QL 401 A513 INVZ AMERICAN MALACOLOGICAL BULLETIN Journal of the American Malacological Society http://erato.acnatsci.org/ams/publications/amb.html VOLUME 19 14 OCTOBER 2004 NUMBER 1/2 Morphology and phylogenetic relationships of genera of North American Sphaeriidae (Bivalvia, Veneroida),TAEHWAN LEE ............. 0.000. c cc cee eee eee nee eens 1 Use of a natural river water flow-through culture system for rearing juvenile freshwater mussels (Bivalvia: Unionidae) and evaluation of the effects of substrate size, temperature, and stocking density. BRAVEN B. BEATY and RICHARD J. NEVES .............0.00.00 ee eee ee 15 Integrating historical and functional data to examine feeding in gastropods. SE TCEPP GURALNICK, coe. uate tain oo Maeda ipa Wadd dai hie SA eee a ak ae Ee EEN ee aD The biology and conservation of freshwater gastropods: Introduction to the symposium. RODE RG DEMEON is .ce oe tea ee ae eee Gwe als 3 ate Ealwsiige ean Gare iian Ging ees 31 Intraspecific competition and development of size structure in the invasive snail Potamopyrgus antipodarum (Gray, 1853). DAVID C. RICHARDS and DIANNE CAZIER SHINN ............33 Behavior, morphology, and the coexistence of two pulmonate snails with molluscivorous fish: A comparative approach. CHRISTINA M. MOWER and ANDREW M. TURNER ............. 32 Effects of pair-type and isolation time on mating interactions of a freshwater snail, Physa gyrina (cay ool a bHONAS IM MCGARUTHY: 44. 25acces neon ssa yeah nae as pe Oe ae tes 47 Comparative conservation ecology of pleurocerid and pulmonate gastropods of the United States. KENNETH M. BROWN and PAUL D.JOHNSON .................0.20202+-57 continued on back cover AMERICAN MALACOLOGICAL BULLETIN BOARD OF EDITORS Janice Voltzow, Editor-in Chief Department of Biology University of Scranton Scranton, Pennsylvania 18510-4625 USA Robert H. Cowie Center for Conservation Research and Training University of Hawaii 3050 Maile Way, Gilmore 408 Honolulu, Hawaii 96822-2231 USA Carole S$. Hickman University of California Berkeley Department of Integrative Biology 3060 VLSB #3140 Berkeley, California 94720 USA Timothy A. Pearce Carnegie Museum of Natural History 4400 Forbes Avenue Pittsburgh, Pennsylvania 15213-4007 USA Angel Valdés, Managing Editor Natural History Museum of Los Angeles County 900 Exposition Boulevard Los Angeles, California 90007-4057 USA Alan J. Kohn Department of Zoology Box 351800 University of Washington Seattle, Washington 98195 USA José Leal The Bailey-Matthews Shell Museum 3075 Sanibel-Captiva Road Sanibel Island, Florida 33957 USA The American Malacological Bulletin is the scientific journal of the American Malacological Society, an international society of professional, student, and amateur malacologists. 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ISSN 0740-2783 Copyright © 2004 by the American Malacological Society AMERICAN MALACOLOGICAL BULLETIN CONTENTS VOLUME 19 | NUMBER 1/2 Morphology and phylogenetic relationships of genera of North American Sphaeriidae (Bivalvia, Veneroida) TAEHWAN LEE ...............0 00 ccc cece eee eee e eee e ee 1 Use of a natural river water flow-through culture system for rearing juvenile freshwater mussels (Bivalvia: Unionidae) and evaluation of the effects of substrate size, temperature, and stocking density. BRAVEN B. BEATY and RICHARD J. NEVES ..................-.00 00005. 15 Integrating historical and functional data to examine feeding in gastropods. ROBERT P: GURALNICK. © ashe tes cachad eteie lalesie ata Fh ers See daw oe ee ZO The biology and conservation of freshwater gastropods: Introduction to the symposium. ROBERT T.. DILLON, Jiri ose eh anSon dane thane es Ae ws Woes Miho Oe nwoiG Hs bade a ae En Leal Intraspecific competition and development of size structure in the invasive snail Potamopyrgus antipodarum (Gray, 1853). DAVID C. RICHARDS and DIANNE CAZIER SHINN ............33 Behavior, morphology, and the coexistence of two pulmonate snails with molluscivorous fish: A comparative approach. CHRISTINA M. MOWER and ANDREW M. TURNER ............. 39 Effects of pair-type and isolation time on mating interactions of a freshwater snail, Physa gyrina (ay,13 21) “EHOMAS M.McCCARTAY 4 cpt sedn.: tauses peek aeee auw bette bk wee Poe mew ews < 47 Comparative conservation ecology of pleurocerid and pulmonate gastropods of the United States. KENNETH M. BROWN and PAUL D. JOHNSON ......................0005. 57 Reproductive isolation between Physa acuta and Physa gyrina in joint culture. ROBERT T. DILLON, Jr., CHARLES E. EARNHARDT, and THOMAS P. SMITH ............ 63 High levels of mitochondrial DNA sequence divergence in isolated populations of freshwater snails of the genus Goniobasis Lea, 1862. ROBERT T. DILLON, Jr. and ROBERT C. FRANKIS, Jr. 0.0... 0. eee 69 Species composition and geographic distribution of Virginia’s freshwater gastropod fauna: A review using historical records. TIMOTHY W. STEWART and ROBERT T. DILLON, Jr. ..... 79 Environmentally and genetically induced shell-shape variation in the freshwater pond snail Physa (Physella) virgata (Gould, 1855). DAVID K. BRITTON and ROBERT F. McMAHON ............. 00000 c eee cee eee 93 A 15-year study of interannual shell-shape variation in a population of freshwater limpets (Pulmonata: Basommatophora: Ancylidae). ROBERT F. MCMAHON ..................-0-- 101 Leopold von Buch’s legacy: Treating species as dynamic natural entities, or why geosraphy matters. MATTHIAS GLAUBRECH TY 2.20)ossexehadasdoesea5 ok eee ye ue ewes 111 Are populations of physids from different hot springs distinctive lineages? AMY R. WETHINGTON and ROBERT GURALNICK FRESE a Cle INO Le ea maar enh acer cee Aun een Meee atv OR eter. KEN I = . 1 1 ae . 1 we, “ns 1 vel tg 1 se ohT hate iy ' ‘ sy sign? rte) eS ve - am ot oy ER, Tad | i ni ‘Lite ae ae AMERICAN MALACOLOGICAL BULLETIN 19° 1/2 * 2004 Morphology and phylogenetic relationships of genera of North American Sphaeriidae (Bivalvia, Veneroida) Taehwan Lee’ Museum of Zoology and Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109-1079, U.S.A., taehwanl@umich.edu Abstract: Recent phylogenetic studies of the Sphaeriidae have produced conflicting results between morphological and molecular data sets for two major topological elements, i.e., monophyly of Pisidium and of asynchronous brooders (Sphaerium and Musculium taxa). Gene trees indicate a paraphyletic Pisidium while morphological trees suggest it is a derived monophyletic clade. Molecular analyses recover a derived monophyletic clade of asynchronous brooders suggesting the evolutionary elaboration of brooding character complexity from synchronous to sequential brooding. Conversely, morphological analyses indicate a sister relationship among Musculium and Pisidium taxa and propose that reduced Pisidium characters are derived from larger plesiomorphic Sphaerium taxa. To test these competing hypotheses of sphaertid relationships, the morphology of North American sphaeriid taxa was studied, and major anatomical and developmental features that have been considered fundamental by previous workers were coded. Parsimony analyses with and without outgroup rooting showed that Sphaerium and Musculium taxa form a monophyletic group, congruent with previous gene trees but not with morphological studies. The conflict between molecular and morphological trees for the monophyly of Pisidium, however, remains unsettled. At present, informative morphological characters appear insufficient to flesh out phylogenetic relationships among infra-generic sphaeriid taxa. Key words: Sphaeriinae, freshwater clams, phylogeny, brooding character evolution Conflicts between the trees inferred from morphological and molecular characters are not rare in phylogenetic litera- tures (see Baker et al. 1998). Gene trees may not reflect species phylogeny when the gene(s) investigated have expe- rienced confounding evolutionary processes such as lineage sorting, horizontal transfer, introgression, and ancestral polymorphism (Doyle 1992, Brower et al. 1996). In many cases, however, levels of incongruence between morphologi- cal and molecular topologies were artificially inflated due mainly to the lack of close examination of either or both types of data (Hillis and Wiens 2000). Recently, independent analyses of morphological (Dreher-Mansur and Meier- Brook 2000, Korniushin and Glaubrecht 2002) and molecu- lar (Cooley and O Foighil 2000, Lee and O Foighil 2003) data sets resulted in incompatible sphaeriid phylogenies. The aim of this study was to test these competing hypotheses of sphaeriid relationships by reevaluating morphology-based phylogeny. The Sphaeriidae (fingernail/pea/pill/nut clams) are known from the Cretaceous (Keen and Dance 1969), repre- senting one of the major molluscan freshwater radiations (McMahon 1991) and presently have a cosmopolitan distri- bution in virtually all kinds of freshwater habitats (Clarke 1973, Burch 1975, Kuiper 1983). Although sphaeriid clams are the smallest bivalves in freshwater, they often constitute ' Mailing address: Museum of Zoology, University of Michigan, 1109 Geddes Avenue, Ann Arbor, Michigan 48109-1079, U.S.A. a large proportion of the benthic fauna in streams and ponds (Avolizi 1976, Eckbald et al. 1977) and are important com- ponents in energy and nutrient cycling (Alimov 1970, Horn- bach et al. 1984, Holopainen and Hanski 1986, Lopez and Holopainen 1987, Way 1988). Sphaertids exhibit a remark- able degree of genome amplification (up to 13n) (Park 1992, Barsiene et al. 1996, Burch et al. 1998, Lee 1999) and a number of North American species may share ancestral ge- nome duplication events that predate their cladogenesis (Lee and O Foighil 2002). Sphaeriid taxonomy Convincing marine outgroups for the Sphaeriidae are presently lacking. The long-assumed monophyly of the su- perfamily Corbiculoidea comprising the Sphaeriidae and Corbiculidae (Newell 1965, Keen and Casey 1969, Taylor et al. 1973, Boss 1982, Morton 1996) has been rejected by both morphological (Dreher-Mansur and Meier-Brook 2000) and molecular (Park and O Foighil 2000, Giribet and Wheeler 2002) studies. Similar brooding characters observed in sphaeriids and freshwater corbiculids are thought to repre- sent convergent adaptations to freshwater habitats rather than shared-derived homologies (Park and O Foighil 2000). Five sphaeriid genera (Byssanodonta d’Orbigny, 1846, Eupera Bourguignat, 1854, Musculium Link, 1807, Pisidium Pfeiffer, 1821, Sphaerium Scopoli, 1777) have been widely recognized based on shell and soft-part morphology and reproductive/developmental characteristics (Odhner 1921, 2 AMERICAN MALACOLOGICAL BULLETIN 1922, Baker 1927, Heard 1965b, Burch 1975, Kuiper 1983, Ituarte 1989, Dreher-Mansur and Ituarte 1999). In the past these genera have been divided into three subfamilies: Eu- perinae, Pisidiinae, Sphaeriinae. The Euperinae are com- prised of Byssanodonta and Eupera (Heard 1965b, Dreher- Mansur and Meier-Brook 2000) and diagnosed by (1) having a functional byssal gland in adults, (2) having well- separated branchial and anal siphons, and (3) lacking a brood-sac for developing embryos (Heard 1965b). They have restricted geographic distribution. Byssanodonta, a mo- notypic genus, is restricted to only a small portion of the upper Parana River, Argentina (Dreher-Mansur and Ituarte 1999). Eupera occurs in Central and South America and Africa/Madagascar, although one species, Eupera cubensis (Prime, 1865), is found in the southern United States (Heard 1965b, Mackie and Huggins 1976, Dreher-Mansur and Itu- arte 1999). Among the remaining three genera, Musculium and Sphaerium were considered to comprise the subfamily Sphaeriinae, separated from the Pisidiinae, which included Pisidium (Baker 1927). The Sphaeriinae were diagnosed by (1) having partially fused siphons, (2) having multiple brood-sacs for developing embryos, and (3) lacking a func- tional byssal gland in adults. The Pisidiinae were diagnosed by (1) having an anteriorly elongated shell, (2) having a greatly reduced branchial siphon (completely absent in some taxa), (3) having only a single brood-sac per brood, and (4) lacking a functional byssal gland in adults (Baker 1927, Heard 1965b, Burch 1975). However, recent studies (Cooley and O Foighil 2000, Dreher-Mansur and Meier-Brook 2000) have shown that the Sphaeriinae and Pisidiinae are not natu- ral groups, and Dreher-Mansur and Meier-Brook (2000) lumped them into the subfamily Sphaeriinae. Musculium, Sphaerium, and Pisidium are cosmopolitan genera with maximum diversities in the Holarctic Region and each genus contains numerous species (Burch 1975, Kuiper 1983). Even though sphaeriid subgeneric classification and re- lationships are still poorly understood, a suite of character reductions apparently related to reduction in shell size oc- curs in the subgenera of Pisidium. The largest subgenus Pi- sidium s. str. has three mantle openings (a pedal slit, an anal siphon, and a reduced branchial opening) and both inner and outer demibranchs, each composed of two lamellae. With reduced shell size, outer demibranchs have only a single lamella in Cyclocalyx Dall, 1903, and the branchial mantle opening and outer demibranchs are completely lost in Neopisidium Odhner, 1921 (Odhner 1921, Heard 1966). Kuiper (1962) further divided Neopisidium into Gondwanan Afropisidium Kuiper, 1962 and Eurasian Odhneripisidium Kuiper, 1962, the former with a protruding ligament, the latter with an introverted ligament. 19° 1/2 * 2004 Sphaeriid reproduction and development Sphaeriid clams have complex reproductive and devel- opmental characteristics, some of which may represent adaptive specializations to freshwater environments. All sphaeriid clams studied to date are simultaneous hermaph- rodites (Woods 1931, Okada 1935a, Heard 1965a, Ituarte 1997, Araujo and Ramos 1997). Sexual maturation occurs remarkably early in sphaertid ontogeny (Burky 1983, Holo- painen and Hanski 1986), and in some cases, gametogenesis is initiated prior to release from the parental clam (Heard 1977). Whereas the male portion takes up the greater part of the gonad in fully-grown individuals of Musculium, Pi- sidium, and Sphaeritum (Okada 1935a, Heard 1977, Araujo and Ramos 1997), the ovarian portion is much larger than the testicular portion in Byssanodonta and Eupera (Ituarte 1997, Dreher-Mansur and Meier-Brook 2000). Histological (Okada 1935c, Araujo and Ramos 1997, 1999), experimental (Odhner 1921, 1929, Thomas 1959, Meier-Brook 1970), and allozyme (Hornbach et al. 1980b, McLeod et al. 1981) stud- ies have indicated self-fertilization in the Sphaeriidae, and the hermaphroditic duct (Okada 1935a, Meier-Brook 1970), inner demibranchs (Araujo and Ramos 1999), and gonads (Araujo and Ramos 1997) have been suggested as sites of fertilization. All sphaeriid clams brood their direct-developing young within the inner demibranchs until they are released as ben- thic juveniles (Gilmore 1917, Okada 1935b, Bonetto and Ezcurra 1964, Heard 1965a, 1965b, 1977, Mackie ef al. 1974b, Ituarte 1997). Sphaeriid genera, however, show dif- ferent degrees of complexity in the details of how brooding is achieved. The simplest form is found in euperine species. Ripe eggs in euperine taxa are large (200-400 um in diam- eter) and have a large amount of yolk. It is generally believed that the euperine embryos are nourished mainly by yolk during development (Ituarte 1997, Dreher-Mansur and Itu- arte 1999, Dreher-Mansur and Meier-Brook 2000). All em- bryos in a brood are synchronously spawned and developed as a single cohort. Developing embryos lie between the ctenidial (gill) lamellae without any link to the parental tis- sues (Heard 1965b, Mackie and Huggins 1976, Dreher- Mansur and Ituarte 1999, Dreher-Mansur and Meier-Brook 2000). Species of Pisidium are also synchronous brooders. Yet, unlike Byssanodonta and Eupera, their embryos develop within a distinct brood-sac (marsupial-sac), which is formed by an outgrowth of the descending filaments of inner demi- branchs (Heard 1965a, 1977, Mackie et al. 1974b). In con- trast, species of Sphaerium and Musculium are sequential brooders, i.e., multiple subsets of embryos in discrete onto- genetic stages are simultaneously present within the inner demibranchs. Each subset results from a distinct spawning event and is sheltered in a separate brood-sac (Heard 1977, SPHAERIID MORPHOLOGICAL PHYLOGENY 3 Mackie 1979). Sphaeriine eggs are much smaller (about 100 um in diameter) and have less yolk than those of euperine species (Raven 1958, Mackie 1978a, Beekey et al. 2000). Transfer of nutrition from the gill to the embryo during development has been indicated in some Musculium species based on detailed histological, cytochemical, and ultrastruc- tural analyses (Okada 1935b, Hetzel 1994). In addition, a high brood mortality rate observed in many sphaeriine spe- cies (Avolizi 1976, Mackie et al. 1976, Meier-Brook 1977, Hornbach et al. 1980b, 1982, Mackie and Flippance 1983) may indicate that successfully developing embryos are nur- tured by the less successful ones (Avolizi 1976). Conflicting views of sphaeriid evolution The systematic validity and relationships of sphaertid genera are controversial although some conclusions have been traditionally held. Early taxonomic studies have sug- gested sister-group relationships between Byssanodonta and Eupera (Klappenbach 1960, Ituarte 1989) and between Mus- culium and Sphaerium (Burch 1975, Heard 1977, Hornbach et al. 1980a) based on morphological and reproductive/ developmental characters. Indeed, many workers (Sterki 1909, Thiele 1934, Haas 1949, Ellis 1962, Herrington 1962, Bowden and Heppell 1968, Gale 1972, Clarke 1973, Dreher- Mansur and Ituarte 1999) have questioned whether or not they are sufficiently distinct to warrant separate generic sta- tus. A number of malacologists (Meier-Brook 1970, 1977, Korniushin 1998a, 1998b, 1998c, Dreher-Mansur and Meier-Brook 2000, Korniushin and Glaubrecht 2002) have recognized a reduction of shell and body size accompanied by a series of conspicu- ous anatomical character losses and/or simplifications in smaller-sized Pisidium taxa, and assumed that miniaturization represents the major evolutionary trend in the Sphaeriinae. However, these phy- logenetic hypotheses were not tested until very recently. Using morphological characters, Dreher-Mansur and Meier-Brook (2000) and Korniushin and Glaubrecht (2002) performed cladistic analyses of the Sphae- riidae, but only the later study tested monophyly of the sphaeriid taxa and ro- a bustness of the recovered clades. Both analyses found two well-supported clades, Euperinae (Eupera, Byssanodonta) and Sphaeriinae ((Sphaerium (Muscu- lum, Pisidium)), and only Pisidium was recovered as monophyletic (Korniushin and Glaubrecht 2002), being a derived <,; clade among sphaeriine genera (Dreher- Origin of asynchronous brooding Pisidium s. lat. Mansur and Meier-Brook 2000, Korniushin and Glaubrecht 2002). In both studies, a sister-group relationship between Musculium and Pisidium was supported based primarily on a suite of micro-scale kidney characteristics. This result, however, was very weakly supported (bootstrap value was less than 50% [Korniushin and Glaubrecht 2002]) and in- congruent with the earlier taxonomic studies contesting the generic distinctiveness of Musculium from Sphaerium (Sterki 1909, Ellis 1962, Herrington 1962, Bowden and Heppell 1968, Gale 1972, Clarke 1973). Although Korniushin and Glaubrecht (2002, Fig. 1B) further suggested some changes, mainly the elevation of taxonomic ranks, to the classification of the Sphaeriidae, many of these changes lack topological and statistical support. Cooley and O Foighil (2000) generated the first com- prehensive sphaeriid gene tree using mitochondrial 16S tDNA sequences. The taxon sampling effort was expanded to incorporate nuclear (ITS1 RNA) and mitochondrial (16S RNA) genomes in Lee and O Foighil (2003). Both studies yielded a paraphyletic Pisidium in which the subgenus Afro- pisidium was sister to all the other sphaeriine taxa consid- ered, either alone (16S data) or together with the Odhneripi- sidium (ITS1-containing data sets). Asynchronous brooders (Sphaerium and Musculium) consistently formed a derived monophyletic group within the Sphaeriinae and it was strongly supported in a combined analysis of 16S and ITS1 (Lee and O Foighil 2003). Basal Pisidium paraphyly and derived sequential brooder monophyly are also apparent in preliminary trees based on nuclear gene fragments: 28S ri- Musculium S. (Musculiun) S. (Amesoda) Sphaerium s. lat. * Sphaerium s. str. S. (Sphaerinova) S.(Herringtoniun) Henslowiana Casertiana eee Cyclocalyx es ee Vusculium Cingulipisidium * Cyclocalyx Pisidium s. str. ; i 5] > Odhneripisidium Pisidium Afropisidium Eupera B Figure 1. Two competing hypotheses of sphaeriid phylogeny. A, Hypothesis of Lee and O Foighil (2003) based on molecular (16S and ITS1) sequence data. B, Hypothesis of Kor- niushin and Glaubrecht (2002) based on morphological characters. While gene trees showed a paraphyletic Pisidium and a derived monophyletic clade of asynchronous brood- ers (Sphaerium and Musculium taxa), morphological analyses recovered a derived mono- phyletic Pisidium clade and a sister relationship among Musculium and Pisidium taxa. An indicates a strong support (bootstrap value >90%) and scale bars represent 2 mm. 4 AMERICAN MALACOLOGICAL BULLETIN bosomal RNA (Park and O Foighil 2000) and 6-phospho- gluconate dehydrogenase (Lee and O Foighil 2002). Sphae- riine classification was also revised based on molecular analyses, and five robust clades (Afropisidium, Odhneripi- sidium, Pisidium, Cyclocalyx, and Sphaerium) were suggested as generic groupings (Lee and O Foighil 2003, Fig. 1A). There are two major topological incongruencies be- tween molecular- and morphology-based sphaeriid phylog- enies. While a derived monophyletic Pisidium clade was strongly supported and diagnosed by a number of morpho- logical characters (Dreher-Mansur and Meier-Brook 2000, Korniushin and Glaubrecht 2002), independent as well as combined analyses of various gene sequence data consis- tently yielded paraphyletic Pisidium (Cooley and O Foighil 2000, Park and O Foighil 2000, Lee and O Foighil 2002, 2003). The other pointed element of incongruence concerns the phylogenetic relationships among the synchronous (Eu- pera and Pisidium) and asynchronous (Sphaerium and Mus- culium) brooding taxa. Molecular studies recovered a de- rived monophyletic clade of asynchronous brooders (Eupera (Pisidium (Musculium, Sphaerium))), but morphological analyses yielded a (Eupera (Sphaerium (Musculium, Pi- sidium))) topology (Fig. 1). The phylogenetic placement of the asynchronous brooders is important because it shapes our view of the primary evolutionary trends in the Sphae- riinae. According to morphological studies, the characters observed in the larger Sphaerium taxa are mostly plesiomor- phic and derived from those are the reduced Pisidium, es- pecially the subgenus Neopisidium, characters. Conversely, molecular data consistently place Sphaerium in a derived sphaeriine clade together with Musculium, revealing the evo- lutionary elaboration of brooding character complexity from synchronous to sequential brooding. The robust topological congruence among independent molecular trees based on diverse gene fragments suggests that a careful reexamination of morphological data is war- ranted. Therefore, the morphology and life history of North American sphaertid taxa was studied, widely accepted char- acters were newly coded, and a parsimony analysis was per- formed. The results showed that Sphaerium and Musculium taxa form a monophyletic group, congruent with previous gene trees but not with morphological studies. The Pisidium monophyly conflict between molecular and morphological trees, however, remains unsettled. At present, informative morphological characters appear insufficient to flesh out phylogenetic relationships among infra-generic sphaeriid taxa. MATERIALS AND METHODS Taxa examined Of the five widely recognized sphaeriid genera, one to six representatives of all four North American genera (Eu- 19° 1/2 + 2004 pera, Musculium, Pisidium, and Sphaerium) were chosen for analyses. Seventeen species selected were also representatives of all sphaeriid subgenera recognized in North America (Table 1). Herein I use the classification system of Burch (1975) and Dreher-Mansur and Meier-Brook (2000), instead of newly suggested ones (Korniushin and Glaubrecht 2002, Lee and O Foighil 2003), to test their competing hypotheses. The marine Astarte sulcata (Da Costa, 1778) and the fresh- water Corbicula North American “Form A” (for form desig- nation see Siripattrawan et al. 2000) and Neocorbicula limosa (Maton, 1809) were also included as outgroups in order to root the sphaeriid phylogeny. It has been widely held that the Astartidae have many plesiomorphic morphological charac- ters, occupying a basal position within the order Veneroida (Taylor et al. 1973, Morton 1996). Indeed, the Astarte species was sister to all the other heterodont taxa studied, including sphaeriids, in a recent molecular phylogenetic analysis (Park and O Foighil 2000). The fresh/brackish water family Cor- biculidae has long been placed in the superfamily Corbicu- loidea together with Sphaeriidae (Newell 1965, Keen and Casey 1969, Taylor et al. 1973, Boss 1982, Morton 1996) although their sister-relationship was rejected recently (Dre- her-Mansur and Meier-Brook 2000, Park and O Foighil 2000). In addition, the two corbiculid species chosen display a number of specialized reproductive and developmental characters similar to the ingroup taxa. Even though I recog- nize this apparent convergent evolution as a potential prob- lem, convincing sister taxa for the Sphaeriidae have not yet been identified. Thus, an unrooted analysis using the in- group taxa only was conducted in order to check if outgroup rooting generates any phylogenetic conflict. Characters Character states for each species were determined from the examination of shells and alcohol-preserved specimens deposited in the University of Michigan, Museum of Zool- ogy and from the previous studies (see references in Table 1). Only the diagnostic characters accepted widely by sphae- riid systematists were coded. Micro-scale anatomical char- acters used in previous cladistic analyses (Dreher-Mansur and Meier-Brook 2000, Korniushin and Glaubrecht 2002) were not included when they were phylogenetically uninfor- mative and/or controversial among studies. Inapplicable characters, such as branchial siphon characters in the species with no branchial siphon, were coded as dashes (-) and a missing character with a question mark (?). The character matrix is shown in Table 2. Characters and character states (an asterisk below de- notes characters that appeared in Korniushin and Glau- brecht [2002], their character states were determined inde- pendently in the present study). SPHAERIID MORPHOLOGICAL PHYLOGENY 5 Table 1. Catalog of the studied taxa, information on specimens examined (Mollusk Division Catalog Number, University of Michigan, Museum of Zoology), and references to anatomical and life-history characters. Specimens were sampled by ‘William H. Heard, *Cristian F. Ituarte, *Renée S. Mulcrone, *Mary Yong, and °the author. Taxon Family Astartidae d’Orbigny, 1844 Genus Astarte J. Sowerby, 1816 *A. sulcata (Da Costa, 1778) Family Corbiculidae Gray, 1847 Genus Corbicula Megerle, 1811 **C. (North American “Form A”) Genus Neocorbicula Fischer, 1887 N. limosa (Maton, 1809) Family Sphaeriidae Deshayes, 1854 (1820) Subfamily Euperinae Heard, 1965 Genus Eupera Bourguignat, 1854 E. cubensis (Prime, 1865) Subfamily Sphaeriinae Baker, 1927 Genus Musculium Link, 1807 *M. lacustre (Miller, 1774) M. partumeium (Say, 1822) M. securis (Prime, 1852) M. transversum (Say, 1829) Genus Pisidium Pfeiffer, 1821 Subgenus Cyclocalyx Dall, 1903 P. adamsi Stimpson, 1851 P. casertanum (Poli, 1791) P. compressum Prime, 1852 P. variabile Prime, 1852 Subgenus Neopisidium Odhner, 1921 *P. conventus Clessin, 1877 Subgenus Pisidium s.str. P. dubium (Say, 1816) Genus Sphaerium Scopoli, 1777 Subgenus Herringtonium Clarke, 1973 *S. occidentale (Prime, 1856) Subgenus Sphaerium s.str. *S. corneum (Linnaeus, 1758) S. fabale (Prime, 1852) S. rhomboideum (Say, 1822) S. simile (Say, 1816) S. striatinum (Lamarck, 1818) *: Type species of the genus and subgenus. **: For form designation see Siripattrawan Saleuddin 1964, 1965, Boss 1982 Britton and Morton 1982, King et al. 1986, Ituarte 1994, Dreher-Mansur and Meier-Brook 2000 Ituarte 1994, Dreher-Mansur and Meier-Brook 2000 Heard 1965b, Mackie and Huggins 1976, Dreher-Mansur and Meier-Brook 2000 Heard 1977, Dreher-Mansur and Meier-Brook 2000 Mackie and Qadri 1974, Mackie et al. 1974a, 1974b, Heard 1977 Heard 1965a, 1966 Heard 1965a, 1966 Heard 1965a, 1966 Heard 1963, 1965a, 1966 Heard 1965a, 1966 Clarke 1973, Heard 1977 Jacobsen 1828, Heard 1977, Dreher-Mansur and Meier-Brook 2000 Drew 1896, Gilmore 1917, Heard 1977 Monk 1928, Heard 1977 Collection Catalog locality number References England 9700 *Michigan, USA 266693 *Argentina 265500 *Cuba 266709 "Michigan, USA 266756 *Michigan, USA 266755 ‘Ontario, Canada. 