimiei in such a derived clade is not well supported, and is only upheld by character 1. However, the resolution for that entire clade is generally low (Figures 6 and 7), indicating it could easily be altered with the addition of new taxa or characters. Despite the lack of support, the placement of P. poindimiei and P. iriomotense is interesting, suggesting a possible reversal from a symbiotic to nonsymbiotic state. This reversal is especially evident on the character trace tree at P. iriomotense, as there are five character state changes seen in this species, three of which are reversals (Figure 7). Despite the general trend showing the advancement of species with symbioses and the clustering of Xenict- mimicking species, the obvious trichotomy and the low decay analysis values indicate that some resolution and robustness is still lacking. Although the results of the permutation tail probability test indicate the tree is informative, the consistency and retention indices are both low, further indicating that the fit of the characters to this tree is not highly robust. PhyUodes- mium rudnunu, which has arguably the most cryptic cerata among all of the Xenia mimics, clusters with P. pecten and P. purcnjgutwn in a more derived portion of the tree. Although the outgroup for this analysis was chosen based on a suspected close relationship with PhyUo- desmiwn and prior usage by Ortiz & Gosliner (2008), the sister group to Phyllodesniiwn has not yet been determined and other outgroups may yield varied results. This problem was especially apparent when mapping the characters on the tree, as many character reversals could not be fully identified due to differing character states within the two outgroup species. In addition, character number 33 was deleted from the character trace tree as the state is unknown in some species and it varies greatly within the ingroup. Regardless of the diversity in the genus Phyl/odesniiuni, there are highly conserved morphological features among the species, and accurately identifying subtle differences is a serious challenge. Molecular investiga- tion would likely shed some light onto the evolutionary history of this group and allow further distinctions between lineages. In addition, many species of Pliy/lo- desniiitm remain to be described, leaving important information missing from a comprehensive phylogeny. Acknowledgments. We would like to thank Robert Bolland for the use of his collected specimens and photographs and Pauline Fiene for the use of her photographs. This research has been funded by the National Science Foundation PEET (DEB 0329054) Phylogeny of the Nudibranchia and the California Academy of Sciences Graduate Assistantship Program in Education. LITERATURE CITED Avila, C., M. Ballesteros, M. Slattery, J. Starmer & V. J. Palil. 1998. Phyllodesmium giuimensis (Nudibran- chia; Aeolidoidea), a new species from Gaum (Micro- nesia). Journal of Molluscan Studies 64:147-160. Baba, K. 1949. Opisthobranchia of Sagami Bay Collected by His Majesty the Emperor of Japan. Iwanami Shoten: Tokyo. 194 pp, 50 pis Baba, K. 1991a. The anatomy of Phyllodesmium serratum (Baba, 1949) from Japan (Nudibranchia: Facelinidae). Venus-50:10F 108. Baba, K. 1991b. Taxonomical study on some species of the genus Phyllodesmium from Cape Muroto-misaki, Shikoku, and Okinawa Province, southern Japan. Venus 50:109-124. Blirghardt, F, j. Evertsen, G. Johnsen & H. Wagele. 2005. Solar powered seaslugs--mutualistic symbiosis of Aeolid Nudibranchia (Mollusca, Gastropoda, Opistho- branchia) with Symhiodiuium. Symbiosis 38:227-250. BiiRGHARDT, I. & T. M. GoSLlNER. 2006. Phyllodesmium rudnumi (Mollusca: Nudibranchia: Aeolidoidea), a new solar powered species from the Indo-West Pacific with data on its symbiosis with zooxantliellae. Zootaxa 1308: 31 47. BtiRGUARDT, I., M. SCURODL & H. WAgele. 2008a. Three new solar powered species of the genus Phyllodesmium Ehrenberg, 1831 (Mollusca: Nudibranchia: Aeolidoidea) from the tropical Indopacific with analysis of their photosynthetic activity and notes on biology. Journal of Molluscan Studies 74:277-292. Biirghardt, I., K. Stemmer & H. WAgele. 2008b. Symbiosis between Symhiodiuium (Dinophyceae) and different taxa of Nudibranchia (Mollusca: Gastropoda) with analyses of long-term retention. Organisms, Diver- sity & Evolution 8:66-67. Blirghardt, 1. & H. WAgele. 2004. A new solar powered species of the genus Phyllodesmium Ehrenberg, 1831 (Mollusca: Nudibranchia: Aeolidoidea) from Indonesia with analysis of its photosynthetic activity and notes on biology. Zootaxa 596:1 18. Biirghardt, I. & H. WAgele. 2006. Interspecific differences in the efficiency and photosynthetic characteristics of the symbiosis of “solarpowered" Nudibranchia (Mollusca: Gastropoda) with zooxanthellae. Records of the Western Australian Museum 69:1-9. Gosliner, T. M., D. W. Behrens & A. Valdes. 2008. Indo- Paeific nudibranchs and sea slugs: a field guide to the world's most diverse fauna. Sea Challengers Natural History Books & The California Academy of Sciences; Gig Harbor, WA & San Francisco, CA. 426 pp. Gosliner. T. M., D. W. Behrens & G. C. Williams. 1996. Coral Reef Animals of the Indo Pacific, Animal Life from Africa to Hawaii Exclusive of the Vertebrates. Sea Challengers: Monterey, California. 315 pp. Kempf, S. C. 1984. Symbiosis between the zooxanthella Svmhiodiniiim (= Gynmoduiium) Alicroadriaticuni (Freu- denthal) and four species of nudibranchs. Biological Bulletin 166:110-126. Kempt, S. C. 1991 . A ‘primitive’ symbiosis between the aeolid nudibranch Berghia vetrucicornis (A. Costa, 1867) and a zooxanthella. Journal of Molluscan Studies 57:75-85. E. Moore & T. Gosliner, 2014 Page 251 Maddison, D. R. & W. P. Maddison. 2005. MacClade 4 [computer program]. Sinauer Associates: Sunderland, Massachusetts. Moore, E. & T. Gosliner. 2009. Three new species of Phyllodesniiuni Ehrenberg (Gastropoda: Nudibranchia: Aeolidoidea), and a revised phylogenetic analysis. Zoo- taxa 2201, 30-48. Moore, E. J. & T. M. Gosliner. 