266757 Heard 1977 °*Michigan, USA 266670 "Michigan, USA 266758 °Michigan, USA 266710 'Ohio, USA 266721 Heard 1977 *Michigan, USA 266722 'Michigan, USA 266764 Heard 1966 °*Michigan, USA 266663 'Michigan, USA 266725 °Michigan, USA 266728 "Michigan, USA 266729 °*Michigan, USA 266714 "Michigan, USA 266740 °Michigan, USA 266665 'Michigan, USA 266742 "Michigan, USA 266746 °Michigan, USA 266715 'Michigan, USA 266753 °Michigan, USA 266752 ‘Ontario, Canada 266760 "Michigan, USA 266748 Heard 1977 Michigan, USA 266747 ‘Ontario, Canada 266763 Heard 1977 °Michigan, USA 266762 "Michigan, USA 266750 *Michigan, USA 266712 'Michigan, USA 266751 °*Michigan, USA 266679 et al. (2000). References for shell characters: Baker (1928), Herrington (1962), Clarke (1973), Burch (1975), Mackie et al. (1980). 6 AMERICAN MALACOLOGICAL BULLETIN — 19+ 1/2 + 2004 Table 2. Data matrix used in the phylogenetic analysis. Toc wll Lally i 2 2 2 22 2 Ds 2 es 12345 67 8 9 0 1.23 4 5 6 7 8 9 0 2 24 5 6 7 8 9-0 Astarte sulcata 000000 0 0 0 - 0 000000000000 - - - - - 0 Corbicula North American “Form A” 0 0 1 1000 100101001 01 101 2121100 - 00 0 Neocorbicula limosa 00 1f1000100T0100212~0 121012 0d21«120 - 0 0 % Eupera cubensis O 1-2-0 0 1 0 1°). 0.1510 °0 0-1 LT 1-07 1h 0 1:0 0° = 00-0 Musculium 022.0 0 0 0 PF 1b. 1.0 0 0-0 1 TL 0.0. 1 1 0 Lf 1 1 1.2 01 Pisidium (Cyclocalyx) 122000001 1 100 1 1 1 1:00 1 10 1 010 0 0 1 Pisidium (Neopisidium) lt 2°2.-0°0 O- Tee) =e Oo=9 0°), = 1) Pol 0) 0 Te) 0 1 0 2 0.0505 1 Pisidium (Pisidium) 122000001-11000211100d1d10101000i~1 Sphaerium (Herringtonium) O22 0 1.0. OT TP a 0h O00! Om 1 TD eb OOr le 0) Te ah Od Sphaerium (Sphaerium) Oo 2 2 70°00 OME HUTS TOie0! OO De DO OSD aly 0) =P Vl le TON a Sphaerium (Sphaerium) corneum Oo 2 2°00 0 00 7. Tat 2e00"-0 (Oele t oP Ovc0 Tete OT 1 ae Tk hed Shell characters 16. Demibranchs behind the foot: 0 = not united, 1= *]. Shell shape: 0 = equilateral to posteriorly elongated, united to each other. 1 = anteriorly elongated. “17. Height of ascending lamellae of inner demi- *2. Number of cardinal teeth in left valve: 0 = three, 1 branchs: 0 = as high as descending lamellae, 1 = = one, 2 = two. lower than descending lamellae. *3. Number of cardinal teeth in right valve: 0 = two, 1 18. Association of ascending lamellae of demibranchs = three, 2 = one. with the mantle and visceral mass, 0 = fused only *4. Morphology of lateral teeth: 0 = smooth, 1 = anteriorly and connected posteriorly by cilia, 1 = serrate. fused along entire length or almost so. 5. Inside of the shell: 0 = not ridged, 1 = ridged. *19. Type of stomach (Purchon 1987): 0 = type IV, 1 = 6. Adventitious maculations on inner shell surface: 0 = type V. absent, 1 = present. *20. Functional byssus in adult: 0 = absent, 1 = present. Gross soft-anatomy characters we . Outer demibranchs: 0 = with two lamellae, 1 Number of mantle openings: 0 = three (pedal slit, branchial, and anal openings), 1 = two (pedal slit and anal openings). . Branchial (incurrent) opening: 0 = not produced into elongate siphon, 1 = produced into elongate siphon. . Morphology of branchial and/or anal (excurrent) siphons: 0 = papillated, 1 = smooth. . Branchial and anal siphons when extended: 0 = well separated, 1 = fused in part. . Pallial fusion ventral to the branchial siphon or aperture (pre-siphonal suture): 0 = absent, 1 present. 2. Morphology of pre-siphonal suture: 0 = short, 1 = elongated. . Morphology of mantle edge ventral to the bran- chial or anal opening: 0 = smooth, 1 = papillated. . Ctenidia: 0 = with outer demibranchs, 1 = without outer demibranchs. II with one lamella. Brooding and life history characters 21 22. i) QW 2/. . Habitat: 0 = marine, 1 = freshwater. Sexual expression: 0 gonochoristic, 1 hermaphroditic. . Type of development: 0 = develop directly as ju- veniles without larval stage, 1 = develop indirectly with larval stage. . Extent of parental brooding: 0 = absent, 1 = present. 25. Type of brooding habit: 0 = synchronous brooding (all embryos in a brood result from a single spawn- ing event and develop as a single embryonic co- hort), 1 = sequential brooding (the products of several distinct spawning events co-exist in the ctenidial marsupia and are composed of develop- mentally discrete subsets of embryos). . Extent of brood-sac (marsupial-sac): 0 = absent, 1 = present. Morphology of the brood sac: 0 = thick-walled and partitioned into smaller chambers, 1 = thin-walled without further partitioning (Heard 1977). . Fully developed juveniles retained within the SPHAERIID MORPHOLOGICAL PHYLOGENY 7 ctenidial marsupia (extra-marsupial larvae sensu Heard 1977): 0 = lying free within the marsupia, 1 = attached to the remnants of the brood sacs or to the descending lamellae of the inner demibranchs (Mackie et al. 1974a, Heard 1977). 29. Precocious maturation (production of gametes by brooded juveniles before being released from the ctenidia): 0 = absent, 1 = present (Heard 1977). *30. Eggs: 0 = large with lots of yolk, 1 = small without sufficient yolk to nourish the embryo until mater- nal deposition of offspring. Phylogenetic Analysis The data were analyzed using PAUP* 4.0b8 (Swofford 2002) under the maximum parsimony optimality criterion. Analyses were performed as branch-and-bound searches us- ing equal character weighting. All characters were treated as unordered and dashes were treated as missing data rather than as a new state. Astarte sulcata, Corbicula North Ameri- can “Form A,” and Neocorbicula limosa were designated as outgroups, and sphaeriid taxa were forced to be monophy- letic to root the phylogeny. An unrooted analysis of ingroup taxa was also conducted and the results were compared with outgroup-rooted topology. Character transformation series were determined on one of the equally parsimonious trees using MacClade 3.07 (Maddison and Maddison 1997) and PAUP*. Branch support levels were calculated with boot- strapping (1000 replications, heuristic searches, 10 random additions each) using PAUP* and with Bremer Decay-Index values (Bremer 1994) using TreeRot (Sorenson 1999), which generates a constraint file for PAUP*. RESULTS Thirty characters were coded, including those from the shell (1-6), gross soft-anatomy (7-20), and brooding/life his- tory (21-30). Within each subgenus, all representative spe- cies had the same character set except for Sphaerium (Sphaerium) corneum (Linnaeus, 1758), which had a differ- ent character state of fully developed juveniles retained within the ctenidial marsupia (Character 28) from the other Sphaerium s. str. taxa. To facilitate a thorough analysis, one character set from each subgenus was included in the data- matrix unless varied taxa existed (Table 1). Of 30 total, 18 characters were found to be parsimony-informative and only 7 were so in ingroup-only analysis. Twelve equally most-parsimonious trees of 37 steps (CI = 0.865, RI = 0.844) were obtained from the analysis of the data matrix including outgroup taxa, and a strict consensus was recovered (Fig. 2). Sphaeriid genera formed a robustly supported monophyletic group and Eupera cubensis was sis- ter to the monophyletic Sphaeriinae. Musculium was Musculium Sphaerium (Sphaerium) S. (Sphaerium) corneum S. (Herringtonium) Sphaeriinae Pisidium (Cyclocalyx) P. (Pisidium) P. (Neopisidium) Eupera cubensis Euperinae Corbicula NA form A Neocorbicula limosa Astarte sulcata Figure 2. Strict consensus of 12 equally parsimonious trees (L = 37, CI = 0.865, RI = 0.844) obtained from the analysis including out- group taxa. Numbers above and below internodes are bootstrap and Bremer values, respectively. grouped with Sphaerium taxa, not with Pisidium, forming an unresolved polytomy, this result was fairly well supported by bootstrap value 85. Three North American Pisidium subgen- era were recovered as monophyletic without further resolu- tion and sister to the Sphaerium s. lat./Musculium clade. The unrooted analysis of ingroup taxa alone resulted in the same topology (12 trees, L = 19, CI = 0.947, RI = 0.923, Fig. 3), but provided a much higher bootstrap value for the Sphaerium s. lat./Musculium clade and the Pisidium clade. Character transformations were depicted on one of the most-parsimonious cladograms obtained from the out- group-rooted analysis (Fig. 4). DISCUSSION Previous morphological trees conflict with gene trees on two major elements of sphaeriid topology, i.e., monophyly of Pisidium and of asynchronous brooders (Sphaerium and Musculium). The present study recovered a monophyletic Pisidium as in the other morphological analyses, but asyn- chronous brooders were found to be monophyletic as in the previous molecular studies (Figs. 2-3). Lee and O Foighil (2003), the most comprehensive mo- lecular study to date utilizing mitochondrial 16S and nuclear ITS1 sequences data, found four strongly supported clades within Pisidium s. lat., and these terminal clades formed a paraphyletic assemblage (Fig. 1A). Paraphyletic Pisidium was also apparent in all the other gene trees generated by diverse nuclear and mitochondrial gene fragments: Mitochondrial 16S ribosomal RNA (Cooley and O Foighil, 2000), nuclear 8 AMERICAN MALACOLOGICAL BULLETIN S. (Sphaerium) corneum S. (Herringtonium) Sphaerium (Sphaerium) Musculium Eupera cubensis Pisidium (Pisidium) P. (Neopisidium) P. (Cyclocalyx) Figure 3. Unrooted strict consensus of 12 equally parsimonious trees obtained from the analysis of ingroup taxa only (L = 19, CI = 0.947, RI = 0.923). Numbers right and left of internodes are boot- strap and Bremer values, respectively. 28S ribosomal RNA (Park and O Foighil 2000), and nuclear single copy 6-phosphogluconate dehydrogenase (Lee and O Foighil 2002). Among the Pisidium terminal clades, Afropi- sidium was consistently positioned basally within the Sphae- riinae (Fig. 1A, Cooley and fe) Foighil 2000, Park and O Foighil 2000, Lee and O Foighil 2003). On the other hand, Korniushin and Glaubrecht’s (2002) morphological analysis recovered a derived monophyletic Pisidium s. lat. clade (Fig. 1B). This was strongly supported by a bootstrap value of 98 and diagnosed by 8 shared derived characters, of which 4 were unambiguous. The present study confirmed Pisidium monophyly (Fig. 2). Six Pisidium species belonging to 3 North American subgenera formed a moderately supported (bootstrap value = 71) monophyletic group. This clade was diagnosed by at least two synapomorphies, anteriorly elon- gated shell shape (character 1) and reduced branchial siphon (character 8) (Fig. 4). Given the strong support and/or consistency in both data sets, the disparity on Pisidium monophyly may origi- nate from the actual differences between molecular and 19+ 1/2 + 2004 Musculium Sphaerium (Herringtonium) S. (Sphaerium) corneum S. (Sphaerium) Pisidium (Pisidium) P. (Cyclocalyx) P. (Neopisidium) Eupera cubensis 3 8 111618212224 Corbicula NA form A Neocorbicula limosa Astarte sulcata Figure 4. One of 12 equally most-parsimonious trees rooted by outgroup showing character transformations. The numbers below the bars denote the character number changing along that inter- node. Character numbers refer to those listed in materials and methods. Black bars indicate character changes from 0 to 1, gray bars from | to 2, and white bars from 1 to 0. An “*” indicates an unambiguous character transformation. morphological phylogenies. However, there are some points that need to be addressed before such a conclusion can be made. First of all, this conflict may be attributable to unde- tected convergent evolution in sphaeriid morphology, al- though molecular sequence data are not free from homo- plasy (Hillis and Wiens 2000). Most morphological synapomorphies for the Pisidium clade are losses and/or simplifications of anatomical characters such as outer demi- branchs and siphons. If some of these character losses were linked to body size reduction and if miniaturization hap- pened independently in each Pisidium s. lat. lineage, conver- gent apomorphies might be accumulated in these already well-separated paraphyletic lineages, preserving misleading phylogenetic information. In addition, sphaeriine lineages are apparently very old—Afropisidium has a Gondwanan distribution (Kuiper 1983). Thus, previous and the present morphological studies that have analyzed living taxa alone may have been misled. Secondly, taxon sampling was not complete in both data sets. Molecular studies lack any Neo- pisidium taxa and have a mere two species each of Afropi- sidium and Odhneripisidium. The number of Neopisidium taxa included was limited in Korniushin and Glaubrecht (2002) and the present morphological study was restricted to North American taxa, lacking any representative species of SPHAERIID MORPHOLOGICAL PHYLOGENY 9 Afropisidium and Odhneripisidium. An extensive taxon sam- pling effort will ultimately improve phylogenetic accuracy (Hillis 1998, Pollock et al. 2002, Zwickl and Hillis 2002). A sister-relationship among asynchronous brooders (Sphaerium s. lat. and Musculium taxa) suggested by mo- lecular studies was confirmed in the present morphological analysis (Figs. 2 and 3). According to the outgroup-rooted analysis, the Sphaerium/Musculium clade was supported by bootstrap value 85 and diagnosed by at least one unambigu- ous and two ambiguous synapomorphies: Short pre- siphonal suture (character 12), sequential brooding (charac- ter 25), and non-partitioned brood sac (character 27) (Fig. 4). When outgroups were excluded from the analysis, all these characters, together with character 10 fused siphon, unambiguously diagnosed the clade and the bootstrap value reached 95. This result is not surprising because morphology-based sphaeriine phylogenies (Sphaerium (Musculium, Pisidium)) recovered previously (Dreher-Mansur and Meier-Brook 2000, Korniushin and Glaubrecht 2002) were far from ro- bust. Dreher-Mansur and Meier-Brook (2000) suggested a sister-relationship for Musculium and Pisidium based on one shell and three kidney characters. However, three of these inferred synapomorphies appear to be controversial: 39, lack of the complex crossed-lamellar structure in the shell (see Mackie 1978b), 40, very long kidney funnel (see Korniushin and Glaubrecht 2002 character 47), and 42, slit-like opening of the excretory sac (see Korniushin and Glaubrecht 2002 character 58). They considered their character 49, reduced number of brood sacs, as an autapomorphy of Pisidium, but since only sphaeriine taxa have brood sacs, having multiple brood sacs would have been a synapomorphy supporting Sphaerium/Musculium clade as it is in the present study (character 27, Fig. 4). Dreher-Mansur and Meier-Brook (2000) also overlooked phylogenetically informative brood- ing characteristics, which have long been recognized as di- agnostic characters for generic-level classification, 1.e., se- quential/synchronous brooding patterns, morphology of brood sacs, and the position of developing embryos. In Kor- niushin and Glaubrecht (2002), only one character, narrow kidney funnel (character 48), ambiguously supported their Musculium/Pisidium clade, the other supporting character, one lamella outer demibranch in the incubated young (char- acter 69), is actually an apomorphy for Musculium because this was inapplicable to Pisidium taxa. The supporting boot- strap value was less than 50% and the alternative topology (Pisidium (Musculium, Sphaerium)) was a mere one step longer (Korniushin and Glaubrecht 2002). Methodological distinctions between the present and previous morphological studies are likely to underlie the differential topological results. While the present study re- stricted the character set to major anatomical and develop- mental features that have been considered fundamental by previous workers, Dreher-Mansur and Meier-Brook (2000) and Korniushin and Glaubrecht (2002) included a large number of fine-scale anatomical features, especially numer- ous, potentially non-independent details of kidney substruc- ture, in their data sets. Although micro-scale anatomical structures have recently been utilized to address taxonomic problems of the Sphaeriidae mainly by malacologists of the Russian school (Korniushin 1991, 1994, 1995, 1998a, 1998b, 1998c, 1999, Piechocki and Korniushin 1994), the plasticity of delicate anatomical differences (Korniushin 1998a) makes the application of these characters to cladistic analyses very difficult at this stage. Because of the potential for significant convergent evolution in brooding character states in corbi- culid outgroup taxa (Dreher-Mansur and Meier-Brook 2000, Park and O Foighil 2000), the present study analyzed ingroup characters without rooting in addition to outgroup rooted analysis. Dreher-Mansur and Meier-Brook (2000) and Korniushin and Glaubrecht (2002), however, included each one of Corbicula and Neocorbicula lineages as an out- group and did not test for outgroup rooting problems. It is noteworthy that the same sphaeriid topology (Euperinae (P7- sidium (Sphaertum, Musculium))) as the present study was recovered when the author analyzed Korniushin and Glau- brecht’s (2002) character matrix without corbiculids. A number of sphaertid systematists (Meier-Brook 1970, 1977, Korniushin 1998a, 1998b, 1998c, Dreher-Mansur and Meier-Brook 2000, Korniushin and Glaubrecht 2002) have proposed that miniaturization represents the predominant evolutionary trend in the Sphaeriinae and that the conspicu- ous character reductions observed in smaller-sized Pisidium taxa are derived from larger Sphaerium as a consequence of shell and body size reduction. However, it is now clear that this evolutionary trend is largely restricted to Pisidium taxa, especially Neopisidium. The present analysis, instead, sup- ports the evolutionary development in brooding character complexity from synchronous to sequential brooding (Kor- niushin 1991, Cooley and O Foighil 2000). Synchronous brooding observed in Eupera and Pisidium is plesiomorphic, rejecting the hypothesis that having a single brood sac per demibranch is derived from a multiple brood sac condition found in Sphaerium and Musculium due to the ctenidial size-reduction (Korniushin 1998b, Dreher-Mansur and Meier-Brook 2000). Previous molecular and morphology-based phylogenies were concordant in that three cosmopolitan genera, Pi- sidium, Musculium, and Sphaerium, form a monophyletic group, the Sphaeriinae, with respect to Eupera species. The present study also supports this conclusion. Eupera was seg- regated from the other sphaeriid clades early at the basal node, and the Sphaeriinae were diagnosed by four unam- biguous synapomorphies (Fig. 4): Two cardinal teeth in the 10 AMERICAN MALACOLOGICAL BULLETIN left valve (character 2), fused siphons (character 10, this is a synapomorphy supporting the Sphaerium/Musculium clade when ingroup taxa only were analyzed), the incubation of developing embryos within brood sacs (character 26), and non-yolky eggs (character 30). The sister-group relationship among asynchronously brooding Sphaerium and Musculium taxa, supported by mo- lecular studies (Cooley and O Foighil 2000, Park and O Foighil 2000, Lee and O Foighil 2002, 2003) and traditionally held by many sphaeriid systematists (Ellis 1962, Herrington 1962, Bowden and Heppell 1968, Clarke 1973), was con- firmed by the present analysis of the morphological charac- ters considered fundamental by previous workers. However, the Pisidium monophyly conflict between molecular and morphological trees remains unsettled. In addition, this study was not able to provide further infra-generic level relationships: Both Sphaerium s. lat./Musculium and_ Pi- sidium clades were polytomous. This may be due to the lack of informative morphological characters. Only seven of a total 30 characters were parsimony-informative in ingroup comparison and no synapomorphy supporting Musculium and Pisidium s. str. was found. A sufficient number of char- acters, of whatever type, as well as taxa are crucial in esti- mating phylogeny (Hillis and Wiens 2000). Adding fossil characters to morphological analyses and incorporating more slowly evolving DNA sequences in the present mo- lecular data set may provide sound basal sphaeriid relation- ships by redeeming ancestral states. Combined analysis of molecular and morphological data collected from global taxonomic sampling appears to be another challenging step toward a comprehensive phylogeny of the Sphaertidae. To do this extensively, identification of convincing marine out- groups as well as a balanced taxon sampling among molecu- lar and morphological data sets and among euperine and sphaeriine lineages are warranted. ACKNOWLEDGMENTS I thank William H. Heard, Cristian F. Ituarte, Renée S. Mulcrone, and Mary Yong for kindly providing specimens. I also appreciate the valuable comments and suggestions made by C. Ituarte, Gerald Mackie, and Janice Voltzow. Their input has significantly improved this manuscript. This study was supported by a Rackham Predoctoral Fellowship from the Horace H. Rackham School of Graduate Studies and Hinsdale/Walker Scholarships from the Museum of Zool- ogy, University of Michigan. LITERATURE CITED Alimov, A. F. 1970. 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Accepted: 17 November 2003 — ie NE ee AMERICAN MALACOLOGICAL BULLETIN 19+ 1/2 * 2004 Use of a natural river water flow-through culture system for rearing juvenile freshwater mussels (Bivalvia: Unionidae) and evaluation of the effects of substrate size, temperature, and stocking density Braven B. Beaty’ and Richard J. Neves” "The Nature Conservancy, Clinch Valley Program, 146 East Main Street, Abingdon, Virginia 24210, U.S.A., bbeaty@tnc.org * Virginia Cooperative Fish and Wildlife Research Unit*, United States Geological Survey, Department of Fisheries and Wildlife Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0321, U.S.A., mussel@vt.edu Abstract: The feasibility of rearing juvenile freshwater mussels using a culture system supplied with natural river water was investigated. Newly transformed juvenile rainbow mussels ( Villosa iris) were reared for approximately 90 days in a flow-through culture system designed to simulate a stream channel. A total of 755 juveniles were reared to at least 90 days old over the course of 3 years. Juveniles were placed in containers partially filled with sieved river substrate, providing both a feeding medium and some protection from physical disturbance. Substrate size and depth were evaluated for influences on growth and survival of juveniles. Juvenile mussels in smaller substrate (<120 jm) grew slightly larger than those in larger substrate (between 120 and 600 um) during one trial (reaching 2.22 mm vs. 1.97 mm in length, respectively [p < 0.10] from a starting size of approximately 0.30 mm), with no difference in survival. Substrate depth, 5 mm or 20 mm, had no effect on either survival or growth. In all experiments, most juveniles were found in the loose, flocculent layer of sediment brought in by the river water. The season when rearing of juveniles was begun had a significant effect on growth and survival of the mussels. Growth and survival were best when rearing was initiated in June and declined as rearing began later in the summer. Differences in water temperature of the culture system explained much of this variation. Separate laboratory experiments suggested that juvenile mussels stopped growing at temperatures below 15°C. When growth data were normalized for degree-days above 15°C, most of the variability in growth was explained (R* = 0.88, p < 0.001). The use of an in situ culture system with river water was shown to be feasible, but seasonal variables must be accommodated. Key words: Unionidae, freshwater mussels, Villosa iris, flow-through aquaculture, juvenile mussels The culture of juvenile freshwater mussels began in the early 20th century to augment populations in the Mississippi River basin that were being harvested for shells to make buttons for clothing. Howard (1916) was successful in cul- turing juvenile yellow lampmussels (Lampsilis radiata luteola Lamarck, 1819) to adulthood and obtained some second generation juveniles. However, other culture trials were not successful (Corwin 1920) and the specific techniques used by the early studies were not clearly reported. Mussel culture efforts were suspended thereafter until efforts to propagate endangered mussels developed in the 1980's. To assist in the recovery of the 70 federally listed species in the U.S., the National Native Mussel Conservation Com- mittee (1998) identified the propagation of juvenile mussels as an important component of the recovery plan for unionid species. Efforts to rear juvenile mussels to a size large enough to avoid many of the perils of early life in the wild were renewed in the last decade. Gatenby et al. (1997) investigated the survival and growth of juvenile rainbow mussels (Villosa * The Unit is jointly supported by the U.S. Geological Survey, Vir- ginia Department of Game and Inland Fisheries, Virginia Poly- technic Institute and State University, and Wildlife Management Institute. iris [I. Lea, 1829]) reared in laboratory upweller dishes, showing that juvenile mussels could be cultured with artifi- cial food and water sources. The effect of algal diet compo- sition and sediment also was tested, confirming that juvenile mussels had specific nutritional needs that were not fully met with just a single food source (Gatenby ef al. 1996). Yeager et al. (1994) showed that juvenile rainbow mussels need a suitable substrate in which to burrow and feed. Suc- cessful culture