201 1. Molecular phylogeny and evolution of symbiosis in a clade of Indopacific nudibranchs. Molecular Phylogenetics and Evolution, 58(1), 116-123. Ortiz, D. M. & T. M. Gosliner. 2003. A new species of Phyllodesmiiim Ehrenberg, 1831 (Mollusca, Nudibran- chia) from the tropical Indo-Pacific. Proceedings of the California Academy of Sciences 54:161-168. Ortiz, D. M. & T. M. Gosliner. 2008. Anatomical review and preliminary phylogeny of the Facelinid nudibranchs (Opisthobranchia: Aeolidina) of the taxon Phyllodesmiwu Ehrenberg, 1831. Veliger 50:1-23. Rudman, W. B. 1980. Aeolid opisthobranch mollusks (Glau- cidae) from the Indian Ocean and the south-west Pacific. Zoological Journal of the Linnean Society 68:139-172. Rlidman, W. B. 1981. The anatomy and biology of alcyonarian-feeding aeolid opisthobranch mollusks and their development of symbiosis with zooxanthellae. Zoological Journal of the Linnean Society 72:219 262. Rudman, W. B. 1991. Further studies on the taxonomy and biology of the octocoral feeding genus Phyllosdesniiuni Ehrenberg, 1831 (Nudibranchia: Aeolidoidea). Journal of Molluscan Studies 57:167-203. SWOFFORD, D. L. 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods) [computer program]. Version 4.0. Sinauer Associates: Sunderland, Massachus- setts. Wagele, H. W. 2004. Potential key characters in Opistho- branchia (Gastropoda, Mollusca) enhancing adaptive radiation. Organisms, Diversity & Evolution 4:175- 188. WAgele, H., I. Burhardt, N. Anthes, J. Evertsen, A. Klussmann-Kolb & G. Brodie. 2006. Species diversity of opisthobranch mollusks on Lizard Island, Great Barrier Reef, Australia. Records of the Western Austra- lian Museum. Supplement 69:33-59. WiLLAN, R. C. 1987. Phylogenetic systematics and zooge- ography of Australian nudibranchs. 1. Presence of the aeolid Godivu ijuadricolor (Barnard) in Western Australia. Journal of the Malacological Society of Australia 8:71- 85. The Veliger 5 1(4);252- 254 (September 16. 2014) THE VELIGER © CMS, Inc., 2014 Effects of Crowding on Survival and Growth of Neonatal Biomphalaria glabrata Snails Maintained on a Nostoc sp. Diet AMANDA E. BALABAN AND BERNARD ERIED* Department of Biology, Lafayette College, Easton, Pennsylvania 18042, USA Abstract. The effects of crowding on the survival and growth of neonatal Biomphalaria glabrata were studied in petri dish cultures containing artificial spring water (ASW) and the filamentous cyanobacteria Nostoc sp. To study the effects of a reduced volume of ASW on a fixed number of neonatals, 25 snails were cultured in either 200, 40, or 1 5 mL of ASW for up to 22 days. The growth of neonatals in 200 mL of ASW was significantly greater (analysis of variance, P < 0.05) than that of snails maintained in either 40 or 15 mL ASW at 10, 17, and 22 days postcultivation. The percent survival by day 22 was 76% for neonatals in 200 mL of ASW, 56% for those in 40 mL of ASW, and 32% for neonatals in 15 mL of ASW. To study the effects of varying the number of snails in a constant volume of ASW, 70 or 30 neo- natals were maintained in petri dish cultures each containing 200 mL of ASW. There was a significant increase in snail growth in the culture with 30 neonates compared to that with 70 snails at 10 and 18 days postcultivation. The percent of surviving snails at 18 days in the culture with 30 snails was 97% compared to 72% in the culture with 70 neonates. The results of our study showed that a reduction in the volume of ASW on a fixed number of neonates and an increase in the number of neonates in a fixed volume of ASW adversely affected the growth and survival of neonatal B. glabrata. INTRODUCTION Biomphalaria glabrata (Say, 1816) is the first interme- diate gastropod hc^st of economically important trem- atodes such as Schisto.soma mansoni and Echinostoma caproui. This snail is also used for basic and applied laboratory and field research on planorbid snails. Laboratory maintenance of B. glabrata is important to conduct experiments on uninfected planorbids and those infected with larval trematodes. Eveland & Haseeb (2010) reviewed the maintenance of adult B. glabrata on various diets but did not include studies on neonatal snails. Although B. juveniles and adults can be maintained on a lettuce diet, this diet is not adequate for neonatals, i.e., newly hatched snails less than 1 mm in diameter (Eewis et al., 1986). Vasta et al. (201 1 ) described a Nostoc sp. diet (Nostoc sp. is a filamentous cyanobacteria), which allows for the growth of neonatals in culture. Although previous studies (Chernin & Michelson, 1957 a, b) have described the effects of crowding on adult B. glabrata, similar studies on neonatals are not available. The purpose of our study was to examine the effects of crowding on neo- natals cultivated on a Nostoc sp. diet. MATERIALS AND METHODS Source of Nostoc sp. Nostoc sp. was purchased from Ward’s biological supply (item no. 86 V 2159) and prepared for use to * Corresponding author, e-mail: friedb(®,lafayette.edu culture neonatal B. glabrata as described in Vasta et al. (2011). Source of Neonatal B. glabrata To obtain neonatals, 10 to 20 adult B. glabrata snails (Naval Medical Research Institute strain) were main- tained in mason jar cultures each with 800 mL of aerated artificial spring water (ASW) at 25 ± 1°C. The composition of ASW and details of the maintenance of adult B. glabrata in our laboratory were described by Schneck & Tried (2005). To obtain egg masses, 4 X 4 cm Styrofoam strips were placed in the cultures as a substratum. Styrofoam with egg masses were trans- ferred to large petri dishes (150 X 25 mm) each containing 200 mE of ASW. Petri dishes were examined every 2 days for up to 7 days to obtain the neonatals. Neonatals began hatching within a week after the egg masses were placed in the cultures. The neonatals were transferred to new petri dishes and placed on the Nostoc sp. diet at the start