trials with young mussels also have been con- ducted in Europe using the pearl mussel (Margaritifera mar- garitifera [Linnaeus, 1758]) (Buddensiek 1995). All of these studies have focused on the goal of developing techniques necessary to make the culture of juvenile mussels a practical component of conservation. Comparing results of culture trials using natural river water to those using laboratory water suggests that culture systems with natural river water have a higher potential for success. Results from early studies of the propagation of juvenile mussels suggest that the best methods are those using natural waters (Howard 1923). Further support for the efficacy of rearing juveniles in stream water is provided by Buddensiek (1995); juveniles held in river water had high survival rates (up to 20% after 12 months) and good growth. The long-term survival rates achieved with culture systems 16 AMERICAN MALACOLOGICAL BULLETIN supplied by natural river water exceed those of laboratory- based systems (Howard 1923, Hudson and Isom 1984, Bud- densiek 1995, Gatenby et al. 1997). These studies clearly indicate that a culture system supplied with natural river water should have a high probability of success. The objectives of this study were to evaluate the feasi- bility of using a flow-through culture system with natural river water for rearing juvenile mussels and to determine the effects of substrate size, substrate depth, and time of year on survival and growth of juvenile mussels. MATERIALS AND METHODS Glochidia of rainbow mussels (Villosa iris) were trans- formed to juveniles on rock bass (Ambloplites rupestris Rafinesque, 1817) in the laboratory. Gravid female mussels were collected from Copper Creek near Nickelsville in Scott County, Virginia, during May 1993 and on 27 May 1994, and 18 and 23 May 1995. Glochidia were flushed from the marsupia using a 5 ml syringe with a 3.8 cm, 21 gauge needle filled with conditioned municipal water (aerated for at least 24 hr with airstone). The glochidia were collected in a petri dish and placed in a 19 | bucket with 15 cm of conditioned municipal water. Three to five rock bass were placed in the bucket for 45 min, and an airstone was placed in the bucket to suspend the glochidia. After the 30-45 min exposure time, the fish were placed in separate 39 | static aquaria until the glochidia transformed and excysted, usually 13-20 days at 23°C. Transformed juveniles were collected by light siphon- ing pressure and filtration through a 120 um sieve within 2 days after excystment. Newly transformed juveniles were held in 7.5 x 7.5 x 3.3 cm polypropylene dishes. The dishes were filled with a 1:1 mixture of conditioned municipal wa- ter and well water with a 3-5 mm layer of fine silt (particle size <120 pm) from the Clinch River immediately upstream of the American Electric Power Clinch River Steam Plant (CRSP) at Carbo, Russell County, Virginia, at river mile 266.1. Within 7 days, the juveniles were placed in the ex- perimental culture systems. The first experiment of this study was conducted in a flow-through culture system with ambient river water on the property of the CRSP. The water for the culture system was taken from the Clinch River immediately upstream of the power plant (pH 7.7-8.4, hardness 124-174 mg CaCO,/l). Mussel populations in this upstream reach of the river ap- peared to be healthy and reproducing, with young adults present, such that water quality was deemed suitable for rearing juvenile mussels. Water from the river was introduced into a U-shaped channel, which served as the flow delivery system for the oval troughs in which the juvenile mussels were reared (Fig. 19° 1/2 * 2004 1A). The channel also served as a settling chamber to remove much of the sediment introduced into the system. This channel had eleven holes drilled through each side at the same height. Each of the 22 holes was fitted with a short tube to guide the flow of water out of this channel and into the oval troughs where the mussels were held. Therefore, the channel served as both a flow regulator and a sediment trap. The flow from the U-channel was directed into oval troughs, capable of holding approximately 75 | of water (Fig. 1B). Each of these troughs was equipped with a raised center that extended above the water level and a standpipe drain to regulate water level. The flow of water into these troughs was directed along the long axis of the oval to promote continu- ous current in a circular fashion. In addition, each of the oval troughs was fitted with a motorized paddle wheel to maintain unidirectional flow. Water velocity ranged from <1-20 cm/sec with a mean of 4.7 (sd = 4.3). This flow simulated the continual current experienced by mussels in a natural riverine setting. Within these troughs, 50 newly transformed juvenile mussels were placed in each 75 x 50 x 50 mm rectangular glass container (during 1993) or 75 x 75 x 33 mm plastic container (during 1994 and 1995). Each of these containers was initially set up with one of two substrate types based on particle sizes. Sieves were used to obtain substrates with two particle size distributions, <120 um and between 120 and 600 um. The two substrates were tested for suitability in rearing juvenile rainbow mussels. In addition, two depths of each substrate were tested, 5 mm and 20 mm. Raw substrate was collected from the Clinch River immediately upstream of the CRSP for the 1993 and 1995 trials and from the Clinch River at Nash Ford for the 1994 trial. The use of river sub- strate provided a natural flora and meiofauna for the culture system. Since the native flora and meiofauna was a desired component of the culture substrate, heat or chemical treat- ments that would kill any potential predators and competi- tors were not used. Once the juveniles were settled in the substrate, the maintenance of a continuous current ensured that the mussels were exposed to a constant supply of sus- pended food and fresh water. Juvenile growth and survival were assessed at the end of the growing season during 1993 (June-October) and 1994 (June-September for batch 1, September-November for batch 2), and at 30, 74, and 94 days during the 1995 trial (August-October). The sampled dishes were removed from the culture troughs and brought back to the Virginia Tech Aquaculture Center to be sampled. The substrate from the dishes was washed through a sieve, leaving the juvenile mus- sels and empty shells. During the 1995 trial, the substrate was sieved and searched in two fractions, the loose flocculent top layer and the more consolidated substrate underneath. The collected juveniles were counted, and the lengths and NATURAL RIVER WATER FLOW-THROUGH UNIONID CULTURE SYSTEM 17 oa A inlet fl U-shaped settling trough outlets standpipes culture dishes oval trough (11) \ center island axle paddlewheel settling trough Figure 1. A, Diagram of the natural river water culture system; juvenile rainbow mussels were placed in 56 cm” dishes in the oval troughs, 16 dishes per trough; the electric motor turned paddlewheels to establish a unidirectional flow; river water continuously flowed through the troughs from the settling trough and out standpipes. B, Cross-sectional view of natural river water culture system; a constant flow of river water was delivered from the settling trough and water levels in the oval culture troughs were maintained by the standpipes; culture dishes were not placed under the paddlewheel, due to turbulence. determine whether juveniles had died or es- caped and examined to estimate the time of mortality based on size or number of growth rings. Comparison of the effect of substrate size, substrate depth, and year on survival and growth was conducted using the t-test procedure with a = 0.05. The ANOVA pro- cedure with a = 0.05 was used to assess the effect of time on juvenile survival during the 1995 trial. A second experiment to investigate the effect of temperature on the growth and sur- vival of juvenile mussels was conducted in environmental chambers in the laboratory using 570 | Living Streams fitted with chiller units (Frigid Units, Inc., Toledo, Ohio) that provided a unidirectional flow, aerated wa- ter, and controlled temperature (Fig. 2). Heaters were placed in one stream to main- tain a constant temperature of 25°C. The bottom of each stream was lined with washed pea-sized gravel to provide a sub- stratum for natural algal and bacterial growth to occur. The streams were filled with Clinch River water approximately 2 weeks before the initiation of the experiment to allow colonization of natural flora. The three streams were maintained at 12°C, 18°C, and 25°C, to simulate late fall/early spring, mid-spring, and summer river tem- peratures, respectively. Light regime for all three streams was 12 hr light and 12 hr dark with the exception that the 25°C stream was covered with opaque foam approximately half of the days to reduce the growth of fila- mentous algae in the culture dishes. Juve- niles were placed in these streams in 75 x 75 x 33 mm plastic containers filled to a depth of 10 mm with fine sediment (<120um). The 18 containers per treatment were placed in the Living Streams with 50 juvenile mussels per container. Subsamples were taken at 30 and 60 days thereafter to determine growth and survival of juveniles at each tempera- ture. Water that evaporated was replaced with distilled deionized water, and the ex- periment was terminated after 60 days. Sta- tistical comparisons of the effects of tem- widths of a subset of 10-15 living juveniles, or of the entire — perature on growth and survival were performed using set if less than 10, were measured using an ocular microm- | ANOVA with a = 0.05. eter in a Zeiss binocular dissecting microscope. The remain- The effect of stocking density on the survival and ing empty shells from mussels that died were counted to growth of juvenile rainbow mussels was tested using an ad- 18 AMERICAN MALACOLOGICAL BULLETIN Chiller unit Mesh dividers 4 Living Stream Water li Flow. 19 * 1/2 + 2004 Figure 2. Diagram of the controlled tem- perature experimental apparatus. Each of three systems was operated at a different temperature, 12°C, 18°C, or 25°C. Juvenile rainbow mussels were held in 18 dishes per system at a density of 50 mussels per 56 cm’. Dishes were filled with fine sediment (<120um) to a depth of 10 mm prior to introduction of juveniles. ee = — a ae a ae i OEE False bottom Impeller ditional laboratory recirculating system constructed to simu- late the Living Stream, generating a unidirectional current by pumping water from underneath a false bottom (Fig. 3). In this system, a false bottom was fitted into a 90 | tank with approximately 2 cm between the downstream end of the false bottom and the end of the tank. A small, external water pump was placed on the wall of the tank to circulate the water. Recirculated water was dispersed at the surface of the tank by means of a distribution header, consisting of a tank- wide, 12.5 mm diameter PVC pipe with drilled holes. Acid- washed rectangular plastic containers were filled with fine sediment (<120 tm) to a depth of 10-15 mm. Three sizes of Pump __-—Pump outlet hose Pump inlet hose _Water distribution header False bottom culture dishes culture dishes containers were used, 75 x 75 mm, 130 x 75 mm, and 130 x 130 mm. Photoperiod was fixed at 14 hr light and 10 hr dark. At the start of the experiment, 6 replicates of each size dish, with 100 juveniles in each, were placed in the culture tank. The tank was filled with an equal mix of conditioned municipal water and well water to obtain a total hardness of 200 mg/l of CaCO. The juveniles fed on the natural flora and detritus that developed in the sediment because no regu- lar supplements of algae were provided to the system. Water temperatures ranged from 22°C to 27°C and were allowed to follow ambient room temperature because it remained suit- able for juvenile mussel culture. The experiment was allowed Figure 3. Diagram of the culture density experimental apparatus. One hundred ju- venile rainbow mussels were placed in 6 replicate dishes of 3 sizes to achieve den- sities of 1.8, 1.0, and 0.6 juveniles per cm?. Water line NATURAL RIVER WATER FLOW-THROUGH UNIONID CULTURE SYSTEM 19 to run for 90 days; then containers were removed from the system, juveniles were sieved from the sediment, and growth and survival were determined. Statistical comparisons of the effects of stocking density on growth and survival were per- formed using ANOVA with a = 0.05. Comparisons among growth data from our study and others were based on cumulative degree-days. Fish hatchery managers have used this concept for predicting growth over the range of water temperatures in their facilities (Piper et al. 1982). The Monthly Temperature Units used in hatchery management are based on the average monthly water tem- perature above 0°C. For our study, comparisons were based on the cumulative degree-days above 15°C, a temperature below which juvenile mussels apparently do not grow ap- preciably. Regression analysis was used to determine the ex- pected relationship between cumulative degree-days above 15°C and length. Outliers were excluded from the calcula- tion of the regression equation based on residuals analysis (p < 0.05). All statistical tests were performed using the statistical analysis software Statistica® (StatSoft 1998). Statistical sig- nificance levels were set at a = 0.05 unless otherwise noted. RESULTS Growth and survival in natural river water flow-through culture system During the 1993 trial, mean survival rate for juveniles was 24.3% and 21.8% to 115 days in the fine and coarse substrate, respectively (Table 1, Fig. 4). The initial sizes of juveniles were not measured, but all individuals were ran- domly taken from the same batch of newly transformed juveniles. Mean lengths of juveniles were marginally differ- ent between the fine and coarse substrates (p < 0.10), with juveniles reaching 2.22 mm (sd = 0.24) and 1.97 mm (sd = 0.35), respectively (Fig. 5). The variation in survival was high for the two treatments, 3-37% for the fine substrate and Table 1. Effects of daily mean temperature on the growth and survival of juvenile rainbow mussels (Villosa iris) held in the culture systems at the Clinch River Power Plant in Carbo, Virginia. Number of days Percent Survival + SE 1993 1994a 1994b Rearing Trial 1995 Figure 4. Mean percent survival per container of juvenile rainbow mussels (Villosa iris) for rearing trials in oval troughs. Error bars are +1 SE and those with different letters are statistically different at the a = 0.05 level. 1-50% for the coarse substrate. However, the fine substrate treatment exhibited more consistent survival rates. Depth of substrate had no effect on survival or growth. Asian clams were found in the dishes at the end of the experiment, with a mean of 8 clams per dish in the large substrate and 2 clams per dish in the fine substrate. Two batches of juvenile mussels were cultured in 1994. The first batch of juveniles (mean initial length = 0.29 + 0.01 mm) yielded a mean survival rate of 3% to 112 days (Table 1, Fig. 4), with one container having 17% survival. The sur- vival rate of juveniles to 93-100 days during the second batch (mean initial length = 0.40 + 0.05 mm) was very low, less than 1% (Table 1, Fig. 4). The empty shells in the containers indicated that death occurred sometime between 14-20 days old, based on the number of fine growth lines. Neither sub- strate size nor depth had an effect on juvenile growth or survival. The 1995 culture trial was sampled at intervals to determine whether mortality oc- curred early or was evenly distributed during the approximately 100-day culture trial. The overall mean survival rate to 94 days was 21.1% for the four substrate treatments combined (Table 1, Fig. 4). Juvenile survival with mean : ; temperature Initial length Final length Percent '0 30 days Nes 40.8% in the fine sediment, Year above 15°C (mm) + SD (n) (mm) + SD (n) survival the only sediment size included in the inter- val sampling. The sample at 74 days yielded 1993 119 NA 2.10 + 0.62 (18) 27.5 a 17.3% survival rate in the fine substrate ee psoas) istsgatie) 22° Ghd conned, hint 1995 79 0.77 + 0.08 (51) 194 number of juveniles. When calculated from 0.25 + 0.02 (20) day 30 to day 74, the juveniles exhibited a 20 AMERICAN MALACOLOGICAL BULLETIN 26 ) | L a J ba] 2.0 7 4) b £ = “1.0 + 4 x =) = Cc (; (2i-n—-1)X; i=] (1-1) > X, i=] G= Here nm equals the number of individual snails and X, is the size of the ith snail when they are sorted from smallest to largest. RESULTS There was a shift from no size hierarchy for Potamopyr- gus antipodarum grown at no-competition to a strong size hierarchy for P. antipodarum grown with-competition (Fig. 3). The no-competition G was 0.251 and the mean with- competition G was 0.461 (95% upper and lower CI = 0.458, 0.465; min. = 0.286; max. = 0.650), which was significantly greater than the no-competition G (one-tailed t test for dif- ferences in means; N = 1000; t = 135.36; p < 0.000). The distribution of growth rates of P. antipodarum at no- competition was normally distributed (N = 20; Shapiro- Wilk W = 0.967; p = 0.696) but those grown with- competition were right-skewed and non-normally distributed (N = 200; Shapiro-Wilk W = 0.852; p < 0.000) (Fig. 3). Individuals of P. antipodarum grown at no- competition (mean growth = 0.338 mm/month, std. dev. = 0.146) grew significantly more (non-parametric Kol- mogoroy-Smirnoy t-test, p < 0.005) than those grown with- competition (median growth = 0.100 mm/month, 25% quartile = 0.050, 75% quartile = 0.200). DISCUSSION Intraspecific competition for limited food resources caused decreased growth of Potamopyrgus antipodarum reared in the laboratory. One snail in our 22 cm” tube trans- lates to roughly 455 snails/ m* and 10-snails/tube translates 36 AMERICAN MALACOLOGICAL BULLETIN 19+ 1/2 + 2004 50 50 | 1-snail/tube 10-sn 25 Percent individuals NO on | 0 L 1.0 0 growth (mm/month) Figure 3. Growth (mm/month) of individuals of Potamopyrgus antipodarum with no competition (1 snail/tube; N N = 200). 20) and with intraspecific competiti to roughly 4555 snails/m’, fairly low compared to naturally- occurring densities of P. antipodarum, which are often be- tween 20,000 and 40,000/m* and occasionally exceed 500,000/m° in rivers in the western USA. Obviously, other environmental and ecological factors affect populations of P. antipodarum in waters in the western USA. It is likely, how- ever, that intraspecific competition occurs in waters infested with P. antipodarum, particularly in late autumn and early winter when primary production is reduced and populations of P. antipodarum are large. Intraspecific competition may partially explain why many researchers report marked de- creases in P. antipodarum densities and a scarcity of smaller individuals in winter (Dianne Shinn, pers. comm., David Richards, pers. comm., Billie Kerans and Chelsea Cada, pers. comm., and Daniel Gustafson, pers. comm.). We have shown that in a freshwater spring with fairly constant year- round temperatures (approx. 14°C) and flow rates that den- sities of P. antipodarum also decrease in autumn and winter, which indicates that temperature and flow are not entirely responsible (Richards et al. 2001). In this experiment, asymmetric competition resulted in a well-defined size hierarchy. These results suggest that size hierarchies in populations of Potamopyrgus antipodarum in the western USA may not be entirely based on age class. Because asymmetric competition affects smaller individuals more than larger individuals, it might also result in repro- ductive hierarchies. Previously we found that in several riv- ers in the western USA, larger individuals of P. antipodarum produce more em- bryos; snails <3.00 mm did not repro- duce at all (Richards et al. 2000). Repro- ductive hierarchies could result in self- thinning populations and/or dominant and suppressed size classes (Ford 1975, Ford and Diggle 1981). We do not know if the effects of inequality in individual reproductive output are greater than the effects of their inequality in size. Even though populations of P. antipodarum in the western USA are clonal, reproductive hierarchies could increase the proportion of genes represented by the larger, more fecund individuals in future generations (Heywood 1986, Damgaard and Weiner 2000). ails/tube 5 = ACKNOWLEDGMENTS on (10 snails/tube; We thank the following for their critical review of this manuscript: David K. Weaver, Billie L. Kerans, Gary T. Lester, Chelsea Cada, Silvia Murcia, Leah Steinbach, and Julianne Zickovich. We also thank Yuliya German for her excellent laboratory assistance and John Borkowski for his helpful statistical advice and development of our Gini coef- ficient program for SAS. LITERATURE CITED Adams, E. S. and W. R. Tschinkel.1995. Density-dependent com- petition in fire ants: Effects on colony survivorship and size variation. Journal of Animal Ecology 64: 315-324. Begon, M. 1984. Density and individual fitness: Asymmetric com- petition. In: B. Shorrocks ed., Evolutionary Ecology, Blackwell, Oxford, Pp. 175-194. Begon, M., J. L. Harper, and C. R. Townsend. 1996. Ecology. 3™¢ Edition. Blackwell, Oxford. Borkowski, J. 2002. Gini. SAS Program. Department of Mathemat- ics, Montana State University, Bozeman, Montana. Bowler, P. A. 1991. The rapid spread of the freshwater hydrobiid snail Potamopyrgus antipodarum (Gray) in the Middle Snake River, southern Idaho. Proceedings of the Desert Fishes Council 21: 173-182. Branch, G. M. 1975. Intraspecific competition in Patella cochlear Born. 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Accepted: 26 October 2003 = AMERICAN MALACOLOGICAL BULLETIN 19° 1/2 + 2004 Behavior, morphology, and the coexistence of two pulmonate snails with molluscivorous fish: A comparative approach* Christina M. Mower! and Andrew M. Turner Department of Biology, Clarion University, Clarion, Pennsylvania 16214, U.S.A., aturner@clarion.edu Abstract: We conducted field, mesocosm, and laboratory studies to compare the behavioral and morphological defenses of two pulmonate snails, Physa acuta and Stagnicola elodes, against predation by pumpkinseed sunfish, Lepomis gibbosus. Field surveys showed that P. acuta occurred in most ponds and lakes in the study region, including those with and without fish. In contrast, S. elodes had a restricted habitat distribution, as it was found only in small ponds without fish. Behavioral plasticity of these two species was evaluated by monitoring their habitat use in mesocosms with and without caged pumpkinseed sunfish. Pumpkinseed sunfish induced a strong habitat shift by P. acuta, but S. elodes did not alter habitat use in the presence of caged pumpkinseeds. Stagnicola elodes has a stronger shell and a larger adult body size than does P. acuta. Handling time experiments showed that pumpkinseed sunfish had more difficulty consuming S. elodes than P. acuta. Despite the endowment of S. elodes with a relatively large, strong shell, it suffered higher mortality than did P. acuta in predation trials with pumpkinseed sunfish. However, the advantage of P. acuta disappeared when covered substrates were removed from the pools, showing that P. acuta rely on habitat shifts to minimize encounters with fish predators. Taken together, these results suggest that the mechanisms allowing P. acuta but not S. elodes to coexist with fish predators are largely behavioral rather than morphological. Key words: anti-predator behavior, chemical cues, Physa, predation, Stagnicola Understanding the mechanisms that underlie shifts in species composition along environmental gradients remains a central challenge of community ecology. The principal gradient in lentic freshwater systems is the environmental continuum from small, temporary ponds lacking fish to deep, permanent lakes containing fish. Freshwater snails, like most aquatic taxa, have characteristic species assemblages over different portions of this gradient (Pip 1986, Jokinen 1987, Dillon 2000). Although the mechanisms responsible for shifts in snail assemblages across this gradient are not well understood, work on other aquatic taxa has highlighted the role of predation in producing predictable patterns of species replacement along the size gradient of ponds (Well- born et al. 1996, Skelly 1997, McPeek 1998). Very simply, smaller ponds tend to be temporary and lack a well devel- oped predator assemblage. Traits that confer protection against the predators of larger ponds and lakes often incur costs that put the bearer of those traits at a disadvantage in small ponds lacking predators or containing different sorts of predators (e. g., Wissinger et al. 1999, Wellborn 2002). Although a large number of studies have produced shopping lists of traits that protect prey against predation, these stud- ' Current address: REI Consultants, PO Box 789, Cool Ridge, West Virginia 25825, U.S.A. ies typically focus on single traits in isolation, and they often fail to link traits to patterns of co-occurrence with predators in the field (Relyea 2004). Thus, while we know what sorts of traits may be used to deal with predators, we have a poor understanding of which sorts of traits are actually most im- portant in allowing prey to coexist with predators and thus drive the process of species replacement. Among the most important predators of pulmonate snails in lakes and ponds are molluscivorous fish (Bronmark et al. 1992, Martin et al. 1992, Bronmark 1994). Molluscivo- rous fish are most numerous in deeper, permanent ponds and lakes and are usually absent from shallow temporary ponds (Lodge et al. 1987). Pulmonate snails have a variety of adaptations that facilitate coexistence with fish predators. Some taxa possess relatively large, thick shells. These taxa are less preferred by fish than are thin-shelled taxa (Stein ef al. 1984, Osenberg and Mittelbach 1989, French and Morgan 1995). Other taxa employ inducible anti-predator defenses. For example, field studies show pulmonates use safe micro- habitats (covered substrates and shallow marginal areas) to a greater degree in lakes and ponds containing fish than in fishless ponds (Turner 1996, Bernot and Turner 2001) and that habitat use is a phenotypically plastic trait. Pulmonates use chemical cues to detect predators (Snyder 1967, Alex- ander and Covich 1991, Covich et al. 1994) and their be- *From the symposium “The Biology and Conservation of Freshwater Gastropods” presented at the annual meeting of the American Malacological Society, held 3-7 August 2002 in Charleston, South Carolina, USA. 40 AMERICAN MALACOLOGICAL BULLETIN havioral responses depend on the type of predator and their spatial and temporal proximity to the predator (Turner ef al. 1999, McCarthy and Fisher 2000, Turner et al. 2000, Bernot and Turner 2001, Turner and Montgomery 2003). Pulmo- nates also respond to predators by altering their reproduc- tive effort (Crowl and Covich 1990, Chase 1999), morphol- ogy (DeWitt 1998), and growth rates (Turner 1997, 2003). In this paper we compare the behavioral and morpho- logical defenses of two species of pulmonate snails, Stag- nicola elodes (Say 1821), synonymous with Stagnicola palus- tris (Miller 1774), and Physa acuta (Draparnaud 1805), synonymous with Physa integra (Haldeman, 1841) and Physa heterostropha (Say, 1817), (Dillon et al. 2002). We present survey data showing that S. elodes is confined to small ponds that lack fish. P. acuta is also found in these same fishless ponds, but it occurs in ponds with fish as well. We hypothesize that P. acuta, but not S. elodes, possess mor- phological or behavioral adaptations that allow coexistence with fish predators. By comparing the antipredator adapta- tions of the two species, we hope to gain some insight into the way in which traits of individuals drive patterns of spe- cies replacement along the size gradient of ponds. METHODS Mollusc surveys We present data extracted from an ongoing survey of snail assemblages of northwest Pennsylvania. We chose to focus our study on Physa acuta and Stagnicola elodes because each often dominates the snail assemblage of ponds in which they occur; more than 90% of the local lakes and ponds contain either P. acuta or S. elodes. Between 1997 and 2002 we sampled snails from 56 lakes and ponds in northwest Pennsylvania, encompassing a wide range of surface areas, depths, and hydroperiods. Sampling was generally con- ducted in May or early June. A D-frame sweep net was used to collect snails, and each pond was represented by at least 20 sweeps. The presence or absence of fish in a pond was as- certained through seining, dipnetting, minnow traps, or a combination thereof. Behavioral plasticity We assessed the relative behavioral responses of Physa acuta and Stagnicola elodes to predation risk by comparing their habitat use in a mesocosm experiment with and with- out caged pumpkinseed sunfish (Lepomis gibbosus). The pumpkinseed sunfish is a specialized molluscivore (Wain- wright 1996, Huckins 1997), can have substantial effects on snail populations (Brénmark et al. 1992), and is ubiquitous in the smaller lakes and ponds of northeastern North America (Scott and Crossman 1973, Lee et al. 1980). Sixteen polyethylene pools (525 1, 1.4 m° surface area) were placed 19° 1/2 * 2004 outdoors at the Pymatuning Laboratory of Ecology in north- west Pennsylvania and were filled with water from Pyma- tuning Reservoir. A square piece of corrugated vinyl (60 cm x 60 cm), raised off the bottom by legs that were 4 cm tall, was added to each pool to serve as habitat structure. Eight randomly selected pools were each stocked with 10 adult P. acuta; the other eight were stocked with 10 adult S. elodes. Both species were collected from an abandoned segment of the Erie Extension Canal near Linesville, Pennsylvania. The sampled reach lacked fish. The species identity treatment was crossfactored with a predation risk treatment. “Fish present” pools received one pumpkinseed sunfish, confined to a cylindrical mesh cage (25 cm diameter x 75 cm length, 1-mm mesh). The mesh cage prevented pumpkinseeds from feeding on experimental snails, but allowed chemical odors emanating from the fish and their prey to disperse through the pool. Each day, caged pumpkinseeds were fed four Physa acuta or Stagnicola elodes, depending on the species treatment to which they were as- signed to. “Fish absent” pools received empty cages. Censuses of habitat use were conducted daily for seven days. We recorded the number of snails occupying near- surface habitats (within 2.5 cm of the water’s surface, or above the water’s surface), the number under the covered substrate, and the number remaining in other portions of the pool. Because earlier studies with Physa acuta show that they respond to fish predators both by moving under cover and by crawling to the water’s surface (Turner 1997), we defined refuge use as the summed proportion of snails using these two habitats. The independent and interactive effects of predation risk and species identity on refuge use were analyzed with a 2 x 2 ANOVA. Preliminary analysis with repeated-measurements ANOVA showed no day-of- experiment effects or interactions of day-of-experiment ef- fects with predator or species effects, so the analyses pre- sented here were conducted on pool means (averaged over the seven day experiment). Shell strength The vulnerability of snails to pumpkinseed sunfish is largely a function of shell strength and body size (Osenberg and Mittelbach 1989). We compared the vulnerability of Physa acuta and Stagnicola elodes to pumpkinseed sunfish by measuring the force necessary to crush the shells of 45 ran- domly selected individuals of each species. Crush resistance was measured by placing a snail at the bottom of an upright cylinder (8 cm diameter) and inserting a piston in the cyl- inder and on top of the snail. The snail’s shell was then subjected to a gradual increase in force by slowly filling the piston with sand until the shell failed. We positioned the snail such that the crushing force was oriented across the shortest shell dimension (dorsal to ventral), thereby best BEHAVIOR, MORPHOLOGY, AND COEXISTENCE WITH PREDATORS 4] mimicking the feeding mode of molluscivorous fish (Wain- wright 1996). The mass of the sand and piston was converted to Newtons, and using body mass as a covariate, we used ANCOVA to test whether size-adjusted shell strength dif- fered between the two species. Studies show that shell strength, measured in this manner, is highly correlated with the time pumpkinseed sunfish spend handling prey (Osen- berg and Mittelbach 1989, Huckins 1997). The ability of gape-limited predators to ingest and crush snails depends on the snail’s body size as well as shell strength (Osenberg and Mittelbach 1989, Nystrém and Perez 1998), so we conducted foraging trials with pumpkinseeds feeding on snails and recorded handling times. Handling times are a good measure of the costs associated with for- aging. Snails to be fed to pumpkinseeds were gathered from a local pond in late June; thus their size distributions re- flected natural patterns. Eight pumpkinseeds, 120-130 mm standard length (SL), were fed Physa acuta and Stagnicola elodes. We recorded the time from ingestion of the snail to shell failure, which was marked by an audible sound. Han- dling times represented the average of approximately 100 individual trials for each species. Predation trials We evaluated the relative vulnerability of Physa acuta and Stagnicola elodes to pumpkinseed sunfish by conducting predation trials that encompassed both the encounter and handling phases of the predation process. To assess the po- tential role of behavioral defenses in minimizing encounter rates and thus ameliorating predation risks, we conducted trials with and without covered substrates present. The ma- nipulation of habitat structure allowed us to assess the role of behavioral defenses and was an effective method of par- titioning the relative roles of behavioral and morphological defenses. Trials were conducted in ten 525 | mesocosms placed outdoors at the Pymatuning Laboratory of Ecology. Each mesocosm was stocked with 10 P. acuta and 10 S. elodes. Half of the ten mesocosms contained a covered sub- strate in the form of a ceramic tile (20 cm square) elevated above the bottom on 2.5 cm tall legs and covering 3% of the pool bottom. One pumpkinseed (120-130 mm SL) was added to each mesocosm, where it was initially confined to a mesh-enclosed cage (30 x 30 x 30 cm). Caged pumpkin- seeds were fed four snails daily for seven days (equally di- vided between S. elodes and P. acuta), thereby acclimating the fish to feeding on snails, and acclimating experimental snails to predation risk. After one week, the pumpkinseed was liberated and allowed to forage for four hours. Counts of snail survivorship were conducted after four hours of pump- kinseed foraging. Because both species were stocked into pools and pumpkinseeds were allowed to choose between them, their probabilities of survivorship were not indepen- dent of each other. Therefore, we analyzed survivorship data by first calculating the mortality rate of S. elodes relative to P. acuta (number of S. elodes eaten/[number of S. elodes eaten + number of P. acuta eaten]). Because the two species were stocked at the same density, this was equivalent to Manly’s index of electivity, which has a value of 0.5 when there is equal preference for the two species (Manly et al. 1972, Chesson 1978). Values greater than 0.5 indicated a prefer- ence for S. elodes and values less than 0.5 indicated that S. elodes were avoided. We then used one-way ANOVA to test for the effect of habitat structure on pumpkinseed electivity. RESULTS Mollusc surveys Field surveys show that Physa acuta occupies a broad range of habitats, whereas the distribution of Stagnicola elodes is quite restricted. Of 56 ponds and lakes surveyed to date, P. acuta was found in 48, but S. elodes occurred in just 5. Physa acuta was found both with and without fish, whereas S. elodes was only found in ponds generally lacking fish (repeated surveys of these ponds have yielded the occa- sional mudminnow, Umbra lima). Each of the five fishless ponds with S. elodes become dry by late summer in most years, but are shaded enough by trees that their soils retain some moisture (A. Turner, pers. obs.). Four of the five tem- porary ponds with S. elodes also contained P. acuta, the fifth contained its congener Physa gyrina (Lea 1838)—we distin- guished P. gyrina from P. acuta by examining penile mor- phology. Both P. acuta and S. elodes could become quite abundant and dominate the snail assemblage of a pond. Sweep net samples taken in early spring, and thus comprised only of overwintering adults, showed that the density of P. acuta could exceed 10 snails per sweep. S. elodes densities could reach two snails per sweep (approximately 0.25 m° sampled per sweep). Behavioral plasticity Pumpkinseeds had a strong effect on habitat use of snails, but these effects were species specific (Fig. 1). Indi- viduals of Physa integra increased their use of covered sub- strates from 41% to 92% when confronted with caged pumpkinseeds. In the absence of pumpkinseeds, covered habitat use by Stagnicola elodes was 42%, but increased to just 47% in the presence of pumpkinseeds, illustrating the differing behavioral responses of the two species to preda- tion risk (species x predation risk interaction: F, ,, = 24.3, P < 0.001). Shell strength Body size, measured as dry mass of soft body parts, had a significant effect on shell strength (Table 1); the slope of 42 AMERICAN MALACOLOGICAL BULLETIN 19+ 1/2 * 2004 1.0 10 (<= Fish absent sae e 6S. elodes wm Fish present e oP. acuta 0.8 oO 84 ° 3 2 ® > 064 Se i) an 3 3 aD) 0.4 oO 4-4 of = ” 02 | ee fle op) 0.0 0 T T T T T T T 0 5 10 1S 20 25 30 35 40 S.elodes _P.. acuta Dry mass, soft tissue (mg) Figure 1. Refuge use by Physa acuta and Stagnicola elodes in the presence and absence of caged pumpkinseed sunfish. Refuge use is Figure 2. Force required to crush the shells of Stagnicola elodes and the summed proportion of snails using covered substrates and near-surface habitats and is averaged over seven days. Vertical bar represents one standard error, n = 4 mesocosms per treatment combination. the relationship was remarkably similar for the two species (homogeneity of slopes test, P > 0.20; Fig. 2). However, species identity did have a significant effect on size-adjusted shell strength (Table 1). Shells of Physa acuta, adjusted for size, failed at an average of 3.18 + 0.16 (+ SE) N, whereas Stagnicola elodes shells of the same size failed at 3.84 + 0.17 N, an increase of 21%. The trials to measure handling time confirmed that pump- kinseeds have more difficulty feeding on Stagnicola elodes than on Physa acuta. Handling time for P. acuta averaged 4.97 s, and handling time for pumpkinseeds feeding on S. elodes averaged 9.14 s (species effect in one-way ANOVA: P < 0.01). This difference may have been due in part to dif- ferences in the body sizes of the two species: shell height of P. acuta averaged 9.80 mm, whereas the shell height of S. elodes collected from the same pond averaged 19.3 mm. Predation trials In the absence of habitat structure, Physa acuta and Stagnicola elodes survived predation by pumpkinseeds at a Table 1. ANCOVA testing the effects of body size and species iden- tity on shell strength. Model R* = 0.36 Source of variation SS df MS F P Body size 44.69 it 44.69 34.2 <0.00001 Species identity 9.68 l 9.68 7.4 0.0078 Error 117.59 90 1.31 Physa acuta as a function of snail body size. Body size is dry mass of soft body parts (excluding shell material). See Table 1 for analysis of size and species effects. similar rate (Fig. 3). Manly’s index of electivity for pump- kinseeds foraging on S. elodes was 0.47 + 0.04 (+ SE), not significantly different from the equal preference value of 0.50. In pools containing habitat structure, however, survi- vorship of P. acuta was elevated more than 3-fold over pools without structure, whereas S. elodes survivorship was virtu- ally unchanged (Fig. 3). Thus, adding habitat structure re- sulted in S. elodes suffering much higher mortality than P. acuta. Manly’s index of electivity for pumpkinseeds feeding 100 [== refuge absent mums refuge present Ce o — 2 ‘cc 60 4 n — S = 40 4 ra 3 0p) 20 7 0 S. elodes P. acuta Figure 3. Survivorship of Stagnicla elodes and Physa acuta in ex- perimental mesocosms when fed upon by pumpkinseed sunfish in the absence and presence of a covered substrate. Vertical bar rep- resents one standard error, n = 8 mesocosms per treatment combination. BEHAVIOR, MORPHOLOGY, AND COEXISTENCE WITH PREDATORS 43 on S. elodes was 0.83 + 0.07, showing strong preference (habitat structure effect on preference: F, 4 = 16.3, P < 0.01). DISCUSSION A number of malacologial studies have sought to link the degree of shell development with the ability to coexist with shell-crushing predators (Vermeij and Covich 1978, Palmer 1979, West et al. 1991). However, a naive malacolo- gist approaching the freshwater habitat gradient and armed only with a knowledge of relative shell strength and predator distributions would fail to correctly predict the habitat dis- tributions of Stagnicola elodes and Physa acuta. Stagnicola elodes has a relatively robust shell but occurs only in envi- ronments largely lacking in shell-crushing predators, whereas P. acuta, with its small thin shell, is most dominant in environments with abundant shell-crushing predators. Only knowledge of the relative behavioral flexibility of these two species allows an accurate prediction of their habitat distributions. In other aquatic taxa, the predictable pattern of species replacement along the pond-size continuum is driven in large part by shifts in the identity of the top predator and tradeoffs involving traits conferring safety against predators (Bendell 1986, Skelly 1997, McPeek 1998). Unfortunately, almost nothing is known about the potential role of such mechanisms in structuring assemblages of snails. Descriptive studies aimed at detecting patterns in the species composi- tion of assemblages of freshwater snails and identifying structuring mechanisms have traditionally focused on the importance of abiotic factors (Okland 1983, Bronmark 1985, Pip 1986, Jokinen 1987). On the other hand, a number of experimental field studies have tested the effects of predators on populations of snails (e.g., Bronmark et al. 1992, Martin et al. 1992, Osenberg et al. 1992, Lodge et al. 1994), but these studies have focused on regulations of populations within lakes, not patterns of replacement among lakes. These results are consistent with the idea that fish pre- vent Stagnicola elodes from occupying a broader range of habitats, but other mechanisms may also be at work. Stag- nicola elodes may be resource limited in permanent ponds (e.g., Eisenberg 1970) or interspecific competition may limit their distribution. Brown (1982) examined the competitive ability of S. elodes in relation to Physa gyrina, and concluded that S. elodes was the better competitor for food resources. In addition, Brown and DeVries (1985) found that S. elodes grew faster in a pond with fish than in a pond without fish. Thus, there is no evidence in this particular case that re- sources or competition limits the distribution of S. elodes. Although this study is the first to compare carefully the traits of these two taxa, our results are consistent with earlier work. Brown (1982) also observed that Stagnicola elodes was restricted to temporary, partially wooded ponds. Brown and DeVries (1985) experimentally demonstrated that the cen- tral mudminnow Umbra limi depresses populations of S. elodes and concluded that fish predation limits their habitat distribution. Interestingly, Brown and DeVries (1985) found that U. limi was capable of feeding only on the very smallest S. elodes (<3 mm shell length), so any population regulation of S. elodes by mudminnows must occur during a short but deadly juvenile bottleneck. Snyder (1967) included S. elodes and Physa acuta in his comparative study of predator avoid- ance and found that although S. elodes showed some re- sponse to intraspecific extract, their reaction was consider- ably weaker than that of P. acuta (Snyder 1967: tables 8-9). Stagnicola elodes in Snyder’s study showed no response to pumpkinseed sunfish, whereas P. acuta responded strongly to the same cues. The fact that our study involved just two taxa limits the generality of our conclusions. There is a clear need for more comprehensive comparative studies of this sort, involving more taxa and perhaps controlling for phylogenetic effects. The available data suggest, however, that behavioral defenses may ultimately prove to play a preeminent role in driving patterns of species replacement. For example, in eastern North America, Helisoma trivolvis (Say 1817) is also broadly distributed across habitat types, and it moves to safe habitats in the presence of predators (Alexander and Covich 1991, Turner 2003). Two studies have shown that the degree of behavioral flexibility is correlated with shell strength such that thin-shelled taxa respond more strongly to predators than do thick-shelled taxa (Snyder 1967, Rundle and Bron- mark 2001). In our study region (northwest Pennsylvania) there occur several species of snails with quite stout shells, including Bithynia tentaculata (Linnaeus, 1758), Campeloma decisum (Say, 1816), and Elimia livescens (Menke, 1830). These taxa are quite restricted in their habitat distributions and are not necessarily found in habitats with abundant shell crushing predators (A. Turner, pers. obs.). Snyder (1967) performed assays of predator avoidance for each of these three thick-shelled taxa and found no evidence for behav- ioral plasticity. On the other hand, taxa with broad habitat distributions tend to be small, relatively thin-shelled taxa (e.g., Planorbidae, Physidae, Lymaeaidae). In fact, we ob- served that P. acuta and H. trivolvis are most dominant relative to other snail taxa in lakes or ponds with abundant predators (A. Turner, pers. obs.). Thus, it appears that it is the behaviorally “reactive” species that are most successful in the presence of predators. A number of studies involving molluscs and their predators have successfully linked interspecific comparisons of shell strength and vulnerability to predators (Stein ef al. 1984, Osenberg and Mittelbach 1989, West et al. 1991, 44 AMERICAN MALACOLOGICAL BULLETIN French and Morgan 1995, Brown 1998). These are short- term foraging trials conducted in aquaria. Perhaps not sur- prisingly, these studies show that prey with poorly developed morphological defenses suffer higher mortality from preda- tors than do well defended prey taxa. However, these studies have quite abbreviated spatial and temporal scales and lim- ited opportunities for prey to use refugia, and thus do not allow for the expression of behavioral or life-historical de- fenses. In addition, few studies have presented survey data in conjunction with measurements of morphology and vulner- ability to predators. It is possible that the traits allowing coexistence with predators are primarily behavioral, life- historical, or physiological, and not morphological, in which case the community ecology of a system is not predictable from measurements of morphology. Investigators have long been puzzled by the general lack of shell development in freshwater snails. With the exception of snail faunas endemic to ancient lakes and rivers, freshwa- ter snails tend to have much thinner shells than do marine snails and tend to be smaller. This pattern has been attrib- uted to differences in predation intensity: perhaps freshwater environments lack specialized shell-crushing molluscivores and contain fewer predators than do marine environments (Vermeij and Covich 1978). However, studies of aquatic food webs show that several widespread and abundant predatory taxa are in fact shell-crushing molluscivores (re- views in Lodge et al. 1987, Dillon 2000, Brown 2001), and a number of experiments now demonstrate that molluscivo- rous fish, crayfish, and other shell-crushing predators can have large effects on the population size and assemblage composition of snails (reviews in Dillon 2000, Brown 2001). In view of the growing literature showing rampant pheno- typic plasticity in the traits of pulmonate snails, the emerging picture is that freshwater snails have quite elaborate de- fenses, but that these defenses are (1) largely inducible, as opposed to constitutive and (2) behavioral, as opposed to morphological. We suggest that it is not differences in mean predation risk but instead differences in the spatial and temporal het- erogeneity of predation pressure in freshwater that may in part account for the reliance of pulmonate snails on induc- ible defenses. Phenotypic plasticity allows simultaneous ad- aptation and cost minimization across broad environmental gradients. In addition, behavioral traits are readily reversible, whereas induced shifts in shell characters are not. One would expect then to see the largest deployment of phenotypically plastic traits to prevail in environments in which predation pressure shows the most spatial and temporal heterogeneity. A full comparative analysis of the concordance of shell de- velopment, phenotypic plasticity, patterns of coexistence with predators, and gene flow will offer valuable insights. 19+ 1/2 + 2004 Finally, we echo Dillon (2000) and point out that com- munity-level studies of freshwater snails lag behind those of other aquatic taxa. This is not because the system is intrac- table. Indeed, given the relative ease with which one can measure their morphological, behavioral, life-historical, and physiological traits, estimate their abundance in the field, and conduct appropriately scaled mesocosm and field ex- periments, freshwater snails clearly present an outstanding opportunity to link mechanistically the traits of individuals to community-level patterns. We look forward with opti- mism to future studies of snail community ecology. ACKNOWLEDGEMENTS Randy Bernot, Nik Mower, Jay DePasse, Erin Kepple, Katy Shopoff, Meredith Burnett, Anita Lahr, the PLE staff, and the students of Clarion University’s ecology group as- sisted with field work and manuscript preparation. Charles Williams, Steve Harris, Roger McPherson, and Sharon Montgomery read early drafts of the manuscript. This work was supported by NSF grant IBN 9982196 to A. Turner, and is contribution number 142 of the Pymatuning Laboratory of Ecology. 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Trade-off between competitive ability and antipredator adaptation in a freshwater amphipod species complex. Ecology 83: 129-136. Wellborn, G. A., D. K. Skelly, and E. E. Werner. 1996. Mechanisms creating community structure across a freshwater habitat gra- dient. Annual Review of Ecology and Systematics 27: 337-363. West, K., A. Cohen, and M. Baron. 1991. Morphology and behavior of crabs and gastropods from Lake Tanganyika, Africa: Impli- cations for lacustrine predator-prey coevolution. Evolution 45: 589-607. Wissinger, S. A., H. H. Whiteman, G. B. Sparks, G. L. Rouse, and W.S. Brown. 