of the growth and survival studies. Snails maintained in ASW without a source of exogenous food were designated as zero day old snails. These snails survived for about 7 to 10 days after hatching, presumably living on intrinsic nutrient reserves. Effects of a Reduced Volume of ASW on a Fixed Number of Neonatals Experiment 1 examined the effects of a reduced volume of ASW on survival and growth of 25 neonatals A. E. Balaban & B. Fried, 2014 Page 253 Figure 1. Mean ± SE of shell diameters (mm) of B. glahrata neonatals maintained on a Nostoc sp. diet in 200 mL (large dish), 40 mL (medium dish), or 15 mL (small dish) of ASW. per culture. In this experiment, 25 B. glahrata neonatals were transferred to either 15 mL of ASW in 60 X 15 mm petri dishes, or 40 mL in 100 X 15 mm dishes, or 200 mL in 150 X 25 mm dishes. The cultures con- taining the neonatals were fed Nostoc sp. ad lihituiii for the duration of the experiment, and the ASW was changed by decantation every other day. The number of surviving snails and measurements of their shell diameters were made at least once or twice a week for up to 22 days postcultivation. Effects of Varying the Nvtmber of Neonatals in a Constant Volume of ASW In Experiment 2, either 70 or 30 neonatals were placed in petri dishes with 200 mL of ASW per dish. Cultures were maintained and fed Nostoc sp. as described in Experiment 1. The number of surviving snails and measurements of their shell diameters were made as described previously. Shell Measurements and Percent Survival In each experiment, the shell diameters of 10 ran- domly selected snails were determined. Snails less than 1.5 mm in diameter were measured with the aid of an ocular micrometer, whereas snails greater than 1 .5 mm were measured with a vernier calipers. The number of surviving snails in each culture was determined at the same time as measurements on shell diameters were made. RESULTS The mean ± SE of the shell diameter of 20 newly hatched unfed neonatals was 0.72 mm ± 0.02. Snails fed Nostoc sp. contained green pigment in the gut within Figure 2. Percent survival of B. glahrata neonatals main- tained on a Nostoc sp. diet in 200 mL (large dish), 40 mL (medium dish), or 15 mL (small dish) of ASW. a few hours of feeding as described by Vasta el al. (2011). In Experiment 1, growth of neonatals in 200 mL of ASW was significantly greater (analysis of variance, P < 0.05) than that of snails maintained in either 40 niL of ASW or 15 mL of ASW at 10, 17, and 22 postcullivation (Eigure 1). The percent survival at day 22 was 76% for neonatals maintained in 200 mL of ASW, 56% for those maintained in 40 mL of ASW, and 32% for snails maintained in 15 mL of ASW (Eigure 2). In Experiment 2, the number of neonatals varied but the volume of ASW was constant. As seen in Figure 3, there was a significant decrease in snail shell diameters (Student’s t test, P < 0.05) in the culture with 70 neonatals compareci to the culture with 30 neonatals at 10 and 18 days postcultivation. Relative to the survival data, as seen in Figure 4, there was a marked decrease in survival as a function of the number of snails in the culture by day 18, at which time the experiment was terminated. Whereas the percent survival in the culture with 30 neonatals was 97%, it was 72% in the culture with 70 neonatals. DISSCUSSION We found that a decreased volume of ASW in cultures with a fixed number of snails (Experiment 1 ) had a det- rimental effect on the survival and growth of neonatal B. glahrata. However, Chernin & Michelson (1957b) reported that a decrease in volume of the culture water by one half did not diminish the growth of adult B. glahrata. Their finding is contrary to our results from Experiment 1, suggesting that adult and neonatal B. glahrata respond differently to crowding as a function of a reduced volume of water. Page 254 The Veliger, Vol. 51, No. 4 Figure 3. Mean ± SE of the shell diameter of neonatals of B. glahrata in cultures maintained in fixed volumes of ASW with either 30 (small population) or 70 snails (large population) per culture. When we increased the number of neonatals in a fixed volume of water, we noted decreased survival and growth of the neonatals. Chernin & Michelson (1957a) noted that the growth of adult B. glahrata was also adversely affected in cultures containing 50 or 1 50 snails compared to those with only 20 snails in a fixed volume of water. Thus, increasing the number of either neonatal or adult snails in cultures with a constant volume of water does adversely affect the growth and survival of B. glahrata. Further research on growth of B. glahrata neonatals should be done. Of particular importance are unknown factors, such as surface area of the culture dishes, optimal volumes of water and amounts of Nostoc sp. needed in the cultures, and other factors that may impact on neonatal growth and survival. Additional studies are needed to obtain a better understanding of growth of neonatals B. glahrata under crowded conditions. Acknowledgments. We are grateful to Dr. Fried A. Lewis, head of the Schistosomiasis Laboratory, Biomedical Research Institute, Rockville, Maryland, for supplying mature B. glahrata snails used in this work through NIH-NIAID contract NOl- AI-55270. Figure 4. Percent survival of neonatal B. glahrata in cultures with a fixed volume of ASW and variation in the number of neonatals, i.e., 30 snails (small population) or 70 snails (large population) per culture. LITERATURE CITED CHERNtN, E. & E. H. Michelson. 1957a. Studies on the biolog- ical control of schistosome-bearing snails. TIE The effects of population density on growth and fecundity of Australorhis glahratus. American Journal of Flygiene 65:57—70. Chernin, E. & E. H. Michelson. 1957b. Studies on the biological control of schistosome-bearing snails. IV. Further observations on the effects of crowding on growth and fecundity in Australorhis glahratus. American Journal of Hygiene 65:71-80. Eveland, L. K. & M. A. Haseeb. 