1999. Foraging trade-offs along a predator- permanence gradient in subalpine wetlands. Ecology 80: 2012- 2116. Accepted: 17 November 2003 AMERICAN MALACOLOGICAL BULLETIN 19° 1/2 + 2004 Effects of pair-type and isolation time on mating interactions of a freshwater snail, Physa gyrina (Say, 1821)* Thomas M. McCarthy’ Department of Biology, University of Kentucky, 101 Morgan Building, Lexington, Kentucky 40506, U.S.A. Abstract: Behavioral tendencies during mating interactions can have important effects on mating patterns. Factors hypothesized to influence mating behavior include the degree of genetic similarity between mates and sexual motivation. This study tested for effects of these factors on mating interactions of the aquatic snail Physa gyrina. Three types of pairs were created from two populations of snails (i.e., two types of intrapopulation pairs and one type of interpopulation pair) and individuals experienced one of three isolation treatments. | recorded behavioral dynamics during mating interactions, including: escalations of mating behaviors, error frequencies, rejection behaviors, and mating frequency. Mating interactions were influenced by both the pairing treatments and isolation treatments. Additionally, there were significant interaction effects between the experimental factors on the behavioral dynamics between individuals during mating interactions. Interpopulation pairs had greater avoidance rates than intrapopulation pairs and differed from control pairs in a variety of ways. Altering the amount of isolation time individuals experienced also significantly affected the behavioral dynamics. Isolation resulted in decreased avoidance responses, with concomitant increases in the escalations of interactions and numbers of copulations. Matings occurred sooner after longer isolation periods, but also experienced higher error frequencies, e.g. misalignment during copulation attempts. These results indicate that the type of potential mate and the context in which interactions occur are important considerations when interpreting observations of mating interactions. Key words: Physa, mating behavior, mate assessment, temporal patterns, body size A major focus in behavioral ecology is the study of mate choice. Most animals do not mate indiscriminately, but pre- fer some potential mates to others (Halliday 1983, Anders- son 1994, Johnstone 1997). Tremendous effort has been made to increase our understanding of mating processes and how mate-choice decisions translate into fitness benefits (Bateson, 1983, Andersson, 1994). Hermaphroditic snails provide an excellent system for examining questions related to mating behaviors during interactions (Leonard 1991, De- Witt 1996, Wethington and Dillon 1996, Baur and Baur 1997, Wethington et al. 1999) and their fitness consequences VJarne and Delay 1990, Chen 1993, Monsutti and Perrin 1999), as well as the effects of contextual influences on both behavior and fitness (Chen 1993, Schrag et al. 1994a, 1994b, De Boer et al. 1997, Locher and Baur 2002). One factor that could potentially affect mate choice is genetic similarity (Bateson 1983). Genetic similarity between mates varies widely (Waser 1993). Individuals may encoun- ter close relatives as potential mates (Barnard and Fitzsi- mons 1988, 1989, Waldman et al. 1992) with genotypes simi- "Current address: Department of Biology, 1600 Burrstone Road, Utica College, Utica, New York 13502, U.S.A. lar to their own (e.g. siblings), or the genotypes of potential mates can differ considerably from that of the individual. Outbreeding may occur between unrelated individuals within a population or between members of different popu- lations via immigration and emigration processes. Pair-types (e.g. inbred versus outbred matings), in conjunction with migration events, might strongly impact the genetic struc- ture of a population. However, surprisingly few studies have examined how mate-choice decisions are mediated by rec- ognition of kinship or how inbreeding or outbreeding affect the fitness consequences of those decisions (Sherman et al. 1997). The contexts in which mating interactions occur also influence mating behavior and mate choice (Halliday 1983, Westneat et al. 2000) and can have important fitness impli- cations (Chen 1993, Keller et al. 1994, Pray et al. 1994, Armbruster et al. 1997, Baur and Baur 1997). Recent sexual experience may influence mating interactions. Some studies show that mate-choice preferences are clear when sexual motivation is low, but with high sexual drive (e.g. virgins or individuals with low sperm stores) choosiness regarding mates may be low, and consequently, mate-choice prefer- ences are not exhibited (Halliday 1983). That is, sexually *From the symposium “The Biology and Conservation of Freshwater Gastropods” presented at the annual meeting of the American Malacological Society, held 3-7 August 2002 in Charleston, South Carolina, USA. 48 AMERICAN MALACOLOGICAL BULLETIN motivated individuals may be less choosy about the quality of potential mates than recently mated individuals (Tomi- yama 1996). For example, Wethington et al. (1999) found no behavioral isolation between individuals from different spe- cies, but they used virgin snails in their study. Discrimina- tion or choosiness might not be expected, even between members of different species, when sexual motivation is high. However, it is also possible that changes in sexual motivation levels, and thus shifts in behavioral tendencies, might occur without significant depletion of sperm stores. Aquatic snails are an ideal model system for studying mating behavior and reproductive fitness. Physa is a genus of aquatic snails common throughout North America (Burch 1989). Individuals of Physa are often easy to collect in the field and maintain in the laboratory. Because individuals of Physa are simultaneous hermaphrodites, every mature indi- vidual, including itself, is a potential mate. Species capable of both cross- and self-fertilization are excellent systems for investigating factors that maintain outcrossing within popu- lations (Schrag et al. 1994a). Additionally, physid snails can store sperm from matings for long periods (>60 days: Weth- ington and Dillon 1991). Individuals of Physa mate readily, produce large numbers of offspring, and their mating be- havior is easily observed (DeWitt 1991). The primary goal of this study was to examine whether two factors, pair-type and isolation time, influenced mating interactions. I also considered potential mechanisms that could have led to observed differences between treatments: changes in encounter probabilities, mating behaviors, or re- jection rates. A second goal of this study was to determine whether there was evidence indicating significant interac- tions between these factors during mating encounters. These questions were addressed by observing mating interactions of aquatic snails in the laboratory. METHODS This experiment examined the effects of pair-type and isolation time (3 x 3 factorial design) on the mating inter- actions of mature individuals of Physa gyrina (Say, 1821). Snails were collected from two local populations: Buck Run Creek (BRC) in Woodford County, KY, and from a small drainage ditch running parallel to train tracks (TT) near the University of Kentucky campus. Snails were held in 38-1 aquaria, fed boiled lettuce ad libitum, and treated for 5 days with an antibiotic (Maracyn®) to minimize disease. Indi- viduals from BRC were generally larger than those from TT (mean shell length + SE: BRC = 10.92 + 0.16 mm, TT = 10.01 + 0.08 mm, t-test: f;7, = 5.20, P = 0.001) but the size ranges did overlap. Ninety snails from each population were randomly as- 19° 1/2 + 2004 signed to one of three pairing treatments: BRC pairs, TT pairs, or interpopulation pairs (BRC x TT). I marked indi- viduals’ shells with a paint pen to indicate the population of origin. I then formed snail pairs by partnering individuals that were similar in size (shell length); partners were closely matched for size and the mean proportional size differences of paired snails did not differ between pairing treatments (percent shell-length differences of partners + SE: BRC = 3.0 + 0.4%, TT = 4.0 + 0.5%, BRC x TT = 4.0 + 0.5%, ANOVA: F, 37 = 1.96, P = 0.15). Because BRC snails were generally larger than TT snails, there were significant differences in mean sizes (= average shell length of pair) of paired snails in the three pairing treatments (ANOVA: F,., = 13.14, P = 0.001), a Tukey post-hoc test indicated that BRC pairs were larger than TT and interpopulation pairs. Pairs were then assigned to one of three time treat- ments: no isolation time (0 day), one day of isolation (1 day), and three days of isolation (3 day). Individuals from pairs that were assigned to the 1- and 3-day isolation treat- ments were isolated in separate containers for the appropri- ate length of time. Snails assigned to the 0-day isolation treatment were used immediately after pair formation. That is, individuals were taken from their holding tanks, marked, assigned a partner, and then observed in the trials without any isolation time. During the experiment, paired snails (n=10 pairs per pairing*time treatment) were placed in Petri dishes contain- ing 50 ml of clean water and observed for 60 minutes. Be- havioral dynamics and interaction outcomes were recorded. DeWitt (1991, 1996) described the behavioral dynamics that occur during mating interactions: snails physically contact each other, one snail will then assume the male gender role by mounting the shell of the second individual, the “male” then crawls in circles on the shell of the “female” until in the proper alignment for attempting a copulation, the “male” will then evert the intromittent organ (the preputium), and a copulation occurs when the preputium contacts and trans- fers sperm to the gonopore of the “female” (sperm can be observed in the preputium). While these are essentially “male” behaviors, DeWitt also described a variety of rejec- tion or resistance behaviors that are gender-neutral (e.g. avoidance response—individual avoids further interaction following a contact by abruptly changing crawling direction or swinging its shell away from the other snail) or conducted by snails occupying the “female” gender role (e.g. lateral shaking of the shell or abruptly drawing shell down to substrate). I quantified the behavioral dynamics by calculating mean conditional frequencies of escalation behaviors for each pair during the observation period as follows: number of contacts per hour, number of mountings per contact, CONTEXT-DEPENDENT MATING BEHAVIOR 49 number of positioning behaviors per mounting, number of preputium eversions per mounting, number of “male er- rors” per mounting (i.e. male everts preputium when not properly aligned along aperture of female’s shell), number of copulations per preputium eversion. I also quantified resis- tance and rejection responses by calculating mean condi- tional frequencies: number of avoidance responses per num- ber of contacts, number of “female” resistance behaviors per mounting, and number of “male rejections” per mounting (i.e. male dismounts female without attempting to copulate and without resistance behaviors from the female). I compared mating behaviors between pair-type and isolation-time treatments with two-way ANCOVAs. Failure- time analyses (Fox, 1993) compared the mean elapsed times until successful copulations occurred in each treatment. Sta- tistical tests were run with SYSTAT 8.0. The ANCOVA mod- els used “mean size” and proportional “size difference” (= percent size difference in shell length) of paired individuals as covariates. The two covariates were used to account for any behavioral differences between pairs (“mean size”) or individuals within pairs (“size difference”) due to differences in body size (DeWitt 1996, DeWitt et al. 1999). I used Pear- son correlation matrices to show the nature of the effect when the covariates were significant. When the ANCOVAs indicated significant treatment effects, I used Tukey post- hoc tests to compare responses between factor-levels within treatments. If the ANCOVAs found significant treatment interaction effects, I used Fisher’s Least Significant Differ- ence (LSD) post-hoc tests and then corrected for the number of multiple comparisons of interest (18 comparisons of in- terest: | compared pairing treatments within time treatments and effects of time within pairing treatments, a ~ 0.003). Failure-time analyses, which compare the rates at which in- teractions occur, may give an indication of mating prefer- ences that are not reflected in behavioral transition rates described above. That is, a treatment factor may influence the speed at which behaviors occur and interactions escalate without affecting the mean conditional frequencies. RESULTS Both the pair-type and isolation-time treatments had significant effects on behavior. Encounter rates differed be- tween pair-types and were influenced by the isolation treat- ments (Table 1, Fig. 1A). The TT pairs had fewer contacts than the other pair-types. Encounter rates also decreased with increasing isolation time. There was no significant in- Table 1. Results of ANCOVA’s that tested for effects of pair-type and isolation time on various aspects of mating interactions in Physa gyrina. The 0-day isolation treatments were dropped from the analyses as indicated below (*) due to low preputium eversion rates. Covariates Treatment effects Mean size Size difference Pair type Isolation time — Pair*Time (df = 1) (df = 1) (df = 2) (df = 2) (df = 4) R-Squared Error df F P F P FP P F P F P. Encounter Rate Contacts/hour 0.371 19 13.73, <0.001 = 1.53 0.22 5.27. <0.01 8.79 <0.001 0.78 0.54 Behavioral Transition Rates Mounting/contact 350 79 7.11 <0.01 0.12 Oi/3"* 72:36 0.101 12.46 <0.001 1.02 0.40 Positioning/mounting 0.330 77 7.07 <0.05 1.62 0.21 3.58 <0.05 7.21 <0.001 2.71 <0.05 Preputium eversion/ mounting 0.298 77 4.37 <0.05 = 2.20 0.14 4.06 <0.05 5.81 <0.005 2.06 0.09 “Errors”/preputium* 0.382 35 0.29 0.60 6.26 <0.05 2.14 0.13 4.72 <0.05 0.56 0.58 Copulations/preputium* 0.197 35 1.20 0.28 1.20 0.28 1.44 0.25 1:97 0.17 151 0.24 Rejection Transition Rates Avoidance/contact 0.288 79 0.86 0.36 0.21 0.65 6.49 <0.005 7.77 <0.001 0.97 0.43 Resistance/mounting 0.242 A7 5.06 <0.05 0.01 0.93 ZS 0.12 3.16 <0.05 2.72 <0.05 “Male rejection”/ mounting 0.433 77 13.24 <0.001 2.97 0.09 4.79 <0.05 12.19 <0.001 3.12 <0.05 Interaction Outcome Copulation number 0.272 79 6.76 <0.05 0.01 0.96 3.44 <0.05 7.31 <0.001 0.97. 0 Copulation duration 0.157 36 0.07 0.79 0.50 0.48 0.17 0.84 0.20 0.82 0.95 0.45 * 0-day isolation treatment excluded from analysis 50 AMERICAN MALACOLOGICAL BULLETIN Avoidance Contacts / Contact / Hour # Preputium evertions / Mount Total # copulations / Hour BRC x TT BRC ay Pair type 0 Day Figure 1. Mean number (+ SE) of (A) physical contacts per hour, (B) avoidance responses per contact, (C) preputium eversions per mounting, and (D) copulations per hour between paired individuals of Physa gyrina in each pair-type (BRC: Buck Run Creek population, TT: train tracks population) and within each isolation time treatment. Bars sharing the same letter within a panel are not statistically different. 1 Day Isolation treatment 19+ 1/2 * 2004 sponses than did the intrapopulation pairs (Fig. 1B). Isolation time also significantly influenced avoidance and mounting behaviors (Table 1). Mounting behaviors increased (0-day = 0.28 + 0.03, 1-day = 0.54 + 0.07, 3-day = 0.76 + 0.09) and avoidance responses decreased (Fig. 1B) in frequency with longer isolation times. Mean size of the paired individuals was positively cor- related with mounting responses (Table 2). That is, snails in larger pairs were more likely to mount. Male positioning behaviors were signifi- cantly influenced by pair-type, isolation time, and the interaction between these factors (Table 1). Although the interpopulation pairs did not differ significantly from the intrapopu- lation pairs, the TT pairs had a significantly higher positioning frequency than did the BRC pairs in the 1-day treatment (Fig. 2A). The po- sitioning frequencies of the snails in the three pairing treatments did not differ significantly in the 3-day treatment. Again, the mean size of the paired snails was positively correlated with the frequency of positioning behaviors (Table 2). “Male rejections” (i.e. dismounting “fe- male” without attempting to copulate and without female resistance) were also signifi- cantly influenced by pair-type, isolation time, and the interaction between these factors (Table 1, Fig. 2B). “Males” in all pairing treat- ments had similar rejection patterns, such that rejection frequencies decreased if snails were isolated (a = 0.003 for multiple comparisons, BRC: 0-day > 3-day, P = 0.001, TT: 0-day > 1-day, P = 0.003, BRC x TT: 0-day > 3-day, P = 0.001). There were no significant differences between pair-types in the 0-day treatment. Al- though the interpopulation pairs did not differ significantly from the intrapopulation pairs, the TT pairs had a significantly lower “male rejec- 3 Day teraction between pair types and isolation times. However, the mean size of the individuals in the pair had a significant effect on the number of encounters (Table 1), there was a negative correlation between size and encounter rate (Table 2). Following an encounter, the probability (mean + SE) of one snail mounting its partner did not differ between the pair types: BRC pairs = 0.49 + 0.05, TT pairs = 0.57 + 0.09, BRC x TT pairs = 0.53 + 0.08. In contrast, avoidance rates differed significantly between the pair types (Table 1). Snails in the interpopulation pairs showed greater avoidance re- tion” frequency than did the BRC pairs in the 1-day treatment. The “male rejection” frequen- cies of the pairing treatments were not significantly different in the 3-day treatment. Furthermore, fewer male rejections were observed in pairs with larger individuals (Table 2). The propensity of males to attempt copulations by everting the preputium differed between pair-type and iso- lation treatments (Table 1). Snails in the BRC pairs at- tempted fewer copulations per mounting than those in the TT pairs. Additionally, snails that had not been isolated (0- day) had significantly fewer preputium eversions than did individuals that had been isolated (Fig. 1C). Mean size was CONTEXT-DEPENDENT MATING BEHAVIOR 5] Table 2. Summary of results from Pearson correlation matrices that examined the effects of covariates (“mean size” and propor- tional “size difference”) on behaviors during mating interactions. Positive values indicate a positive relationship between the covari- ate and the response variable, while negative values indicate a nega- tive relationship (ns = non-significant). Pearson correlation coefficients Size Response variable # obs. Mean size difference Contacts/Hour 90 —0.321 ns Mounting/Contact 90 0.245 ns Positioning/Mounting 88 0.179 ns Preputium eversion/Mounting 88 0.131 ns “Errors”/Preputium 43 ns —0.414 Copulations/Preputium 43 ns ns Avoidance/Contact 90 ns ns Resistance/Mounting 88 0.149 ns “Male rejection”/Mounting 88 —0.259 ns Copulation number 90 0.18 ns Copulation duration 47 ns ns positively correlated with the frequency of preputium ever- sions (Table 2). Interestingly, increasing “error” rates (Le. misalignment during preputium eversions) were observed the longer snails were isolated (Table 1: 0-day = 0.06 + 0.06 [note that 0-day treatments were omitted from the analyses due to the extremely low frequency of preputium eversions and are only included here for comparison], 1-day = 0.11 + 0.05, 3-day = 0.24 + 0.05). “Error” rates were also influenced by the proportional size differences between individuals within pairs, such that similarly sized snails had higher error rates than pairs with greater size discrepancies (Table 2). The rates of successfully copulating, given an attempt (as above, 0-day treatments were omitted), were not influenced by ei- ther pair type or isolation time (Table 1). “Female resistance” behaviors (lateral shell shaking or quickly drawing shell to substrate following a mounting by a “male”) were significantly influenced by isolation time and the interaction between pair-type and isolation time (Table 1). Females in the TT and interpopulation pairs had similar resistance patterns. The frequency of resistance behaviors by females in the BRC pairs was significantly lower than those of females in the other pairing treatments (Fig. 2C). Addi- tionally, the mean size of the paired individuals was posi- tively correlated with female resistance (Table 2). The total number of matings that occurred during the 1-hour observation period was influenced by both the pair- type and the isolation time (Table 1). Interpopulation pairs had a greater number of copulations than did BRC pairs and the number of copulations increased with longer isolation times (Fig. 1D). Copulation number was positively corre- lated with the mean size of the pairs (Table 2). Failure-time analyses indicated that matings also occurred sooner with increasing isolation time (Fig. 3). However, duration of copulation was not influenced by any of the treatments (Table 1). DISCUSSION The results of this study indicate that the context in which mating interactions occur has important implications for behavioral dynamics and interaction outcome. The ex- perimental treatments significantly affected mating interac- tions of Physa gyrina. The types of individuals present in the pairs influenced behavioral transitions during interactions. Additionally, short-term periods of isolation had strong ef- fects on mating interactions. There were also significant in- teraction effects between the pair-type and isolation-time treatments. Furthermore, both body size and the magnitude of differences in body sizes of pair mates influenced mating interactions. Pair-type treatments were constructed to examine whether the presumed genetic similarities of pair-mates would influence mating interactions. Typically, individuals will interact with others from their own population, but there may be opportunities to interact with immigrants (or emigrate themselves). | assumed that individuals within the intrapopulation pairs (i.e. BRC pairs and TT pairs) would be more similar genetically than those in interpopulation pairs (Dillon and Wethington 1995). However, it is important to note that the snails may not be assessing the degree of ge- netic similarity of potential mates, but rather some other aspect of the phenotype (e.g. environmentally influenced traits). As a whole, the results suggest that potential mates are being assessed and treated differently. Interpopulation pairs had the greatest mean number of copulations (significantly greater than BRC pairs, Fig. 1). Snails acting in the male gender role in interpopulation pairs tended to have higher frequencies of positioning behaviors and fewer “male rejec- tion” behaviors (note that the lack of significant differences between interpopulation pairs and intrapopulation pairs for these behaviors is likely due to the low a-value [a = 0.003] resulting from the corrections for multiple comparisons, e.g. test comparing positioning behaviors of BRC pairs versus BRC x TT pairs was not significant in 1-day treatments: P = 0.008). This differs from previous experiments that have demonstrated that snails prefer individuals from their own populations as mates (Baur and Baur 1992, Rupp and Wool- house 1999). In support of these studies, I also found that interpopulation pairs had the highest frequencies of avoid- ance behavior and tended to have higher frequencies of “fe- wn ie) AMERICAN MALACOLOGICAL BULLETIN # Male rejections # Positions / Mount # Female resistance 0 Day 1 Day 3 Day Isolation treatment Figure 2. Behavioral responses of individuals of Physa gyrina observed during mating interactions: (A) mean number (+ SE) of positioning behaviors per mount- ing, (B) the proportion (+ SE) of individuals acting in male gender role that rejected their partner without attempting to copulate, and (C) mean number (+ SE) of resistance behaviors per mounting by individuals acting in the female role. See text for descriptions of “male rejection” and “female resistance.” Bars sharing the same letter within a panel are not statistically different (ns = non-significant dif- ferences within isolation treatment, BRC: Buck Run Creek population, TT: train tracks population). 19+ 1/2 + 2004 ing interactions between hermaphroditic indi- viduals, especially with regards to gender allocation, choice, or conflict (e.g. Leonard 1991, DeWitt 1996, Wethington and Dillon 1996, Crowley et al. 1998, Angeloni et al. 2002, Locher and Baur 2002). The behavioral tenden- cies discussed above suggest that individuals may have asymmetrical gender-based mating preferences. In this experiment snails seemed to prefer interpopulation partners while acting in the male gender role, but also resisted inter- population partners while occupying the female gender role. The avoidance of interpopulation partners during gender-neutral situations (fol- lowing a contact) may be a mechanism to avoid becoming the “female” during an interaction. While it is beyond the scope of this study to discuss in any detail the possible mechanisms underlying population-level differences, there are several lines of evidence suggesting that the populations may have subtle differences in their mating behaviors. First, the population pairs differed significantly in contact rates (BRC > TT) and the frequency of preputium eversion (TT > BRC). Secondly, the patterns of response to isolation treatments differed between the population pairs for a number of the response variables. For instance, after one day of isola- tion the TT pairs had higher positioning fre- quencies than BRC pairs, and TT “females” also had higher frequencies of resistance than did BRC snails. However, neither of these behaviors differed significantly between intrapopulation pairs in the 0- and 3-day isolation treatments. Interestingly, this suggests that individuals from separate populations may behave similarly un- der one set of circumstances but exhibit dis- similar behavioral patterns in a slightly differ- ent context. Isolation periods were intended to ma- nipulate the sexual motivation of the snails (Wethington and Dillon 1996, De Boer et al. 1997) and might simulate low population den- sity situations in the field. As expected, there were strong temporal effects on behavior, iso- lation reduced avoidance, increased frequencies of mounting and preputium eversion, and in- creased the number of copulations. Matings oc- male resistance” (significantly greater than BRC pairs in curred sooner the longer snails were isolated (Fig. 3), but 1-day isolation treatments, Fig. 2). surprisingly there was also a greater error rate during pre- Numerous recent studies have theoretically or empiri- putium eversion. These behavioral patterns are consistent cally addressed some of the interesting complexities of mat- with the expectations for increased sexual motivation. CONTEXT-DEPENDENT MATING BEHAVIOR Proportion of mated pairs 0 10 20 30 40 50 Time (min) Figure 3. Mating distribution functions of time depicting the proportions of pairs of Physa gyrina that had mated within each isolation-time treatment. (ns = non-significant, ™ = P< 0.01). (onl QW tional levels may be caused by distinct mechanisms. For example, “male” moti- vation can be influenced by the build-up of sperm (De Boer et al. 1997), whereas “female” motivation might be influenced by the quantity or maturity of eggs. Individual body sizes and the size differences between mates were impor- tant factors in mating interactions. Theory predicts that both the size of an individual and the size of its mate should influence gender strategies during mating interactions of simultaneous hermaphro- dites (Angeloni et al. 2002). Both mating behaviors during interactions and copu- lation frequency were positively corre- lated with the mean body sizes of paired Au individuals in this study. This suggests mating effort increases with body size. Surprisingly, the proportional size differ- ences of mates were negatively correlated with errors during preputium eversion. Isolation time also had unexpected effects on behavior. First, the numbers of contacts decreased with increasing iso- lation time. The simplest explanation for this effect is that the durations of interactions increased with isolation time so that there was less time during the observation period for subsequent interactions. Second, the behavioral patterns, while often not statistically significant, suggest that isolation time might differentially influence motivational levels of the gender roles. For instance, the data indicate that “male” escalation behaviors typically increased after one day of iso- lation, which suggests elevated motivational levels. But snails acting as females generally had greater frequencies of resis- tance behaviors in 1-day treatments, which implies that “fe- male” motivational levels had not been influenced to the same degree as “male” sexual motivation. A possible expla- nation for the observed effects is that there is a trade-off between mating preferences and sexual motivation, such that, choosiness decreases as motivation increases (Halliday 1983, Tomiyama 1996). Consequently, the sexual motiva- tion of a snail (as a “male”) increased after one day of iso- lation but it was still relatively choosy (when acting as “fe- male”) about the quality of potential mates. Interestingly, resistance behaviors were not as frequent in the 3-day treat- ments suggesting that “female” motivation had increased even though it is unlikely that the snails were experiencing sperm depletion. Again, this suggests gender-differences within individuals, and the asynchrony of increased motiva- That is, closely size-matched pairs had higher error rates than pairs in which in- dividuals were more disparate in size. It is also interesting to note that as size increased the frequencies of “male” rejection decreased while those of “fe- male” resistance increased. These phenomena could be in- terpreted in a number of ways. For example, the findings might imply that as an individual’s body size increases, it is less likely to reject potential mates when occupying the male role and more likely to resist as a female. However there is an alternative explanation for these findings since paired indi- viduals were similar in size. Individuals acting in the male role may be less likely to reject large females and females may be more likely to reject large males. For instance, DeWitt (1996; also see Angeloni et al. 2002) found that smaller snails typically occupied the male gender role while larger snails acted as females. DeWitt also observed greater rejection rates when males were larger than females. This study demonstrates that both the type of individual encountered and isolation time can influence the behavioral dynamics and outcomes of mating interactions. It also illus- trates some of the gender complexities of hermaphroditic systems. There are likely to be many factors, such as migra- tion rates and population densities, influencing mating in- teractions in wild populations. Furthermore, there may be unexpected interactions between these factors. Therefore, the context-dependent nature of the interactions suggests that we must be careful about how experimental findings are related both to other laboratory studies and to field observations. 54 AMERICAN MALACOLOGICAL BULLETIN ACKNOWLEDGMENTS Discussion and comments by T. DeWitt, A. Wething- ton, A. Sih, D. Westneat, P. Crowley, E. McCarthy, and an anonymous reviewer greatly improved this work. A. Sih pro- vided statistical assistance. The numerous efforts of A. Weth- ington and R. Dillon were greatly appreciated. This paper was based on dissertation work submitted in partial fulfil- ment of the requirements for a Ph.D. from the University of Kentucky. Support was generously provided by the Graduate School and the Biology Department at the University of Kentucky, Ribble Enhancement Funds, Robert A. 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Alternative mechanisms of nonindependent mate choice. Animal Behaviour 59: 467-476. Wethington, A. R. and R. T. Dillon. 1991. Sperm storage and evi- dence for multiple insemination in a natural population of the freshwater snail, Physa. American Malacological Bulletin 9: 99- 102. Wethington, A. R. and R. T. Dillon. 1996. Gender choice and gen- der conflict in a non-reciprocally mating simultaneous her- maphrodite, the freshwater snail, Physa. Animal Behaviour 51: 1107-1118. Wethington, A. R., E. R. Eastman, and R. T. Dillon. 1999. No pre- mating reproductive isolation among populations of a simul- taneous hermaphrodite, the freshwater snail Physa. In: First Freshwater Mollusk Conservation Society Symposium, Ohio Biological Survey. Pp. 245-251. Accepted: 5 December 2003 on mn i AMERICAN MALACOLOGICAL BULLETIN 19° 1/2.° 2004 Comparative conservation ecology of pleurocerid and pulmonate gastropods of the United States” Kenneth M. Brown! and Paul D. Johnson? Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, U.S.A., kmbrown@Ilsu.edu * Tennessee Aquarium Research Institute, 5385 Red Clay Road, Cohutta, Georgia 30710, U.S.A., paul.johnson@tnari.org Abstract: To understand better the conservation status of freshwater pleurocerid gastropods in the southeastern United States, we contrasted their distribution and biology with pulmonate snails, a group far less imperiled. With 157 taxa in North America, the Pleuroceridae have a similar species richness to all pulmonate families combined (153 taxa). The Pleuroceridae has more recently extinct species (38) and 66% of the remaining taxa are considered imperiled (G Rank =2). Pleurocerid species richness is greatest in the Alabama and Tennessee River basins in the southeastern United States, with only 25 species outside the southeastern United States. We argue that the endemic nature of their distributions, limited dispersal, and poor abilities to colonize heighten chances of extinction for pleurocerids in comparison to pulmonates. We also outline our approach to conservation, which involves delineating appropriate habitat parameters for successful re-introductions using artificial propagation. We call for a national conservation strategy for pleurocerids, emphasizing threats to lotic pleurocerids and how management agencies can surmount them, similar to the strategy already developed for unionid bivalves. Key words: Re-introduction, Artificial Propagation The pleurocerids (Gastropoda: Cerithioidea) are an im- periled group of freshwater snails limited to streams and rivers in the southeastern United States. Their biology con- trasts with that of a sister taxon, pulmonate snails, which have comparatively healthy populations and are found throughout the United States. Critical differences in pleuro- cerid ecology make these snails more susceptible to extinc- tion, with recent extinctions of 38 species. Conservation of remaining populations of pleurocerids will involve assess- ment of their habitat requirements, artificial propagation, and reintroduction of populations to suitable habitats. METHODS Taxonomic nomenclature used in this manuscript fol- lows Turgeon et al. (1998). We based our assessment of the original distribution and diversity of gastropods in part on the monograph by Burch (1989), augmented with recent field data for many species in the Tennessee and Mobile River basins. Burch’s monograph includes collection data from malacologists in the nineteenth and twentieth centuries and was compiled from data originally assembled by Calvin Goodrich from 1922-1944. Burch reviewed all species’ names for synonomy and grouped taxa that were obvious examples of excessive “splitting.” We therefore consider Burch’s monograph to be the best available document to assess the original distributions of gastropods before exten- sive anthropogenic impacts. Data on the number of extinctions in each group are from Neves et al. (1997) and the conservation status of re- maining populations are primarily drawn from databases initially prepared for The Nature Conservancy and now maintained by NatureServe (2004). For designating conser- vation status of extant species, we use the G ranking system developed by Heritage/Conservation Data Center Network and The Nature Conservancy (Stern 2002). The higher the G rank a species receives, the greater the number of known populations. We consider that any G rank of two or less constitutes an imperiled species. A brief explanation of the conservation rank categories are: GX—Presumed Extinct: Species is believed to be extinct throughout its historic range. G1—Critically Imperiled: Species with 5 or fewer oc- currences, and few remaining individuals. G2—Imperiled: Species occurrences are rare (6-20) throughout the known range. G3—Vulnerable: Species typically has 21-100 occur- rences within known range. *From the symposium “The Biology and Conservation of Freshwater Gastropods” presented at the annual meeting of the American Malacological Society, held 3-7 August 2002 in Charleston, South Carolina, USA. 58 AMERICAN MALACOLOGICAL BULLETIN G4—Apparently Secure: Species is uncommon but not rare, with more than 100 occurrences in a wide geographic range. G5—Secure: Species is common with wide geographic range and abundant occurrences within range. We consider species with a GX rank to also be imper- iled. In the absence of complete field data on North Ameri- can freshwater gastropods, current information on species distribution is limited and many areas remain unsampled. Future sampling may locate species now considered extinct, especially those that are extremely rare or have small geo- graphic ranges. This exact scenario has occurred twice in the last 12 years, with the rediscovery of Tulotoma magnifica (Conrad, 1834) (Hershler et al. 1990) and Leptoxis downiei (Lea, 1843) (P. Johnson, pers. comm.), both of which were thought extinct in the Coosa River basin. The NatureServe (2004) data bases themselves were originally based on Burch’s monograph, but distributional data and G rankings for southeastern “prosobranch” species in particular have been evaluated and modified at 4 meetings of field investigators sponsored by The Nature Conservancy and the Alabama Division of Wildlife and Freshwater Fish- eries (Mirarchi et al. 2004). These databases undoubtedly contain limitations, but again are probably the best indicator we have of the current distributions and diversity of gastro- pods. For pleurocerids, distributional data from the Tennes- see and Mobile River basins are the most exhaustive and current, and information about species endemic to the Gulf Coastal drainages are the probably the most unreliable, since these populations have received less sampling effort. RESULTS Conservation status of freshwater gastropods Extinction rates in molluscs are higher than more- publicized rates in vertebrate groups like fish, birds, reptiles, or mammals. There have been 51 recent gastropod, and 34 recent unionid mussel extinctions, in comparison to 30 re- cent extinctions in the vertebrate groups combined. Of the remaining molluscan populations, a much higher percentage are imperiled than in other freshwater groups such as fish, amphibians and crayfish. Sixty percent of extant gastropod, and 49 percent of extant unionid mussel species are at risk, in comparison to values of 21-33% for the other aquatic taxa. Two families of North American freshwater gastropods, the Hydrobiidae and the Pleuroceridae, appear to be the most at risk (Table 1). Hydrobiids are currently considered to be the most diverse group of freshwater gastropods in the United States, based on recent species descriptions from springs and headwaters in the southeastern and western 19+ 1/2 + 2004 Table 1. Comparative statistics on diversity, extinctions, and con- servation status of North American freshwater “prosobranchs” and pulmonates. Data adapted and updated from NatureServe data- bases and Turgeon et al. (1998). Imperiled species are those with rankings of G2 or less. Percent Number Number of imperiled Family of species extinctions species “Prosobranchia:” Hydrobiidae 239 8 85 Neritinidae 1 0 0 Pleuroceridae 157 38 66 Pomatiopsidae 6 0 67 Viviparidae 25 0 28 Pulmonata: Ancylidae 13 0 31 Lymnaeidae 56 0 23 Physidae 40 0 23 Planorbidae A4 5 5 Valvatidae 10 0 40 Total 590 51 60 United States. The endemic nature of their distributions ex- plains why a high proportion of these populations are at risk, especially if a species has been collected at only a few sites. Pleurocerids are the second most diverse group of freshwater snails in the United States and the southeast is the center of their diversity. However, the family Pleuroceridae also has the greatest number of recently extinct species for any group of North American snails (Table 1). Of the eight genera comprising the Pleuroceridae, one is extinct and two others have lost half of their species (Table 2). In comparison, most pulmonate families have few if any species that have gone extinct and extant populations are usually large, as is also the case for the families Valvatidae and Viviparidae. Currently, 21 species of freshwater gastropods are fed- erally listed as threatened or endangered (Table 3) and most have had recovery plans formulated. Alabama has the great- est number (10) of federally listed species. However, 95 taxa (51% of extant species) in Alabama were recognized as being in need of conservation at a recent Alabama Division of Wildlife and Freshwater Fishes (ADWFF) state wildlife meeting (Mirarchi et al. 2004). In general, endangered spe- cies are clustered either in the rivers in the southeastern United States (both pleurocerids and hydrobiids) or in iso- lated springs in the western states (hydrobiids). Based on distributional data from Burch’s (1989) monograph, pleurocerids are obviously most diverse in river systems in the southeastern United States (Fig. 1). Alabama is home to 101 species, Tennessee 41, Georgia 24, and Ken- PLEUROCERID CONSERVATION Table 2. The conservation status of the North American Pleuroceridae by genus. Data were updated from NatureServe (2004) databases and a recent assessment by the Alabama Division of Wildlife and Freshwater Fishes (Mirarchi et al. 2004). only on differences in environmentally plastic traits like shell structure. This may also explain the bimodal distribution pat- tern in pulmonates to some extent. nat Percent In light of this distributional differ- Genus Species Gl G2 GX imperiled moe ie ence between the two groups, it is not Athearnia Morrison, 1971 2 l 0 l 100 surprising that G rankings also differ (Fig. Elimia H. and A. Adams, 1854 85 20 15 15 59 4). The modal G ranking for pleurocerids Gyrotoma Shuttleworth, 1845 6 0 0 6 100 1S Gl and the median 1S G2,compared to Io Lea, 1831 1 0 0 0 0 a mode and median of G5 for pulmo- Juga H. and A. Adams, 1854 ? 2 BZ nates. These data indicate clear differ- Leptoxis Rafinesque, 1819 24 5) 3 12 83 Weer, s mde te ae ences in conservation status between the Lithasia Haldeman, 1840 11 2 5 I 72 Grates GE Some Ghee ain ees Pleurocera Rafinesque, 1818 19 4 8 0 63 Ser age sag Career ecg ot camo Pre otoil 157 37 35 35 cerid species much more at risk. Pleuroceridae percent total 235 20.3 223 66.1 Distributional patterns within river tucky 20. The Coosa River in northeastern Alabama origi- nally had 45 species, followed in diversity by the Tennessee, Cahaba, Cumberland, and Ohio Rivers. The Coosa, Cahaba, and Tallapoosa Rivers are in the Alabama River drainage system, probably the world’s most diverse system for fresh- water gastropods. In comparison, pulmonate species are much more widely dispersed (Fig. 2). Pulmonates are most diverse in the northern tier of states in the United States. Even the tenth most diverse assemblage, in Wisconsin, has over 30 species, and pulmonates are quite diverse and abundant in Canada as well (Clarke 1981). Pleurocerids and pulmonates also differ greatly in the sizes of their geographic ranges (Fig. 3). Most pleurocerids are found in only one state. Pulmonates show a bimodal distribution, with some species restricted to only a few states and others being widespread. Most pulmonate species are found in at least three states. These data must be taken with the caveat that some “species” probably have restricted dis- tributions simply because they are not true species but are eco-morphs described in a particular stream or pond based Table 3. Distribution of federally listed freshwater gastropods as of 2002. Number of State species listed Families represented Alabama 10 Hydrobiidae, Pleuroceridae, Viviparidae Idaho 6 Hydrobiidae, Lymnaeidae, Physidae, Valvatidae Missouri 1 Hydrobiidae New Mexico 2 Hydrobiidae Tennessee 2 Hydrobiidae, Pleuroceridae systems have not been studied extensively in either group. However, one study sug- gests that the groups may differ in micro-habitat use as well. Brown et al. (1998) sampled gastropod assemblages from headwater sites in the Salt River system in central Kentucky to the confluence of the Salt River with the Ohio River. All sampling sites were grouped by river order (e.g., the number of tributaries flowing into them). For example, a first order stream is a headwater stream, which is often ephemeral in this system, drying late in the summer to form a series of non-connected pools. Pulmonates (Physa spp. and Helisoma spp.) were only found in low order systems (Fig. 5). Elimia semicarinata (Say, 1829) was widely distributed and oc- curred in all but the largest river reaches. The other two pleurocerid genera, Plewrocera Rafinesque, 1818 and Lithasia Haldeman, 1840, occurred only in the higher-order reaches. DISCUSSION Differences in “prosobranch” and pulmonate biology Why do “prosobranch” and pulmonate gastropods dif- fer so dramatically in conservation status? First, these two groups of gastropods have quite different evolutionary path- ways. Freshwater “prosobranch” gastropods apparently evolved from species physiologically adapted to the lower osmotic concentrations in estuaries and then dispersed into freshwater environments (McMahon 1983, Brown 2001). This adaptive radiation occurred before continental drift had separated modern continents. In contrast, terrestrial pulmo- nates apparently evolved from species living in the high in- tertidal zone that adapted to semi-terrestrial environments. The evolution of a simple lung allowed pulmonates to be- come completely terrestrial. Pulmonates then secondarily in- vaded freshwater habitats. As one might expect from these divergent evolutionary histories, the biologies of pulmonates and freshwater “pro- 60 AMERICAN MALACOLOGICAL BULLETIN Alabama '#'/]/ Tennessee V7V7 Georgia 1/7777] Kentucky (77/77 Virginia (77 Ohio 77] Florida (7 North Carolina (77 Indiana (7 Illinois [3 ce) 20 40 60 80 100 Number of Species Figure 1. Number of pleurocerid taxa found in the ten states with the greatest biodiversity. Data compiled from Burch (1989). California (27777 Wyoming New York Michigan |Z Minnesota [ZZ Oregon Idaho Ohio Wlinois (ZZ Wisconsin (7 0) 10 20 30 40 50 Number of Species Figure 2. Number of pulmonate taxa found in the ten states with the greatest biodiversity. Data compiled from Burch (1989). sobranchs” are quite different. Pulmonates are mostly an- nual species, with high fecundities and hermaphroditic re- production (Brown 1983, Dillon 2000). Pleurocerids, on the other hand, are perennial, iteroparous, and dioecious (Hu- ryn et al. 1994, Brown 2001). Its lung evidently allows a juvenile pulmonate to be passively and widely dispersed in the mud clinging to birds’ feet (see references in Brown 2001). Freshwater prosobranchs, in contrast, have much slower and more restricted adult dispersal along river courses, perhaps explaining higher levels of genetic differen- tiation and endemism (Davis 1982). Another major differ- ence involves the degree of risk of predation. Pleurocerids have thick shells and opercula that decrease their risk to invertebrate predators and fish (Lodge et al. 1987). Pulmo- nates have thin shells that are easily crushed by fish (Stein et al. 1984) or crayfish (Brown 1998) and the lack of an oper- culum increases predation risk to invertebrate predators as well (see references in Brown 2001). 19 * 1/2 * 2004 Pulmonates Pleurocerids Number of Species 1 2 3 4 5 Number of States per Species Figure 3. Distribution of species ranges for pleurocerid and pul- monate gastropods in the United States. Data plotted are the total number of states in which each species occurs. Data compiled from Burch (1989). or Pleurocerids Will Pulmonates 3 s0 a 5 30 ] : / he : ee 7 GX G1 G2 G3 G4 G5 Conservation Ranking Figure 4. G rankings for extant populations of pleurocerid and pulmonate gastropods in the United States. GX stands for extinct. Rankings with higher numbers refer to species with greater num- bers of known populations. Data compiled from NatureServe (2004). Populations of both gastropod groups are food limited, based on a number of studies in which periphyton resources were experimentally manipulated. In pond-dwelling pulmo- nates and lotic “prosobranchs,” either adult density or indi- vidual growth rates respond positively to food additions or density reductions (see references in Brown 2001). Some lotic pleurocerids (i.e., Elimia spp. and Pleurocera spp.) have lower adult abundances and smaller individual sizes in swift- flowing streams, evidently because higher flow rates dislodge larger snails or interfere with movement and feeding (Johnson and Brown 1997). In Lithasia spp., Leptoxis spp., and Io fluvialis (Say, 1825), in contrast, larger snails are common in areas with high current velocities (P. D. Johnson, pers. comm.). PLEUROCERID CONSERVATION 61 ut ZZ cs BEE pp ZZ ©) Cl ec fae p> ee Be Number Collected as River Order Figure 5. Distribution patterns of freshwater gastropods in the Salt River system in central Kentucky. Number of specimens collected for each species are arrayed against river order. Note that pulmo- nates (Ph = Physa hendersoni [Clench, 1925], Ht = Helisoma triv- olvis [Say, 1817]) are common in headwaters, while pleurocerids (Es = Elimia semicarinata, Lo = Leptoxis obovata [Say, 1829], Pa = Pleurocera acuta Rafinesque, 1831, Pc = Plewrocera canaliculatum [Say, 1821]) are common in larger rivers. Data from Brown et al. (1998). With their more “r-selected” life histories, higher dis- persal rates, and hermaphroditic habits, pulmonates are ide- ally suited to colonize and survive in ephemeral habitats like temporary ponds, ditches, and other lentic environments (Brown et al. 1998). Pleurocerids and other prosobranchs are restricted to rivers and lakes because of lower dispersal rates or are more common in these permanent habitats be- cause they are more resistant to predation (Brown et al. 1998). Recovery efforts A two-pronged approach to conservation of imperiled populations of gastropods is necessary. First, detailed infor- mation on distributions and habitat requirements is neces- sary. Such data can be used to assess whether sites within the species’ historic range are still suitable for re-introduction of artificially propagated populations. Important micro-habitat characters include, but are not limited to, water depth and hardness, current velocity, substrate particle size, periphyton production, water quality, and habitat stability (Johnson and Brown 1997, Brown 2001). Second, adequate propagation methods must be devel- oped. Although translocations have been successful with Io fluvialis (Ahlstedt 1991), this option works only when the extant population is large. For many pleurocerids, especially in the Alabama River basin, sufficient individuals cannot be translocated without endangering the original population, and captive propagation is necessary. For each species, tem- perature and flow regimes must first be evaluated to deter- mine the best combination to induce egg laying and juvenile growth. Propagation techniques for several genera of pleu- rocerids (Elimia H. and A. Adams, 1854, Io Lea, 1831, Lep- toxis Rafinesque, 1819, and Pleurocera) are under develop- ment at the Tennessee Aquarium Research Institute in Cohutta, Georgia. In 2002, over 8000 juvenile Leptoxis pli- cata (Conrad, 1834), a federally listed species, were pro- duced from 100 adults held for 3 months. In October 2002, 2734 lab-propagated I. fluvialis were released into the Ten- nessee River below Nickajack Reservoir, in Marion County, Tennessee. In summary, pleurocerids are most at risk because of their limited dispersal powers. The limited movement by adults has undoubtedly played a role in geographically iso- lating populations, promoting speciation and resulting in the high diversity of the group. Pulmonates, by comparison, are fairly cosmopolitan, but less diverse. Their shorter life cycles (allowing rapid population growth), hermaphrodit- ism, and high fecundity promote greater dispersal and suc- cessful colonization across river basins. The more endemic nature of pleurocerids has thus tar- geted them for increased chances of extinction. But what are the ecological threats to persistence? They are probably much the same as those responsible for high extinction rates in unionids (Williams ef al. 1993, Vaughn and Taylor 1999). Many pleurocerids are abundant in shallow riffles (shoals) in rivers, where conditions are quite favorable: warm, oxygen- ated water, large substrate particle sizes (providing refugia where snails can survive spates), sediment-free substrates, and maximal periphyton production. Impoundments change micro-habitats dramatically; the same sites often be- come hypoxic, with extremely cold water, high siltation rates, and loss of periphyton food. Large impoundments may also limit adult migration among the last remaining pristine stretches of rivers and thus increase fragmentation of populations. We suggest that a national strategy for freshwater snail conservation is needed, similar to the one developed for unionid mussels (National Native Mussel Conservation Committee 1997). Information is needed on which species are most in peril and what plans resource managers should implement to assure survival of these species, including habitat restoration, conservation of existing populations, captive propagation, and re-introduction. The strategy should emphasize threats to extant populations and stress both public outreach and inter-agency cooperation. ACKNOWLEDGEMENTS We would like to thank Rob Dillon, for inviting us to give this presentation at the symposium on freshwater gas- 62 AMERICAN MALACOLOGICAL BULLETIN tropod ecology at the 2002 meetings of the American Mal- acological Society in Charleston, South Carolina, and Jay Codeiro, of NatureServe, for his help in compiling and ref- erencing data bases on freshwater snail conservation. LITERATURE CITED Ahlstedt, S. A. 1991. Reintroduction of the spiny river snail Jo flu- vialis (Say, 1825) (Gastropoda: Pleuroceridae) into the North Fork of the Holston River, southwest Virginia and northeast Tennessee. American Malacological Bulletin 8: 139-142. Brown, K. M. 1983. Are life histories real? Data from freshwater snails. American Naturalist 121: 871-879. Brown, K. M. 1998. The role of shell strength in the foraging of crayfish for gastropod prey. Freshwater Biology 40: 1-6. Brown, K. M. 2001. Mollusca: Gastropoda, Chapter 10. In: J. H. Thorp and A. P. Covich, eds. The Classification and Ecology of North American Freshwater Invertebrates. Academic Press, Or- lando, Florida. Pp. 297-325. Brown, K. M., J. E. Alexander, and J. H. Thorp. 1998. Differences in the ecology and distribution of lotic pulmonate and proso- branch gastropods. American Malacological Bulletin 14: 91- 101. Burch, J. B. 1989. North American Freshwater Snails. Malacological Publications, Hamburg, Michigan. Clarke, A. H. 1981. The Freshwater Molluscs of Canada. National Museums of Canada. Ottawa. Davis, G. M. 1982. Historical and ecological factors in the evolu- tion, adaptive radiation, and biogeography of freshwater mol- luscs. American Zoologist 22: 375-395. Dillon, R. T., Jr. 2000. The Ecology of Freshwater Molluscs. Cam- bridge University Press, Cambridge. Hershler, R., J. M. Pierson, and R. S. Krotzer. 1990. Rediscovery of Tulotoma magnifica (Conrad) (Gastropoda: Viviparidae). Pro- ceedings of the Biological Society of Washington 103: 815-824. Huryn, A. D., J. Koebel, and A. C. Benke. 1994. Life history and longevity of the pleurocerid snail Elimia: A comparative study of eight populations. Journal of the North American Bentho- logical Society 13: 540-556. Johnson, P. D. and K. M. Brown. 1997. The role of current and light in explaining the habitat distribution of the lotic snail Elina semicarinata. Journal of the North American Benthologi- cal Society 15: 344-369. Lodge, D. M., kK. M. Brown, S. P. Klosiewski, R. A. Stein, A. P. Cov- ich, B. K. Leathers, and C. Bronmark. 1987. Distribution of freshwater snails: Spatial scale and the relative importance of physicochemical and biotic factors. American Malacological Bulletin 5: 73-84. Mirarchi, R. E., J. T. Garner, M. F. Mettee, and P. E. O'Neil. 2004. Alabama Wildlife, Volume 2: Imperiled Aquatic Mollusks and Fishes. University of Alabama Press, Tuscaloosa, Alabama. McMahon, R. H. 1983. Physiological ecology of freshwater pulmo- nates. In: W. D. Russell-Hunter, ed. The Mollusca, Volume 6, Ecology. Academic Press, Orlando, Florida. Pp. 359-430. 19° 1/2 + 2004 National Native Mussel Conservation Committee. 1997. National strategy for the conservation of native freshwater mussels. Journal of Shellfish Research 17: 1419-1428 NatureServe 2004 An online encyclopedia of life. Available at: http://www.natureserve.org/explorer February 2004 Neves, R. J., A. E. Bogan, J. D. Williams, S. A. Ahlstedt, and P. W. Hartfield. 