2010. Laboratory rearing of Biomphalaria glahrata snails and maintenance of larval schistosome in vivo and in vitro. Pp. 33-55 in R. Toledo & B. Fried (eds.), Biomphalaria Snails and Larval Trem- atodes. Springer: New York, NY. Lewis, F. A., M. A. Stirewalt & C. P. Souza. 1986. Large- scale laboratory maintenance of Schistosoma man.soni, with observation on three schistosome/snail host combi- nations. Journal of Parasitology 72:813-829. SCHNECK, J. L. & B. FRtED. 2005. Growth of Biomphalaria glahrata (NMRI strain) and Helisoma trivolvis (Colorado strain) under laboratory conditions. American Malaco- logical Bulletin 20:71-73. Vasta, J. D., K. E. Lesage & B. Fried. 2011. A simple method for culturing neonatal Biomphalaria glahrata snails. Journal of Parasitology 91\1A6-1A1. The Vehger 51(4):255-260 (September 16, 2014) THE VELIGER < CMS, Inc., 2014 Effects of the Invasive Alga Grad /aria salicornia on Molluscan Species Abundance, Richness, and Diversity in Sheltered Shoreline Pools in East Hawaii ROBERT D. STROHL AND MARTA J. deMAINTENON* University of Hawaii at Hilo, Hilo, Hawaii 96720, USA Abstract. Mollusks comprise diverse taxa that provide crucial ecological links in coastal marine habitats. These organisms are highly sensitive to the environment around them and thus any changes can greatly affect molluscan species diversity. Gracilaria salicornia (C. Agardh) E.Y. Dawson, 1954 is an invasive alga species in Hawaii, which has become prevalent in many coastal marine ecosystems. Its interactions with native species are not well documented. In this study, we investigated the relationship between abundance of G. salicornia and benthic nearshore molluscan species abundance and diversity. Three levels of G. salicornia abundance (low, intermediate, high) were surveyed at two sites in Hilo, Hawaii Island. Each site was sampled five times. The total abundance of mollusks was highest in the area of low G. salicornia abundance, but there was no significant difference. The total number of mollusk species was significantly higher in the area of high G. salicornia abundance. The fact that more mollusk species occupy the area of high G. salicornia abundance suggests that this invasive species of alga docs have a positive effect on species diversity in at least some taxa. INTRODUCTION Invasive species are increasingly common in tropical marine communities. Several species of macroalgae are invasive in Hawaii, including the rhodophyte Gracilaria salicornia (C. Agardh) Dawson, 1954. This hardy red alga, native to the western Pacific, arrived on the Island of Hawaii before the 1950s (Smith et ak, 2004; Nelson et ak, 2009). It was purposely introduced from Hawaii to Oahu, and is now common in nearshore protected subtidal habitats of Hawaii and Oahu, and dominant in some areas on Oahu. On Hawaii island, it has been documented as occurring on the windward side, from Hilo area to Kapoho on the southeast tip of the island (Hunter, 2000). It is fast-growing, and capable of forming thick monospecific mats and overgrowing reef corals. G. salicornia monopolizes nutrients from the sediments and excludes endemic algae species that arc primary food sources for other species, thus decreasing local biodiversity (Smith et ak, 2004). In Hawaii, G. salicornia is also collected as a food item and to produce agar. In spite of its invasive nature, however, its role in local nearshore benthic communities is not well documented. Invasive algae can have beneficial effects on near- shore communities, by increasing habitat complexity and food resources. Viejo ( 1999) noted such an effect in the case of the invasive algae Sargassnni ninticnnu because the epifaunal assemblages in those habitats * Corresponding author, e-mail: demainle(§)hawaii.edu were not host-specific and so readily utilized the invasive Sargassnni. However, the generally positive effect of increased complexity on diversity was coun- teracted in some cases, in which increased algal density past a threshold value apparently excluded many potential resident molluscs (Chemello & Milazzo, 2002; Kelaher, 2003). Different algal architectures have also been shown to differentially affect the diversity of associated molluscs, with degree of branching, algal width, and stem width positively correlated with diversity (Chemello & Milazzo, 2002). Invasive algae can provide a novel food source for marine species, and indeed green sea turtles in Kaneohe Bay have been found to have shifted their diet to include large amounts of invasive algae species, including G. salicornia (Russell & Balazs, 2009); whereas the local collector urchin Tripnenstes gratilla will eat G. salicor- nia if it is the only focid available, but do not prefer it given a choice (Stimson et ak, 2007). Generally, molluscs associated with macroalgae do not feed on the alga itself, though some feed on algal epibionts such as diatomaceous films or hydroids. Mollusks are good test organisms for changes in marine benthic community structure because they are one of the most highly diverse taxa in nearshore communities, taxonomically well delimited, and occupy a variety of trophic levels. In this stmly. we investigated the effects of the invasive algae Gracilaria salicornia on nearshore benthic molluscan diversity and community structure. Page 256 The Veliger, Vol. 51, No. 4 METHODS Benthic molluscan species richness and abundance were compared between three different levels of invasive alga Gracilan'a salicorniu (high, >50% cover; moderate, 25- 50% cover; and low, <25% cover) in two sheltered coastal sites in Hilo, Hawaii, Onekahakaha Beach Park (I9°44.3'N, I55°2.4'W) and Chocks (19°44.2'N, 155°2.6'W). These consist of nearshore habitats par- tially enclosed behind natural basaltic rock breaks with depths of 0 to 2 m. Though often called tide pools, they still remain connected to the ocean at low tide and physical conditions tend not to vary as much as a true