1997. Status of aquatic mollusks in the Southeastern United States: A downward spiral of diversity. In: G. W. Benz and D. E. Collins, eds., Aquatic Fauna in Peril, The Southeast- ern Perspective. Special Publication 1, Southeast Aquatic Re- search Institute, Lenz Design and Communications, Decatur, Georgia. Pp. 43-86. Stein, R. A., C. G. Goodman, and E. A. Marschall. 1984. Using time and energetic measures of cost in estimating prey value for fish predators. Ecology 65: 702-715. Stern, B. A. 2002. States of the Union: Ranking America’s Biodi- versity. PDF Version Available at: http://www.natureserve.org/ publications/statesUnion.jsp February 2004 Turgeon, D.D., J. F. Quinn, Jr., A. E. Bogan, E. V. Coan, F. G. Hochberg, W. G. Lyons, P. M. Mikkelson, R. J. Neves, C. F. E. Roper, G. Rosenberg, B. Roth, A. Scheltema, F. G. Thompson, M. Vecchione, and J. D. Williams. 1998. Common and Scien- tific Names of Aquatic Invertebrates from the United States and Canada: Molluscs, 2°* Edition. American Malacological Union, Council of Systematic Malacologists. Bethesda, Mary- land. Vaughn, C.C., and C.M. Taylor. 1999. Impoundments and the decline of freshwater mussels: A case study of an extinction gradient. Conservation Biology 13: 919-920. Williams, J. D., M. L. Warren, Jr., K. S. Cummings, J. L. Harris, and R. J. Neves. 1993. Conservation status of freshwater mussels of the United States and Canada. Fisheries 18: 6-22. Accepted: 10 February 2004 AMERICAN MALACOLOGICAL BULLETIN 19+ 1/2 * 2004 Reproductive isolation between Physa acuta and Physa gyrina in joint culture” Robert T. Dillon, Jr., Charles E. Earnhardt, and Thomas P. Smith Department of Biology, College of Charleston, Charleston, South Carolina 29424, U.S.A., dillonr@cofc.edu Abstract: Recent laboratory tests of postzygotic reproductive isolation in physid snails, although providing fresh insight into the evolution of an important model organism, have focused on reproductively compatible populations of Physa acuta. Here we extend such studies to include a population of Physa gyrina known to be incompatible with P. acuta. Reared in pairs, the median age of first reproduction in a laboratory population of P. acuta originating from Charleston, South Carolina, USA was nine weeks. Over the next ten weeks of reproduction, the laboratory population of P. acuta posted a mean fecundity of 61.9 embryos per pair per week, with a mean F, viability of 63% and 100% F, fertility. Individual P. acuta reproduced by self-fertilization when reared with P. gyrina in no-choice mating experiments. Their median age at first reproduction was delayed to 10.5 weeks, their fecundity was 36.4 embryos per parent per week, and F, viability reduced to 26%. These figures were not significantly different from the reproductive success of individual P. acuta self-fertilizing in isolation (median 11 weeks at first reproduction, 37.4 embryos per parent per week, 37% hatchling viability, 88% F, fertility). Laboratory populations of P. gyrina originating from Hot Springs, Virginia, USA, were not as well adapted to our culture conditions as P. acuta. Pairs did not initiate egg laying until a median age of 11.5 weeks, after which their mean fecundity was only 21.2 per pair per week over ten weeks, with an F, viability of 33.5% and 100% F, fertility. When reared with P. acuta in joint culture, individual P. gyrina did not reproduce successfully. Thus the effects of joint culture with P. gyrina were negligible for P. acuta but ruinous for P. gyrina reared with P. acuta. These results have important implications for the interpretation of experiments involving postmating reproductive isolation with no-choice design. Key words: Physa, Basommatophora, Pulmonata, mating, speciation Freshwater pulmonates of the family Physidae may si- multaneously be counted among America’s best-known and least-known gastropods. Their adaptability to laboratory culture has led to great strides in our understanding of ge- netics (Dillon and Wethington 1992, 1994, Monsutti and Perrin 1999), morphology (DeWitt et al. 1999), life history (Rollo and Hawryluk 1988, Crowl and Covich 1990, McCol- lum et al. 1998), ecology (Brown ef al. 1994, Turner ef al. 2000, Bernot and Turner 2001), reproductive biology (Jarne et al. 2000, Wethington and Dillon 1993, 1997), and behav- ior (Covich et al. 1994, Wethington and Dillon 1996, DeWitt 1996, Turner et al. 1999, McCarthy and Fisher 2000). For a review see Dillon (2000). Yet their genetic diversity and phe- notypic plasticity has resulted in confusion regarding the specific identity of even the most widespread American physid taxa. Recently we have initiated a program of laboratory breeding experimentation designed to assess reproductive isolation among a variety of physid populations worldwide. We have established that two of the nominal species most common in North America, Physa heterostropha (Say, 1817) and Physa integra (Haldeman, 1841), are conspecific with European populations of Physa acuta (Draparnaud, 1805) (Dillon ef al. 2002). Our no-choice mating experiments yielded no evidence of delay in maturity or reduction in fecundity, F, viability, or F, fertility among hybrids of six populations (two of each species) below incross controls. Experimental crosses such as these will, however, be more reliably interpreted given the benefit of a “negative control.” Thus the purpose of the present experiment was to docu- ment the reproductive activity of pairs of physids known to be reproductively isolated when cultured in a no-choice design. Physa acuta is a member of the subgenus Costatella Dall, 1870 (Burch and Tottenham 1980), characterized by a two- part penial sheath. Physa gyrina (Say, 1821), a member of the subgenus Physa (s.s.), has a three-part penial sheath. The animals are similar in their overall morphology; P. gyrina maturing at a slightly larger size and bearing a more rounded shell with more convex apical whorls. Individuals of the two species will copulate, although our preliminary observations have suggested that the only progeny born are the products of self-fertilization, rather than hybridization (Wethington et al. 2000). The most likely outcome of jointly culturing a pair of non-hybridizing physids in a no-choice design would seem *From the symposium “The Biology and Conservation of Freshwater Gastropods” presented at the annual meeting of the American Malacological Society, held 3-7 August 2002 in Charleston, South Carolina, USA. 64 AMERICAN MALACOLOGICAL BULLETIN to be reproduction below that posted by control pairs of either species. Self-fertilization, which would be the only reproductive option expected in this experimental situation, is known to engender delayed age at first reproduction, re- duced fecundity, and reduced hatchling viability in physids generally (Jarne et al. 1993, 2000, Wethington and Dillon 1997). Moreover, a pair of mismatched Physa Draparnaud, 1801 might be expected to compete with each other for food and other resources, and perhaps interfere with the self- fertilization process through false copulation, yielding a re- duction in fecundity below even control individuals self- fertilizing in isolation. It is also possible, however, that self-fertilization in one or both individuals might be “socially facilitated” by a sec- ond snail present in joint culture, even if not conspecific. Vernon (1995) observed that, although self-fertilization re- duces reproductive success in the planorbid Biomphalaria glabrata (Say, 1818), the reproductive output of snails reared in pairs but prevented from cross fertilizing by a nylon mesh barrier may approach that of outcrossing pairs. The ordi- narily self-fertilizing terrestrial pulmonate Balea perversa (Linné, 1758) enjoys increased longevity and reproductive success when cultured with a partner, even though paired snails do not copulate (Baur and Baur 2000). Such social facilitation might also occur between snails as similar as Physa acuta and Physa gyrina. Our investigation thus included four treatments: an ex- periment and three controls. The reproductive success of the acuta X gyrina experiment was compared to acuta x acuta controls, gyrina X gyrina controls, and a self-fertilizing con- trol of Physa acuta reared in isolation. This design allowed us to identify social facilitation even in the reduced reproduc- tive output expected from a pair of non-hybridizing species. METHODS Our population of Physa acuta was collected at Charles Towne Landing State Park (32°49'N, 79°59'W), west of the Ashley River within the city limits of Charleston, South Carolina. Animals from this population (previously identi- fied as Physa heterostropha [Say, 1817]) have been the subject of many of our past studies on the genetics (Dillon and Wethington 1994, 1995) and reproductive biology (Weth- ington and Dillon 1991, 1993, 1997) of the genus Physa. Our population of Physa gyrina was collected in the town of Hot Springs, Virginia, approximately 100 meters downstream from the origin of naturally-heated waters inside “The Homestead” resort (38°36'N, 79°30'W). This is the type lo- cality of Physa aurea (Lea, 1838), now recognized as a sub- species of P. gyrina (Burch and Tottenham 1980). Our standard culture vessel was a transparent polyeth- 19° 1/2 + 2004 ylene 295.73 ml (10 US oz.) drinking cup, which we filled with approximately 210 ml of aerated, filtered pond water and covered with a 95 x 15 mm polystyrene Petri dish lid. The food was O. S. I. Spirulina Aquarium Flake Food, sold in pet stores primarily as a diet for herbivorous aquarium fishes. All experiments took place at room temperature, ap- proximately 23°C. We isolated ten wild-collected snails from each of the two study populations in separate cups, collected egg masses, and reared the offspring to 3 mm shell length, approximately three weeks post-hatching, with weekly water change. These two sets of ten wild-conceived but laboratory-born sibships (A1-A10 and G1-G10) constituted the P generation for the four treatments (one experiment and three controls) we re- port here. Each treatment was composed of ten replicates. Control A was a set of ten pairs of unrelated Physa acuta (Al x A2, A2 x A3,..., Al0 x Al). Control G was similarly consti- tuted for Physa gyrina (G1 x G2, G2 x G3, ...). The AG experiment was a set of ten cups of P. acuta paired with P. gyrina (AGI, AG2, ..., AG10). The As control was a set of ten cups with isolated P. acuta snails (Al, A2,..., A10). Each replicate received a water change and fresh food every seven days, at which time the sides of the cup were inspected for egg masses. If egg masses were present, we counted all embryos and transferred the adults to a fresh cup. Eggs were monitored until hatching, generally about two weeks, and all viable, crawling F, juveniles were counted. Replicates were terminated upon the mortality of either adult individual. For statistical analysis of fecundity (egg production) and F, viability (hatching success), week 1 was set separately for each treatment as the first week in which eggs were laid in three or more replicates. Fecundity and F, viability were subsequently recorded for ten weeks. The central tendency of age at first reproduction was compared among the A, AG, and G treatments by dividing at the pooled median and testing the resulting 3 x 2 con- tingency table using chi-square. The AG experiment was compared to the As control in age at first reproduction using a similar median-based approach, although a Fisher’s exact test was employed rather than chi-square, because of the former test’s improved power. We compared the fecundity of the A, AG, and G treatments using two-way analysis of variance, with week and treatment the independent variables and embryos as the dependent variable (Statistica release 5.5, StatSoft 1994). Overall (ten-week) F, viability was compared among these treatments using analysis of covariance, with treatment the independent variable, viable hatchlings the dependent variable, and embryos the covariate. Post hoc tests were performed using Tukey’s “highly significant difference” (HSD) tests for unequal sample sizes (Spjotvoll and Stoline 1973). A second ANOVA and a second ANCOVA were used REPRODUCTIVE ISOLATION IN PHYSA 65 to compare the fecundity and F, viability of treatment A to treatment As. Leading (pre-maturity) zeros were not in- cluded in any ANOVA or ANCOVA, nor were post-mortem zeros included, although internal zeros (i.e., reproductive failure by mature, apparently healthy snails) were analyzed. To assess F, fertility in the AG, A , and G treatments, F, hatchlings were reared from each of three separate unrelated replicates to size 3 mm (AGI, AG2, AG3, Al2, A34, A56, G12, G34, G56). These were paired across replicates within treatment in time series—one early pair from eggs laid around week 1, one middle pair produced around week 5, and one late pair produced around week 10. Each treatment thus yielded nine F, pairs. For example, treatment A yielded: Al2 x A34 early, Al2 x A34 middle, Al2 x A34 late, Al2 x A56 early, ..., A34 x A56 late. Nine pairs were likewise constituted for treatments G and AG, and the total of 27 pairs of F, snails were reared to adulthood with weekly feed- ing and water change. We recorded the date at which em- bryos and viable F, hatchlings were produced by each pair. A larger sample of 56 F, progeny from the AGI, AG2, and AG3 treatments was reared to 4-5 mm shell length, at which time they were frozen in 100/ul of tissue buffer for analysis by protein electrophoresis. Populations of Physa acuta and Physa gyrina share no alleles at six of the seven polymorphic allozyme loci routinely surveyed in our labo- ratory, including 6-phosphogluconic acid dehydrogenase (6Pgd) and isocitrate dehydrogenase (Isdh). We used hori- zontal starch gel electrophoresis in an aminopropylmorpho- line pH 6 buffer system (Clayton and Tretiak 1972) to assess the allozyme phenotype of all putative F, hybrids at these two loci. Details regarding our electrophoretic methods, in- cluding a description of our equipment and recipes for stains and buffers, have been previously published (Dillon 1992, Dillon and Wethington 1995). RESULTS Data on the production of F, progeny by the 10 pairs of parents in the A control, the G control, and the AG experi- ment are compared in Figure 1. The first pair of A parents laid eggs in week 6, although the median age at first repro- duction was 9 weeks. Setting the ninth week of the treatment as week 1 for the purpose of analysis, over 10 weeks the mean weekly production of embryos was 61.9 per pair, and for hatchlings 39.1 per pair (63.0% viability). All 9 pairs of F, progeny successfully reproduced, laying eggs at a median age of 8 weeks that hatched to viable F, progeny a median of 2 weeks later. The reproductive success of our G control of Physa gy- rina was not as great as typically posted by Physa acuta under our culture conditions. Egg laying commenced at week 10 ez) hatchlings —9- embryos = —8— parerits 111 8#910101010%9 9 9 A coma] Pared #. avuta 4 io | 46 Eperment = ee PF acutax F pina | ne a 5 butt 6 q 0) B & rae aT) EI 8 dg i 20 2 a ba o 0 a a ee G Comino] TEINS cae A yo F ts ft at > Say fleet = OY Pared # emma lh a a 9 i Gb b YF PB Parent age (areeks) Figure 1. Production of embryos and viable hatchlings as a func- tion of parental age (weeks post hatching) for ten pairs of Physa acuta (A control), ten pairs of Physa gyrina (G control) and ten pairs of P. acuta * P. gyrina (AG Experiment). The bars are stan- dard errors of the mean. The number of reproducing pairs is given with parental survivorship (right axis). Asterisks* denote week 1 for analysis of variance. and reached a median between weeks 11 and 12 of the treat- ment. Setting week 11 to start, the mean fecundity over 10 weeks was only 21.2 embryos per pair per week, and the mean weekly F, hatchling yield was only 7.2 (33.5% viabil- ity). One F, pair was terminated by mortality, but the re- maining 8 pairs laid viable eggs at a median age of 8 weeks that hatched to viable F, progeny at week 10. Reproductive success in the AG experiment was gener- ally intermediate between the A control and the G control. Egg laying began at week 9 and reached a median between weeks 10 and 11, yielding a mean fecundity of 36.4 per pair per week over 10 weeks. Hatchling production averaged only 9.3 per pair per week, for a 25.6% F, survival rate over that period. One pair of F, snails retained for testing ultimately proved sterile, but the remaining 8 pairs reproduced on the 66 AMERICAN MALACOLOGICAL BULLETIN same schedule as the A and G controls: egg laying at a median of 8 weeks and viable F, hatchlings at week 10. Our comparison of age at first reproduction in treat- ments A, AG, and G (Fig. 2) revealed a significant difference in central tendency (x* = 9.02, p = 0.011). Seven of the pairs of Physa acuta in control A reproduced before any of the pairs of P. gyrina in control G, with the AG experiment intermediate. Analysis of variance also uncovered a signifi- cant difference in fecundity (Table 1), HSD post hoc tests confirming that the A control produced significantly more embryos than the AG experiment (p = 0.0149), which yielded more embryos than the G control (p = 0.0001). Analysis of covariance (Fig. 3) returned a significant differ- ence between treatments (F = 16.8, p = 0.000). HSD post hoc tests showed that the 63% F, viability posted by the A con- trol was significantly greater (p = 0.0001) than either the 33% of the G control or the 26% of the AG experiment, which did not differ (p = 0.8196). Protein electrophoretic analysis of F, progeny from the AG experiment revealed all viable offspring to be the prod- ucts of self-fertilization by the Physa acuta parent. The 56 3 A control [_ AG expt (ee | Geontrol ESSE As Onset of maturity (replicates) i | | | p bo oe — 4 | 1 * | oo 3 | oH Bs | | oa | oy | | | ead | oe el L_| | 12 9 9 10 11 Age (weeks) Figure 2. The number (of 10) replicates laying their first eggs as a function of parental age (weeks post-hatching) for pairs of Physa acuta (A control), pairs of Physa gyrina (G control), isolated indi- vidual P. acuta (As) and the P. acuta x P. gyrina experiment (AG). Table 1. Results of analysis of variance comparing the fecundities measured over 10 weeks in the A control, the G control, and the AG experiment. df MS df MS Effect effect effect error error F p-level treatment 2 36,399 211 1,301 27.97 0.000 week 9 35/91 211 1,301 2.9] 0.003 tx w 18 4,266 9211 1,301 3.28 — 0.000 19° 1/2 + 2004 | Embryos Ee] Hatchlings 600 b 400 200 | Offspring (per ten weeks) A AG As G Figure 3. Total ten-week fecundity and yield of viable hatchlings (mean + sem) for pairs of Physa acuta (A), pairs of Physa gyrina (G), the P. acuta x P. gyrina experiment (AG), and isolated indi- vidual P. acuta (As). Values significantly different by post hoc tests are designated with different lower case letters in Arabic for em- bryos or Greek for hatchlings. offspring we examined were distributed evenly across the ten weeks of reproduction in three replicates and were entirely homozygous for the P. acuta markers 6pgd100 and Isdh100 (Dillon and Wethington 1995). No hybrid progeny were recovered, nor did the Physa gyrina parent apparently re- produce successfully by self-fertilization. Reproduction in the ten individual Physa acuta isolated for the As control is shown in Figure 4. Egg laying com- menced at week 10 and reached a median at week 11. Taking week 10 as a start, the mean fecundity over ten weeks was 37.4 embryos per parent per week, yielding a weekly average of 13.9 hatchlings per parent for a 36.9% F, viability. Com- parison of the AG experiment to the As control showed no difference in age at first reproduction (Fisher’s exact prob- ability = 0.65), fecundity (p = 0.6795, Table 2), or hatchling viability (F = 0.82, p = 0.38, Fig. 3). DISCUSSION The reproductive success we recorded for pairs of Physa acuta in these experiments (a median age of 9 weeks at first reproduction and 61.9 embryos per pair per week, 63% vi- ability) was similar to that recorded for the Charleston population by Dillon et al. (2002). The present figures are lower than those reported for Charleston P. acuta by Weth- ington and Dillon (1997), but the 1997 experiments involved mated singletons (rather than pairs) and took place over the lifetime of the animals, rather than ten weeks. Apparently our Virginia population of Physa gyrina is not as well-adapted to standard culture conditions as is REPRODUCTIVE ISOLATION IN PHYSA 67 {J hatchlings —O—_ embryos — @— parents 120 2° © __@ _@_® _e_@ o—e — + 10 3% -4' 10) 10> 7 -6 fF As Control | e 100 | P. acuta isolates | —— $ 2 t | 6 5\4 z Zz g e Oh = 80 F | q s = L } | 6 2 op i | | = & | a & 60 | | | Ih S . L | | | 3 c | | Oo 4 5 S 40 F | etl ° = [ i | | | 2 2 20 + ? | | - “ f — 0 bod n 4 i Pe ee ee 0 5 6° 7% 8 9 10° Th-12 18 14-15 16 17 18) 19 Parent age (weeks) Figure 4. Production of embryos and viable hatchlings as a func- tion of parental age (weeks post hatching) for ten individual Physa acuta reared in isolation (As control). The bars are standard errors of the mean. The number of reproducing individuals is given with parental survivorship (right axis). Asterisk* denotes week 1 for analysis of variance. Table 2. Results of analysis of variance comparing the fecundities measured over 10 weeks in the As control and the AG experiment. df MSs df MS Effect effect effect error — error F p-level treatment 1 475 132 274 0.17 0.68 week 9 2;922 132 25774 1.05 0.40 tx w 9 3,942 132 2/71 1.42 0.18 Physa acuta. Its reproductive record, which began at a me- dian age of 11 weeks and featured only 21.2 embryos per pair per week with a 33.5% viability, was significantly below A control P. acuta. Indeed, the fecundity and F, viability posted by outcrossing pairs of P. gyrina was even below figures posted by our As self-fertilizing P. acuta. Although copulation has been observed between Physa acuta and Physa gyrina, our results suggest that postmating reproductive isolation between the two species is complete. We cannot rule out the possibility that subviable F, hybrids (and pure P. gyrina as well) may have been born but were unable to compete with contemporaneous cohorts of pure P. acuta. In any case, all the progeny we recovered from ten weeks of joint culture were the products of self-fertilization by the P. acuta parent. As has been previously reported (Wethington and Dil- lon 1997), individual Physa acuta isolated in our As control and forced to self-fertilize displayed delayed age at first re- production (median age 11 weeks) and much-reduced F, viability (36.9%). There was no significant difference in the reproduction of As isolates and the self-fertilizing P. acuta cultured jointly with Physa gyrina (10.5 weeks, 36.4 em- bryos/week, 25.6% viability). Apparently, joint culture has neither a positive nor a negative effect on P. acuta. Culture with an individual P. acuta seems to be quite deleterious for P. gyrina, however, effectively shutting down whatever (rela- tively low) reproduction it would have otherwise achieved. The results of this investigation offer no evidence of social facilitation between the two species of Physa. They do, however, contain an important cautionary message for fu- ture studies of postmating reproductive isolation in labora- tory cultures of pulmonates. By none of the measures of fitness we employed here—age at first reproduction, fecun- dity, F, viability, and F, fertility—were the results of the AG experiment significantly depressed below either of the two controls. Our finding that the survivorship of the outcrossed F, progeny of Physa gyrina from the G control was not significantly greater than that of the selfed progeny of Physa acuta from the AG experiment was especially surprising. Had we not examined the allozyme phenotypes of the F, progeny via protein electrophoresis, and discovered that no hybrids were being produced, the reproductive isolation dis- played between species as different as P. acuta and P. gyrina might have been missed entirely. ACKNOWLEDGMENTS We thank our colleagues Matt Rhett, Amy Wethington, and Chuck Lydeard for assistance with this project. Tom McCarthy and Bob McMahon provided excellent sugges- tions on the manuscript. Funding was provided by a grant from the National Science Foundation, DEB-0128964. LITERATURE CITED Baur, B. and A. Baur. 2000. Social facilitation affects longevity and lifetime reproductive success in a self-fertilizing land snail. Oikos 88: 612-620. Bernot, R. J. and A. M. Turner. 2001. Predator identity and trait- mediated indirect effects in a littoral food web. Oecologia 129: 139-146. Brown, kK. M., K. R. Carman, and V. Inchausty. 1994. Density- dependent influences on feeding and metabolism in a fresh- water snail. Oecologia 99: 158-165. Burch, J. B. and J. L. Tottenham. 1980. North American freshwater snails: Species Jist, ranges, and illustrations. Walkerana 3: 1-215. Clayton, J. W. and D.N. Tretiak. 1972. Amine-citrate buffers for pH control in starch gel electrophoresis. Journal of the Fisheries Research Board of Canada 29: 1169-1172. Covich, A. P., T. A. Crowl, J. E. Alexander, and C. C. Vaughn. 1994. 68 AMERICAN MALACOLOGICAL BULLETIN Predator-avoidance responses in freshwater decapod- gastropod interactions mediated by chemical stimuli. Journal of the North American Benthological Society 13: 283-290. Crowl, T. A. and A. P. Covich. 1990. Predator-induced life-history shifts in a freshwater snail. Science 247: 949-951. DeWitt, T.J., A. Sih, and J. A. Hucko. 1999. Trait compensation and cospecialization in a freshwater snail: Size, shape, and antipredator behaviour. Animal Behavior 58: 397-407. DeWitt, T. J. 1996. Gender contests in a simultaneous hermaphro- dite snail: A size-advantage model of behavior. Animal Behav- tour 51: 345-351, Dillon, R. T., Jr. 1992. Electrophoresis IV, nuts and bolts. World Aquaculture 23: 48-51. Dillon, R. T., Jr. 2000. The Ecology of Freshwater Molluscs. Cam- bridge University Press, Cambridge. Dillon, R. T., Jr. and A. R. Wethington. 1992. The inheritance of albinism in a freshwater snail, Physa heterostropha. Journal of Heredity 83: 208-210. Dillon, R. T., Jr. and A. R. Wethington. 1994. Inheritance at five loci in the freshwater snail, Physa heterostropha. Biochemical Genetics 32: 75-82. Dillon, R. T., Jr. and A. R. Wethington. 1995. The biogeography of sea islands: Clues from the population genetics of the fresh- water snail, Physa heterostropha. Systematic Biology 44: 400- 408. Dillon, R.T., Jr., A. R. Wethington, J. M. Rhett, and T. P. Smith. 2002. Populations of the European freshwater pulmonate Physa acuta are not reproductively isolated from American Physa heterostropha or Physa integra. Invertebrate Biology 121: 226-234. Jarne, P., M-A. Perdieu, A-F. Pernot, B. Delay, and P. David. 2000. The influence of self-fertilization and grouping on fitness at- tributes in the freshwater snail Physa acuta: Population and individual inbreeding depression. Journal of Evolutionary Bi- ology 13: 645-655. Jarne, P., M. Vianey-Liaud, and B. Delay. 1993. Selfing and out- crossing in hermaphrodite freshwater gastropods (Basom- matophora): Where, when and why. Biological Journal of the Linnean Society 49: 99-125. McCarthy, T. M. and W.A. Fisher. 2000. Multiple predator- avoidance behaviours of the freshwater snail Physella heter- ostropha pomila: Responses vary with risk. Freshwater Biology 44: 387-397. McCollum, E. W., L. B. Crowder, and S. A. McCollum. 