tide pool might. For each station type at both sites, five replicate 0.5-m- quadrats were sampled at low tide. Sites sampled for this study were generally 0.5-m depth or less at low tide. The substrate was bedrock with gravel or sand to basalt cobbles and boulders. Molluscs were collected by hand and by brushing rocks and cobbles into nets or bags. Larger, common and sessile species were identified and counted in situ, and smaller species were identified in the lab using a stereomicroscope. Results were analyzed using Model III factorial ANOVAs (Minitab version 16) to compare species abundance and species richness amongst the three levels of the invasive alga and two sites. To compare diversity, Shannon-Weiner diversity indices were com- puted for each site and each station type. RESULTS Sixty species were identified overall across all samples. Mollusc species richness varied between sites (Figure 1; F = 13.88 with 1,24 df, P < 0.05) and levels of Grucilaria abundance (Figure 2; F = 38.81 with 2,2 df, P < 0.05), but there was no interaction between factors {F = 1.43 with 2,24 df, P > 0.25). Species richness was higher at Onekahakaha and increased with increasing cover of Gnicilaria. Abundance, in contrast, showed no significant differences between sites (Figure 3; F = 0.45 with 1,24 df, P > 0.25) or levels of Gracilaria cover (Figure 4; F = 3.32 with 2,2 df, P > 0.25) and no interaction (F = 1.92 with 2,24 df, P > 0.10). Diversity indices (Figure 5) varied from 1.05 to 2.78 and increased with Gracilaria cover, and evenness followed a similar pattern. DISCUSSION The results suggest that G. salicornia has a positive effect on mollusk species diversity in the habitats investigated. Many species of mollusks, mostly gastro- R. D. Strohl & M. J. deMaintenon, 2014 Page 257 Low Intermediate High GracHaria abundance Figure 2. Species ricliness by level of Grucilaria cover (mean per quadrat ± SE); letters indicate significantly different groupings. Figure 3. Mollusc abundance by location (mean per quadrat ± SE). Shannon Wiener Diversity Mollusc abundance Page 258 The Veliger, Vol. 51, No. 4 Low Intermediate High Gradtaria abundance Figure 4. Mollusc abundance by level of Graciluria cover (mean per quadrat ± SE). Low I nte r m ed i ate High Gracjfaria abundance Figure 5. Shannon -Wiener diversity indices by site and level of Gracilaria cover. R. D. Strohl & M. J. deMainlenon, 2014 Page 259 Table 1 Four most common mollusc species per site and level of Gracilaria. Gracilaria level Onekahakaha Chocks Low Theodoxus neglect us (420) I.sogiwinon perna (85) Dolahrifera dolahrifera (37) Serpulorhis variahilis (33) Theodoxus negleeliis (304) Isognunion perna (33) Serpulorhis variahilis (32) Dolahrifera dolahrifera (8) Intemiediate Hipponix iinhricatus (86) Serpulorhis variahilis (43) Bittinni zehruni (15) Bracliidoiites erehristrialus (13) Theodoxus neglect us (85) Dolahrifera dolahrifera (80) Serpulorhis variahilis (62) I.sognuinon perna (21 ) High Hipponix inihricatns (59) Serpulorhis variahilis (42) I.sognonion perna (29) Dolahrifera dolahrifera (23) Hipponix inihricatns (99) Serpulorhis variahilis (52) Isognunion perna (39) Dolahrifera dolahrifera (25) pods, were found in the areas of highest G. saliconiia abundance. The Hawaiian molluscan fauna is known to have a higher relative proportion of gastropods to bivalves than many tropical continental faunas (Kay, 1979). These organisms may use the invasive alga as a shelter from predation and the physical elements, and directly or indirectly as a food source. The predomi- nant native alga present at both sites was Padiiui spp., so the Gracilaria may have aided in increasing habitat complexity considerably. There is a difference in diversity between sites as well; this may be due to the larger size and greater depth of the sheltered pool at Onekahakaha, potentially allowing a greater number of species to be present. Molluscan abundance between levels of G. saliconiia varied greatly. Though Figure 4 suggests that the low Gracilaria samples had higher molluscan abundance, variability was very high. Some samples had high numbers of Theodoxus ueglcctus^ a species present in the low Gracilaria samples in numbers from 1 1 to almost 1 80. The random large numbers of T. neglectas, a shoreline species tolerant of a wide range of salinities, may indicate that the sampling areas lacking G. saliconiia represent a slightly different habitat. How- ever, the overall molluscan community composition found in these samples was otherwise similar to that in the high Gracilaria samples, whereas true brackish and shoreline habitats locally tend to contain almost exclusively T. iiegleciiis. Exclusion of T. neglect us does not affect the results. The four most common species per each site and level of algae are listed in Table 1. There was a high degree of similarity between sites relative to the most common species present, and they are typical windward tide pool species as reported by Kay (1979). The composition of the less common species varied considerably. Several of the most common species are filter feeders that live attached to rocks, e.g., the brown purse shell Isognonion penun the hoof shell Uipponix imhricaliis, the mussel Bracliklontes crehristriatus, and the worm shell Scrpiilorhis variahilis. Three are herbivores, the neritid Tlieocloxiis neglectas, the com- mon sea hare Dolahrijera dolahrifera, and small zebra horn shell Bittinni zehrnin. Larger herbivorous species such as the snake head cowrie Cypraea capntserpentis and the top shell Trochns intcxfns were also common. In areas with low Gracilaria cover, the most common species was Theodoxus neglectas, whereas the most common species in sites with more Gracilaria was Hipponix inihricatns. Certain mollusc species in this study, including Bittinni spp., were observed to consume G. saliconiia. Possibly Bittinni could be used as a native control for G. saliconiia. However, more studies are needed to determine mollusk diets and how fast they are capable of consuming the algae, given the snails’ small size. Fish have been considered for use as bio-control agents for G. .saliconiia but do not prefer it over native algae (Smith et af, 2004). The native sea urchin Tripnenstes gratilla is also being explored as a bio-control agent. Molluscan herbivores such as Bittinni might be valuable aids to help in the control of this and other invasive species. LITERATURE CITED Chemf.llo, R. & M. Mu.azzo. 2002. Effect of algal architectLire on associated fauna: some evidence from phytal molluscs. Marine biology 140:981 990. Fry, B. & E. B. Shekr. 1989. ”C measurements as indicators of carbon flow in marine and freshwater ecosystems. Bp. 196 229 in P. W. Rundel, .1, R. Ehleringer & K. A. Nagy (eds.). Stable Isotopes in licological Research. Springcr-Verlag Publishing: New York. NY. Hlinter, C. L.. 2000. Hawaii Coral Reef Initiative research program web site. http://www. botany. hawaii.edu/GratlStud/ smith/websites/Gsal-home.htm. Accessed January 15. 201 I. Kay. E. a. 1979. Hawaiian Marine Shells. Reef and Shore Fauna of Hawaii. Section 4: Mollusea. Bernice P. Bishop Page 260 The Veliger, Vol. 51, No. 4 Museum Special Publication 64(4). Honolulu: Bishop Museum Press. 653 pp. Kelaher, B. P. 2003. Changes in habitat complexity negatively affect diverse gastropod assemblages in coral- line algal turf. Oecologia 135:431-441. Michener. R. H. & D. M. Schell. 1994. Stable isotope ratios as tracers in marine aquatic food webs. Pp. 1 39-157 in K. Lajtha & R. H. Michener (eds.). Stable Isotopes in Ecology and Environmental Science. Blackwell Scientific Publications: Oxford. Nelson, S. G., E. P. Glenn, D. Moore & B. Ambrose. 2009. Growth and distribution of the macroalgae GracHaria salicornia and G. parvispora (Rhodophyta) established from aquaculture introductions at Molokai, Hawaii. Pacific Science 63:383-396. Russell, D. J. & G. H. Balazs. 2009. Dietary shifts by green sea turtles (Cheloiiiu mydas) in the Kane’ohe Bay region of the Hawaiian Islands: a 28-year study. Pacific Science 63: 181-192. Smith, J. E., C. L. Hunter, E. J. Conklin, R. Most, T. Sauvage, C. Squair & C. M. Smith. 2004. Ecology of the invasive red alga Gracilaria salicornia (Rhodophyta) on 0‘ahu, Hawaii. Pacific Science 58:325-343. Stimson, j., T. Cunha & J. Philippoff. 2007. Pood preferences and related behavior of the browsing sea urchin Tripjieustes gratilla CLxxnvcXSUS) and its potential for use as a biological control agent. Marine Biology 151:1761-1772. Viejo, R. M. 1999. Mobile epifauna inhabiting the invasive Sargasswii miiticum and two local seaweeds in northern Spain. Aquatic Botany 64:131-149. The Veliger 5l(4):26l--264 (September 16, 2014) THE VELIGER • CMS, Inc., 2014 A Test for Mucus Removal in the Chiton Lepidochitona cinerea (Linnaeus, 1767) (Polyplacophora: Chitonida: Ischnochitonidae) LAUREN H. SUMNER-ROONEY Queen’s University Belfast, Marine Laboratory, 12-13 The Strand, Portaferry, Co. Down, Northern Ireland BT22 IPF, and Queen’s University Belfast, School of Biological Sciences, Medical Biology Centre, 97 Lisburn Road, Belfast, Northern Ireland BT9 7BL SHAUN CAIN Eastern Oregon University, La Grande, Oregon 97850, USA GERARD P. BRENNAN Queen’s University Belfast, School of Biological Sciences, Medical Biology Centre, 97 Lisburn Road, Belfast, Northern Ireland BT9 7BL AND JULIA D. SIGWART Queen’s University Belfast, Marine Laboratory, 12-13 The Strand, Portaferry, Co. Down, Northern Ireland BT22 IPF, and Queen’s University Belfast, School of Biological Sciences, Medical Biology Centre, 97 Lisburn Road, Belfast, Northern Ireland BT9 7BL Abstnid. Several methods have been proposed to ’’clean” the soft tissues of molluscs of mucus, so that the surface cilia can be examined microscopically. We report the first empirical test of the effectiveness of methods for removing mucus in the pallial cavity surface of chitons. Three methods were compared, at several time intervals: the enzyme hyaluronidase, the mucolytic agent N-acetyl cysteine (NAC), and seawater washing via the natural action of cilia in excised tissue. Treatment in NAC for 10 min produced the best results, and we recommend this protocol as a starting point for further investigation on mucus removal in a broader suite of taxa. We present the first description of the pallial surface cilia in the chiton Lepidochitona cinerea. During the course of this study, we also determined that these chitons were frequently infested with a ciliate protozoan parasite, Trichodina sp., which have been historically reported from chitons but never studied in detail. The parasites were absent where antimucus treatments were effective, but their abundance and large size (about 30-pm diameter) in less successful treatments obscured the view of the pallial cavity surface. INTRODUCTION Traditionally, molluscan systematics has relied largely on analyses of shell anatomy. More recently, with the improvement of visualization techniques such as SEM imaging, shell features have been extended to micro- structural elements in gastropods (e.g., Geiger, 2012), bivalves (e.g.. Turner, 2008) and chitons (e.g., Sigwart & Sirenko, 2012). However, another potentially rich source of information that has still not fully exploited for systematics is microanatomy of the soft parts of the body. Previous work on the visualization of soft parts in other molluscan taxa using a scanning electron microscope (SEM) has required the removal of mucus (e.g., Nezlin et al., 1994), but there has been no definitive protocol reported for use on adult chitons, nor any comparison of methods in any taxon. To determine the best way forward to visualize cilia in the pallial cavity of chitons, we reviewed published techniques for a variety of invertebrates and directly tested the effect on the chiton Lepidochitona cinerea. The enzyme hyaluronidase has been reported to digest mucus and similar materials in a variety of taxa including bivalves (Calabro et al., 2005), proso- branchs (Nezlin et al., 1994), caenogastropods (Ron- kin, 1952), and onychophorans (Mayer & Harzsch, 2007). However, the efficacy of proteolytic enzymes has been refuted (e.g., Stamm & Docter, 1965), and Page 262 The Veliger, Vol. 51, No. 4 hyaluronidase has not been widely used. N-acetyl cysteine (NAC) has also been reported to function as a mucolytic agent in both vertebrate and invertebrate animals, including a marine bivalve (Moraes & Lopes, 2003), a sacoglossan (Pierce et ah, 1996), and planarians (Bocchinfuso et ah, 2012), but its primary use has been in mammals (e.g., Stamm & Docter, 1965). In the absence of a chemical treatment, motor cilia may continue to beat in excised tissue. So by excising tissue blocks and maintaining them in cold seawater, the mucus could clear “naturally” (SC, personal observation). This is also analogous to washing in seawater or phosphate-buffered saline, which is another treatment suggested previously (e.g., Buckland-Nicks, 1993). In order to test and optimize a treatment effective for chitons, we experimentally compared these three approaches. MATERIALS and METHODS Fourteen treatment regimes were tested for their efficacy in mucus removal. These were all variants on the three techniques outlined above (hyaluronidase, NAC, and surgical separation) using different time and temperature intervals. Live specimens of Lepidochitoua cinerea collected in Strangford Lough (County Down, Northern Ireland) were used for each treatment. All animals were held in aquaria How-through seawater at the Queen's University Marine Laboratory (QML), where one of the authors (JS) has successfully kept long-term captive specimens. All experiments were performed on site in laboratory facilities at QML. Following treated incubation with gentle agitation, specimens were Hushed with a pipette. All specimens were fixed in 4% glutaraldehyde in cacodylate buffer (pH 7.4) and post-fixed in 1% osmium tetroxide in cacodylate buffer in preparation for SEM visualization. Tissues were then dehydrated in acetone and prepared for histology as described by Ruthensteiner (2008). Specimens were dried overnight using hexamethyldisi- lazane (HMDS, Sigma-Aldrich), sputter-coated with gold using a Polaron ESI 00 SEM Coating System (Quorum Technologies) and visualized in a FEI Quanta 200 scanning electron microscope operating at 10 kV. Bovine hyaluronidase (Sigma-Aldrich) was applied at 4.46 e mM in low-calcium artificial seawater (420 mM NaCI, 10 mM KCl, 50 mM MgCL, 10 mM Trizma, 10 mM MnCl). Specimens were incubated for periods of 5 min, 10 min, or 1 hr, and at temperatures of either 10°C or room temperature (six treatment regimes, two specimens each). Another set of specimens were treated with 500 mM NAC in an 2-( A-morpholino) ethanesulfonic acid ( MES) buffer (50 mM MES, 10 mM sucrose, 90 mM sorbitol, all Sigma-Aldrich, pH titrated to 5.5 using NaOH, adapted from Pierce et ak, 1996). These specimens were incubated for intervals of 5 min. 10 min, or 30 min, and also at temperatures of either 10°C or room temperature (six treatment regimes, two specimens each). Four L. cinerea were decapitated and kept in seawater at 5°C for either 1 hr or for 24 hr before fixation (two treatment regimes, two specimens each), a method to induce “natural” clearing of the tissue by the action of still-active cilia, which has given good results in Tritonia diomedea (SC, personal observation). Finally, one specimen of L. cinerea was fixed without mucus removal treatment, as a control. RESULTS AND DISCUSSION The development of SEM techniques has been a crucial step forward in the study of microanatomy across all taxa, yet recent attempts to characterize the pallial cavity of another chiton, Leptochiton rugatus via SEM were hampered by excessive mucus obscuring the surface epithelium (JS, SC, personal observation). The SEM images here (Eigure 1) show that the pallial cavity of Lepidochitona cinerea is carpeted with microvilli, with large tufts of cilia (6 pm in diameter) of approximately 15-20 pm in length at regular intervals (approximately 25 pm apart. Figure lA, B). This pattern persisted throughout the pallial cavity and into the gill row with no visible change in density or frequency. These are the first images to illustrate the surface features of the pallial cavity in L. cinerea. The “success” of our treatments was assessed qualita- tively by visualization under SEM, to determine whether cilia were clearly visible, or whether tissue appeared badly dehydrated, or covered in mucus sheets obscuring some or all of the cilia in the pallial cavity. Specimens were examined whole; that is, the entire pallial cavity on both sides was visually examined under SEM although only small portions are illustrated herein (Eigure 1 ). Temper- ature did not have any substantive influence on tissue quality, and we conclude that perfomiing these chemical treatments at room temperature does not decrease quality. The different treatments for mucus removal had varying rates of success, with NAC generally producing the best results. Incubation of specimens with hyal- uronidase did not effectively remove mucus from the pallial cavity when incubated for less than an hour (Eigure 1C, D), but after longer incubations both cilia and microvilli were visible and only small spheres of mucus were present. NAC preserved cilia and microvilli in good condition, and left little mucus when applied for 10 min or more (Eigure lA, B). In addition, the use of hyaluronidase appears to have disrupted the tissue, with cracks appearing in the epithelium in hyaluroni- dase-treated samples (not shown), this effect was not present under NAC treatment despite identical dehy- dration and preparation for SEM. The difference in mucus coverage observed between the decapitated specimens (i.e., natural clearing of the tissue) L. H. Sumner-Rooney et al., 2014 Page 263 Figure I. SEM images of the anterior pallial cavity of treated L. ciiicrca. A, B, NAC, IO C, 10 min; C, D. Hyaluronidase. 10 C, 5 min; E, F, Decapitated, I hr. Abbreviations: cil, cilia tuft; mic, microvilli; pai', TrichoJiiin sp. parasite; muc, mucus. Scale bars: A, C, E = 100 pm: B, D, F = 10 pm. and the untreated control specimen was minimal. In both cases, the cilia could not be clearly observed and the discrete cilia tufts, visible in other specimens, could not always be clearly identified beneath the mucus “sheet” (Figure 1 E, F). An additional novel observation is the presence of a ciliate protozoan parasite, Triiluulina sp., which also obscured the view of the pallial cavity surface. These protozoa, visible at low magnification, had not been noted Page 264 The Veliger, Vol. 51, No. 4 during specimen preparation, but on subsequent investi- gation were found to infest the majority of specimens available for study. Therefore, we do not believe the influence of the parasite confounded our results here by intluencing either mucus production by the chitons or its removal in our experiments. Under treatment conditions where the mucus was completely or partially removed, these parasites were also cleared. However, they were observed in the untreated, decapitated, and the shorter hyaluronidase-treated specimens and caused a significant obstruction to our view of the pallial cavity due to their large size (around 30 pm across). Their presence here is particularly notable as they have only been reported twice in the literature for chitons: once in our study species, L. cinerea (Plate, 1898) and one other report in the Pacific chiton Kaflian'iui tunicata (Kozloff, 1961). Although the NAC treatment was the most effective for Lepiilochitona, it may not be a panacea and its effectiveness requires further investigation across a broad range of molluscan taxa. Two additional specimens of Leptoc/iiton asellits (Leptochitonidae) were treated with NAC at room temperature for 30 min as described above. Unlike Lepidochitoua cine)-ea. which is a member of the suborder Chitonida, Leplocliitoii aseUus belongs to the other major clade of living chitons, Lepidopleurida. However, in this case, mucus deposits continued to obstruct the structures. Ronkin ( 1952) noted that the susceptibility of mucus to digestion by hyaluronidase varied in different areas in Busycon caiuilicii/atiim, and postulated that variation in chemical composition might explain this. It may be that the taxonomic differences between chitons extend to the composition of mucus, with dissimilar proportions of glycoproteins and proteoglycans being affected differently by the NAC and hyaluronidase chemical treatments, and thus giving results of variable quality. Although the applicability of this treatment requires further investigation, our recommended treatment on the basis of our empirical tests is NAC, effective after 10 min as a starting point for other investigators. LITERATURE CITED Bocchinfliso, D. G.. P. Taylor. E. Ross, A. Ignatch- ENKO, V. iGNATCHENKO, T. KlSLINGER. B. .1. PEARSON & F. Moran. 2012. Proteomic profiling of the planarian Schmidtea niediterranea and its mucous reveals similarities with human secretions and those predicted for parasitic tlatworms. Molecular and Cell Proteomics 11:681-691. Buckland-Nicks, J. 1993. Hull cupules of chiton eggs: parachute structures and sperm focusing devices? Biolog- ical Bulletin 184:269-276. Calabro, C., M. P. Albanese, S. Martella, P. Licata, E. R. Lauriano, C. Bertuccio & A. Licata. 2005. Glycoconjugate histochemistry and nNOS immunolocal- ization in the mantle and foot epithelia of Tapes philippinarwn (bivalve mollusc). Folia Histochemica et Cytobiologica 43:151-156. Geiger, D. L. 2012. Monograph of the little slit shells. Vol. 1: Introduction, Scissurellidae. Santa Barbara Museum of Natural History: Santa Barbara. CA. 728 pp. Kozloff, E. N. 1961. A new genus and two new species of ancistrocomid ciliates (Holotricha: Thigmotricha) from sabellid polychaetes and from a chiton. Journal of Eukaryotic Microbiology 8:60-63. Mayer, G. & S. Harzsch. 2007. Immunolocalization of serotonin in Onychophora argues against segmental ganglia being an ancestral feature of arthropods. BMC Evolutionary Biology 7:118. Moraes, D. T. D. E. & S. G. B. C. Lopes. 2003. The functional morphology of Neoteredo reynei (Bartsch, 1920) (Bivaliva, Teredinidae). Journal of Molluscan Studies 69:31 1-318. Nezlin. L. P., R. Elofsson & D. A. Srharov. 1994. Transmitter-specific subsets of sensory elements in the prosobranch osphradium. Biological Bulletin 187:174- 184. Pierce, S. K., R. W. Biron & M. E. Rumpho. 1996. Endosymbiotic chloroplasts in molluscan cells contain proteins synthesized after plastid capture. Journal of Experimental Biology 199:2323-2330. Plate, L. H. 1898. Uber primitive Organisationsverhiiltnisse, Viviparie und Brutpflege bei Chitonen. Sitzungsberichte der Koniglich Preussischen Akademie der Wissenschaften 14:213-217. Ronkin, R. R. 1952. Cytological studies of mucus formation and secretion in Busycon. Biological Bulletin 102:252-260. Rlithensteiner. B. 2008. Soft part 3D visualization by serial sectioning and computer reconstruction. Zoosymposia 1: 63-100. SiGWART. J. D. & B. I. SiRENKO. 2012. Deep-sea chitons from sunken wood in the West Pacific. Zootaxa 38:1-38. Stamm, S. J. & J. Docter. 1965. Clinical evaluation of acetylcysteine as a mucolytic agent in cystic fibrosis. Diseases of the Chest 47:414—420. Tl'RNER, j. A. 2008. Digital imaging of micro bivalves. Zoosymposia 1:47-61. Instructions to Authors The Veliger puhWshts original papers on any aspect of malacology. All authors bear full responsibility for the accuracy and originality of their papers. Presentation Papers should include an Abstract (approximately 5% of the length of the manuscript), Introduction, Materials and Methods, Results, and Discussion. 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