1998. Com- plex interactions of fish, snails, and littoral zone periphyton. Ecology 79: 1980-1994. Monsutti, A. and N. Perrin. 1999. Dinucleotide microsatellite loci reveal a high selfing rate in the freshwater snail Physa acuta. Molecular Ecology 8: 1076-1078. Rollo, C. D. and M. D. Hawryluk. 1988. Compensatory scope and resource allocation in two species of aquatic snails. Ecology 69: 146-156. Spjotvoll, E. and M. R. Stoline. 1973. An extension of the T-method of multiple comparison to include the cases with unequal sample size. Journal of the American Statistical Association 68: 976-978. 19+ 1/2 * 2004 StatSoft. 1994. Statistica, General Conventions and Statistics I. Stat- soft, Inc., Tulsa, Oklahoma. Turner, A. M., R. J. Bernot, and C. M. Boes. 2000. Chemical cues modify species interactions: The ecological consequences of predator avoidance by freshwater snails. Oikos 88: 148-158. Turner, A. M., S. A. Fetterolf, and R. J. Bernot. 1999. Predator iden- tity and consumer behavior: Differential effects of fish and crayfish on the habitat use of a freshwater snail. Oecologia 118: 242-247. Vernon, J.G. 1995. Low reproductive output of isolated, self- fertilizing snails: Inbreeding depression or absence of social facilitation? Proceedings of the Royal Society of London (B) 259: 131-136. Wethington, A. R. and R. T. Dillon, Jr. 1991. Sperm storage and evidence for multiple insemination in a natural population of the freshwater snail, Physa. American Malacological Bulletin 9: 99-102. Wethington, A. R. and R. T. Dillon, Jr. 1993. Reproductive devel- opment in the hermaphroditic freshwater snail, Physa, moni- tored with complementing albino lines. Proceedings of the Royal Society of London (B) 252: 109-114. Wethington, A. R. and R. T. Dillon, Jr. 1996. Gender choice and gender conflict in a non-reciprocally mating simultaneous hermaphrodite, the freshwater snail, Physa. Animal Behavior 51: 1107-1118. Wethington, A. R. and R. T. Dillon, Jr. 1997. Selfing, outcrossing, and mixed mating in the freshwater snail Physa heterostropha: Lifetime fitness and inbreeding depression. Invertebrate Biol- ogy 116: 192-199. Wethington, A. R., E.R. Eastman, and R. T. Dillon, Jr. 2000. No premating reproductive isolation among populations of a si- multaneous hermaphrodite, the freshwater snail Physa. In: R. A. Tankersley, D. I. Warmolts, G. T. Watters, B. J. Armit- age, P. D. Johnson, and R. S. Butler, eds., Freshwater Mollusk Symposia Proceedings, Ohio Biological Survey, Columbus. Pp. 245-251. Accepted: 20 February 2004 AMERICAN MALACOLOGICAL BULLETIN 19° 1/2 + 2004 High levels of mitochondrial DNA sequence divergence in isolated populations of freshwater snails of the genus Goniobasis Lea, 1862* Robert T. Dillon, Jr. and Robert C. Frankis, Jr. Department of Biology, College of Charleston, Charleston, South Carolina 29424, U.S.A., dillonr@cofc.edu Abstract: In addition to their utility for phylogenetic reconstruction, mitochondrial sequence data have increasingly been applied to studies of species-level systematics. We amplified and sequenced a 709 bp fragment of the mitochondrial gene encoding cytochrome oxidase 1 and an approximately 530 bp fragment of the ribosomal large subunit (16S) gene for three individuals from each of three populations representing geographic races of the well-studied freshwater “prosobranch” snail Goniobasis proxima. By comparing intraspecific divergence to divergence in these same genes among G. proxima and the related Gontobasis semicarinata and Goniobasis catenaria, our purpose was to calibrate mitochondrial sequence data for application in future systematic studies of isolated, poorly-mobile molJuscan populations in which genetic relationships may be less well understood. We identified four distinct haplotypes in the nine mitochondrial genomes of G. proxima amplified for each gene fragment, with a maximum likelihood sequence difference of 8.6%-16.9% for CO1 and 5.7%-18.7% for 16S. These levels of intraspecific divergence overlapped extensively with interspecific maximum likelihood differences, which ranged from 11.4%-17.7% for CO1 and 9.5%-16.5% for 16S. The extreme fragmentation that typically characterizes the population structure of freshwater gastropods, together with the ability of such populations to reach large size and great age, must be taken into consideration before systematic inference can be made on the basis of sequence divergence for these genes. Keywords: 16S, CO1, Elimia, Virginia, Carolina All major invertebrate taxa include poorly known groups in which the relationships between species are not clear. In freshwater and terrestrial molluscs, for example, species ranges may be fragmented into isolated populations and variation in shell morphology and other traditional characters may be negligible or subject to phenotypic plas- ticity. Thus as the tools of molecular genetics have become more accessible, malacologists have turned to DNA sequence data as a source of evidence by which biological species may be distinguished. Before any new measurement tool can be employed ef- ficiently, however, it must be calibrated. Levels of DNA se- quence divergence should first be examined within and among populations for which specific relationships have previously been established by breeding studies or similarly direct means. If molecular data are to achieve ideal utility as criteria for species recognition, the maximum levels of se- quence divergence among populations known to be conspe- cific should be less than the minimum sequence divergence between known, closely related species. Among the most commonly sequenced genes in studies of molluscan population divergence are the mitochondrial genes encoding cytochrome oxidase I (CO1) and the large ribosomal subunit (16S). Sequence variation for the CO1 gene distinguishes unambiguously among species of the ma- rine vesicomyid clams (Baco et al. 1999), the freshwater bivalve genera Lasmigona Rafinesque, 1831 (King et al. 1999) and Corbicula Megerle von Miuhlfeld, 1811 (Renard et al. 2000), and the marine gastropod genera Crepidula Lamarck, 1799 (Collin 2000), Notoacmaea (Simison and Lindberg 1999), and Hydrobia Hartmann, 1821 (Wilke and Davis 2000, Wilke et al. 2000). Sequence variation for the 16S gene effectively discriminates among species in the marine bivalve genera Ostrea Linnaeus, 1758 (O Foighil et al. 1995, 1998, Jozefowicz and O Foighil 1998), and Mercenaria Schuma- cher, 1817 (O Foighil et al. 1996), and in the freshwater bivalve genera Amblema Rafinesque, 1820 (Mulvey ef al. 1997) and Dreissena van Beneden, 1835 (Stepien et al. 1999). The 16S gene has also proven useful to distinguish species of land snails in the genera Candidula Kobelt, 1871 (Pfenninger and Magnin 2001), Discus Fitzinger, 1833 (Ross 1999) and Cepaea Held, 1837 (Thomaz et al. 1996). In some situations, however, the level of sequence di- vergence within molluscan species has been found to exceed divergence among species. In the special case of doubly- uniparental inheritance, the male and female mitochondrial genomes within populations of Mytilus spp. and Anodonta spp. have diverged more than between same-sex compari- *From the symposium “The Biology and Conservation of Freshwater Gastropods” presented at the annual meeting of the American Malacological Society, held 3-7 August 2002 in Charleston, South Carolina, USA. 70 AMERICAN MALACOLOGICAL BULLETIN sons of valid species (Rawson and Hilbish 1995, Hoeh et al. 1996). Sequence divergence among conspecific populations of the land snail Mandarina sp. from remote Pacific islands has proceeded to the extent that between-species variance seems to have been swamped (Chiba 1999). We are aware of four prior works comparing the levels of DNA sequence divergence within and among conspecific populations of freshwater gastropods to divergence between related species. In three of these cases, researchers have re- ported an overlap between the maximum levels of diver- gence within species and the minimum divergence between species. In the pleurocerid fauna of Alabama, Lydeard et al. (1998) reported that 16S divergence ranged from 0% to 3.93% within species of Goniobasis Lea, 1862 (or Elimia H. and A. Adams, 1854) and from 0.3% to 11.08% between species. Populations representing different subspecies of the oriental pomatiopsid Oncomelania hupensis (Gredler, 1881) may show COI sequence differences exceeding those re- ported between the related pomatiopsid genera Gamma- tricula Davis and Liu, 1990 and Tricula Benson, 1843 (Davis et al. 1998). Hershler et al. (1999b) reported that the CO1 divergence between two Death Valley populations of the hydrobiid Tryonia variegata Hershler and Sada, 1987 was equal to or greater than the level observed in most other comparisons among eight species of Tryonia Stimpson, 1865. The subsequent results of Hershler et al. (1999a) on a larger sample of hydrobiids from the American southwest seemed to cast doubt on previous assumptions regarding specific relationships in this group. In any case, it is possible that the evolution of DNA sequences in freshwater gastro- pods may be more similar to that described by Chiba (1999) for island populations of the land snail Mandarina sp. than to the bivalves, marine gastropods, or even most terrestrial gastropods that have attracted the bulk of previous study. At the level of its population genetics, the pleurocerid Goniobasis proxima (Say, 1825) is among the best known of all freshwater gastropods. The purpose of this paper was to assess sequence variation for the mitochondrial CO1 and 16S genes within and among populations of G. proxima and compare the intraspecific values obtained to interspecific values from two other well-characterized Goniobasis species known to be related. Our purpose is to confirm the small body of previously published evidence suggesting that popu- lations of freshwater snails may be so old, large, and isolated that intrapopulation sequence divergence is liable to swamp interpopulation sequence divergence in two of the mito- chondrial genes most commonly examined by malacologists using the tools of molecular genetics. The Pleuroceridae is a holarctic family of freshwater “prosobranch” gastropods that has diversified extensively in the rivers and streams of the American southeast. Popula- tions are perennial and may reach great densities, locally 19° 1/2 + 2004 hundreds per square meter. Reproduction is entirely sexual, as far as is known. The biology of the Pleuroceridae has been reviewed by Dillon (2000). Goniobasis proxima is a common pleurocerid inhabitant of small softwater streams in the piedmont and mountains from southern Virginia to north- ern Georgia, on both sides of the eastern continental divide. Populations are isolated both by intervening mountain ranges and by larger rivers, to which the snail does not seem adapted. A sample of 25 populations from a 20,000 km? area straddling the borders of Virginia, North Carolina, and Ten- nessee showed extreme divergence in both morphology and allozyme frequencies; some pairs of populations sharing no alleles at multiple enzyme loci (Dillon 1984a). Three races of G. proxima have been recognized (A, B, and C) on the basis of shell morphology and allozyme divergence inhabiting dif- ferent parts of the range (Dillon and Davis 1980, Dillon 1984b). Transplant experiments (Dillon 1988a) and artificial introductions (Dillon 1986) have, however, uncovered no evidence of reproductive isolation among any of these populations. As genetically diverse as Goniobasis proxima may be, its levels of interpopulation divergence do not approach those recorded among other well-characterized species, such as Gontobasis semicarinata (Say, 1829) (Dillon and Davis 1980) or Goniobasis catenaria (Say, 1822) (Dillon and Reed 2002). Goniobasis semicarinata is primarily an inhabitant of the American interior, ranging through Ohio, Indiana, and Ken- tucky. Its biology is similar to that of G. proxima, although it bears a heavier shell and is restricted to harder water and lower elevations. The southern limit of G. semicarinata con- tacts the northern border of the range of G. proxima in the New River drainage of Virginia. Goniobasis catenaria inhab- its streams and rivers on the southern and eastern borders of the G. proxima range (Dillon and Keferl 2000). There are several subspecies, one of which (G. catenaria dislocata) bears a shell distinguishable from that of G. proxima only by faint axial costae. The 2n = 36 karyotype of G. catenaria is not strikingly different from the 2n = 34 karyotype of G. proxima (Dillon 1989, 1991), but there is no evidence of hybridization between the two species, even in close contact (Dillon and Reed 2002). So analyzed together, the Goniobasis fauna of the southeastern United States would seem an ex- cellent model upon which to gauge the utility of mitochon- drial sequence data for species discrimination in freshwater gastropods, and perhaps in poorly-mobile freshwater inver- tebrates more generally. METHODS Study populations Although separated by less than 120 km over land, our three populations of Goniobasis proxima shared no freshwa- MITOCHONDRIAL DIVERGENCE IN POPULATIONS OF GONIOBASIS SPP. 71 ter connection (Fig. 1). Our Race A sample came from a tributary of the Yadkin River, which drains south to the Atlantic through the Pee Dee system, our sample of Race B was from a tributary of the New River, flowing west to the Mississippi through the Ohio River system, and our sample of Race C was from a small tributary of the Dan River, flowing east to the Atlantic through the Roanoke River sys- tem. Our sample of Goniobasis semicarinata was collected from a small tributary of the New River only 50 km east of our G. proxima Race C. Our Goniobasis catenaria came from a tributary of the Santee River, which flows through South Carolina to the Atlantic approximately 350-400 km south of the other four populations. Detailed locality data are as follows: Gontobasis proxima Race A-Naked Creek at NC 1154 bridge, 5.2 km N of Fur- guson, Wilkes Co., NC. Goniobasis proxima Race B-Cripple Creek at Va 671 bridge, 3.7 km E of Cedar Springs, Wythe Co., VA. Goniobasis proxima race C-Nicholas Creek at Va 100 km Zz Figure 1. A portion of the southeastern United States, showing drainage relationships among sample sites. The circles are popula- tions of Goniobasis proxima (A, B, C), the square is a population of Goniobasis semicarinata (Gs), and the triangle is a population of Goniobasis catenaria (Gc). 623 bridge, 5.2 km SW of Ferrum, Franklin Co., VA. Go- niobasis semicarinata-Little Pine Run at Va 100 bridge, 12 km S of Pulaski, Pulaski Co, VA. Goniobasis catenaria dislo- cata-the head of Chapel Branch, Santee, Orangeburg Co., SC. Allozyme data and maps locating these populations have been published as follows: Race A is “Yad” of Dillon and Davis (1980) and Dillon and Reed (2002) or “Yad1” of Dil- lon (1984a, 1988b). Race B is “Crip” of Dillon and Davis (1980) and Dillon (1984a, 1988a). Race C is “Phlp” of Dillon (1984a). Our Goniobasis semicarinata is population “Pine” of Dillon and Davis (1980) and our G. catenaria dislocata is “Sant” of Dillon and Reed (2002). We analyzed three individuals from each race of G. population proxima and one individual from each of the other two species. Thus a total of 11 fragments were amplified and sequenced for each of the two mitochondrial genes exam- ined here. Laboratory methods Total cellular DNA was obtained using either fresh or previously frozen samples of whole buccal mass (~20 mg). Following proteinase K digestion of the tissue, the DNA was extracted using either a DNeasy Tissue Kit (Qiagen) or by two phenol and two chloroform extractions followed by ethanol precipitation. The optimum DNA concentration for PCR of each of the samples was determined empirically. We amplified a 709 bp fragment of the mitochondrial cytochrome oxidase I gene using the “universal” CO] prim- ers of Folmer ef al. (1994): 5'-ggtcaacaaatcataaagatattgg-3' and 5’-taaacttcagggtgaccaaaaaatca-3’. Our 525-532 bp frag- ment from the 5’ half of the 16S mitochondrial rDNA gene was amplified using primers “SNLOO2” and “SNL448” of Lydeard et al. (1997, 1998), the former trimmed slightly to afford a better match of annealing temperatures: 5’- aaatgattatgctaccttt-3’ primer “l6sar-L” (or L2510) commonly used as a starting and 5'-gaaatttcattcgcactag-3’. The point to amplify the 3’ half of the mitochondrial 16S gene (Palumbi et al. 1991) is encountered around bases 410-430 of the sequences we determined in the present study. Lydeard et al. (1998) reported that the 5’ half of the pleu- rocerid 16S gene shows greater variability than the 3’ half more usually sequenced by other workers. A typical amplification reaction of 50 tL contained 1.5 uL of DNA (the optimum amounts generally ranged be- tween 50 and 200 ng, determined empirically), 100 mM Tris (pH 9), 50 mM KCl, 1.5 units of Taq DNA polymerase, 150 uM of each of the four deoxynucleotide triphosphates, and 0.2 uM of each primer. PCR amplification was by a 15 min 95°C activation step followed by 30 cycles, each consisting of 45 sec at 94°C, 1 min at 45°C, and 1 min at 72°C. Upon completion of the 30 cycles a 10 min 72°C incubation was performed to extend uncompleted strands. Amplification of 72 AMERICAN MALACOLOGICAL BULLETIN fragments of the expected size was verified by agarose gel electrophoresis. The PCR-amplified DNA was prepared for sequencing using a QIAquick PCR purification kit (Qiagen). Cycle sequencing was performed by the Medical University of South Carolina Biotechnology Resource Laboratory. The PCR product was sequenced twice for both strands. Analysis Our initial alignments of the four sequence fragments obtained for each individual were performed using the “Web align” feature of Biowire Jellyfish (version 1.5, Biowire.com) with default settings. Final alignments (between individuals) were performed online with program blastn through the “Blast two sequences” utility available from the National Center for Biotechnology Information (Tatusova and Mad- den 1999, NCBI 2003). The apparently high frequency of indels in our 16S data prompted us to lower the gap opening penalty from 10 to 2 and the gap extension penalty from 2 to 1. The strong A/T bias also observable in our 16S data prompted us to de-select the low complexity filter. We translated our CO1 sequence fragments with the invertebrate mitochondrial code and a +3 lag using Biowire Jellyfish, then aligned the resulting amino acid sequences pairwise with the blastp program, also available online through the NCBI “Blast two sequences” utility (NCBI 2003). All blastp parameters were set to default, except that the low complexity filter was de-selected. We recorded the simple percent nucleotide difference (“p distance”) between each unique pair of sequences as one minus the identity returned by the pairwise BLAST utility, extending through the entire (unvaried) primer region on each end. Where indels had apparently yielded two se- quences of different lengths, the length of the larger se- quence served as denominator in the calculation of identity. Thus the impact of each indel was weighted by its length. We also calculated maximum likelihood (ML) distances among our sequence fragments using the DNADIST pro- gram available in PHYLIP (version 3.573c, Felsenstein 1995). Both the base composition frequencies and transition:trans- version ratios were determined empirically. A single joint analysis was performed for the CO] data. Because indels are scored as missing data (rather than as mismatches) in the calculation of ML distances, however, their accumulated ef- fects across the diverse 16S sequences we ultimately obtained would have resulted in substantial loss of data upon multiple alignment. We therefore elected to calculate ML distances among 16S sequences pairwise in multiple separate runs, rather than in a single joint analysis. For parsimony analysis, we performed a conventional multiple alignment of our 16S sequences using BioEdit ver- sion 5.0.9 (Hall 1999) and concatenated each 16S sequence (elongated by multiple insertions) to its corresponding CO1 19° 1/2 + 2004 sequence. We then analyzed the combined data set using PAUP* version 4.0b10 (Swofford 2002), setting Goniobasis semicarinata as root, Goniobasis catenaria as root, and semi- carinata + catenaria as root, with 1,000 bootstrap replicates. RESULTS Results for the CO1 and the 16S genes were similar in many respects. All three individuals of Goniobasis proxima from population A yielded identical CO1 and 16S haplo- types, as did all three individuals from population B. Popu- lation C yielded two strikingly different CO1 haplotypes and two strikingly different 16S haplotypes. For both genes, the haplotype carried by two individuals was designated “C1” and the haplotype carried by the third snail was designated “C2.” The two other species, Goniobasis semicarinata and Gontobasis catenaria, also yielded distinct haplotypes, result- ing 1n six unique sequence fragments for each gene. The total of 12 unique sequences has been entered in GenBank, ac- cession numbers AY063464-AY063475. The total length, from the 5’ beginning of the first primer to the 3’ end of the second primer, was 709 bp for all six unique CO1 sequence fragments. Table 1 shows that the simple uncorrected nucleotide difference between the four sequences of Goniobasis proxima ranged from 8.0% between populations A and B to 14.7% between the two sequences identified in population C. This translated to an amino acid difference of 1.3%-5.5%. Interspecific divergence was not strikingly different from intraspecific divergence, evaluated at the maximum. The uncorrected difference between Go- niobasis semicarinata and the other species, and between Goniobasis catenaria and the other species, ranged from 10.2%-15.2% as nucleotides or 0.4%-5.1% as amino acids. Combined over the six unique CO] sequences we ob- tained, the base frequencies were 24%A, 19%C, 20%G, and Table 1. Comparisons of six mitochondrial CO1 sequence frag- ments amplified from three species of Goniobasis. Above the di- agonal are the percent differences (p distances) of nucleotide bases (709 in the denominator, including both primers) and below the diagonal are percent amino acid differences (235 in the denominator). A B Cl C2 G.s. G.c. G. proxima A 8.0 14.0 14.1 11.7 10.9 G. proxima B 4.3 V7 14.5 10.2 10.3 G. proxima Cl Zeil 3:9 14.7 13:5 15:2 G. proxima C2 1.7 51 1.3 13.5 14.4 G. semicarinata 0.4 3.8 1.7 1.3 12.1 G. catenaria 0.9 5.1 3.0 21 1.3 MITOCHONDRIAL DIVERGENCE IN POPULATIONS OF GONIOBASIS SPP. 73 37%T. Pairwise transition : transversion (Ts : Tv) ratios are shown in Table 2, the overall empirical ratio being 4.14. Corrected by the base frequencies and the Ts : Tv ratio, the six sequences are arranged by their maximum likelihood distances in Fig. 2. The distance from the 5’ end of the leading primer to the 3’ end of the trailing primer for the 16S fragment am- plified in this study ranged from 524-532 bp for the six unique sequences we obtained. Table 3 shows that uncor- rected sequence differences ranged from 6.1%-17.1% among the populations of Goniobasis proxima, with 1-4 indels ap- parent for each comparison. Evaluated at the maximum, this is again not strikingly different from the levels of divergence between species, which ranged from 9.3%-17.9%. Variable bases did not seem equally distributed across the approximately 530 bases of the 16S fragment we ampli- fied, but rather seemed localized, as one might expect from the stem-and-loop structures assumed by ribosomal sub- units. Apparent Ts : Tv ratios were generally low, approach- ing unity in several instances (Table 2). Across all six unique 16S sequence fragments (3,170 bases) the base frequencies were 35% A, 36% T, 13% C, and 16% G. Corrected by base composition and pairwise Ts : Tv ratios, the sequences are diagramed by their maximum likelihood distances in Fig. 3. Multiple alignment elongated the joint 16S product to 545 bases by multiple insertion, which when concatenated with the 709 CO1 bases yielded a combined sequence of 1,254 characters. Of these 928 were constant, 207 were par- simony-uninformative, and 119 were parsimony- informative. Phylogenetic analysis with both Goniobasis semicarinata and Gontobasis catenaria specified as roots re- turned single trees of length 478 (CI = 0.8159, HI = 0.1841), both depicting Goniobasis proxima as paraphyletic. The third analysis, combining G. semicarinata and G. catenaria, could not be rooted such that the outgroup was monophyletic, and collapsed to yield the single tree rooted by G. semicarinata alone (Fig. 4). Table 2. Apparent transition : transversion ratios in comparisons of six mitochondrial sequence fragments amplified from 3 species of the genus Goniobasis. Data for the CO1 gene are above the diagonal, and those for the 16S gene are given below. A B Cl C2 G.s G.c G. proxima A 16.7 3.50 3.50 4.57 3.81 G. proxima B 6.00 3.05 3.86 3.80 3.50 G. proxima Cl 3.86 8.00 2.92 2:25 312 G. proxima C2 2:35 2.39 25 2.56 2.33 G. semicarinata 3.42 2.83 2.38 1.31 3.05 G. catenaria 4.18 3.46 2.86 1.18 129 Figure 2. The six unique mitochondrial CO] sequence fragments diagramed by their maximum likelihood distances. The circles are Goniobasis proxima (A, B, C1, C2), the square is Gontobasis semt- carinata (Gs), and the triangle is Goniobasis catenaria (Gc). Thick segments join haplotypes that are less than 10% different, thin segments connect haplotypes ranging from 10%-15% different, and dashed segments join haplotypes of greater than 15% maximum- likelihood distance. DISCUSSION The level of sequence divergence we observed within and among conspecific populations of Goniobasis proxima was exceptionally high. A review of the molluscan literature suggests that intraspecific divergence in either of the genes we examined here typically ranges no higher than 5%. This generalization holds true for marine gastropods (Simison and Lindberg 1999, Collin 2000, Hamm and Burton 2000, Wilding et al. 2000, Wilke and Davis 2000), marine bivalves (Geller et al. 1993, @ Foighil et al. 1996, Chase et al. 1998, O Foighil et al. 1998, Baco et al. 1999), and freshwater bivalves (King et al. 1999, Stepien et al. 1999, Renard et al. 2000). A notable exception occurs in the sex-specific haplotypes of certain bivalves, which may differ by as much as 30% (Hoeh et al. 1996, 1997). Levels of sequence divergence among con- specific populations of land snail are also generally reported to reach maxima higher than the 5% typical for most mol- luscs: 5.3% in Partulina Pfeiffer, 1854 (Thacker and Hadfield 2000), 8% in Candidula (Pfenninger and Magnin 2001), 8.4% in Discus (Ross 1999), 9.5% in Euhadra Pilsbry, 1890 74 Table 3. Comparisons of six mitochondrial 16S sequence fragments amplified from three species of Goniobasis. The diagonal gives the sequence length, including both primer regions. Above the di- agonal are percent nucleotide differences (p distances), where the denominator is the sum of the sequence length and its corresponding apparent number of indel bases. Below the diagonal is the number of indels, recorded as the apparent number of deletions in the column sequence (total bases) over the number of deletions in the row sequence (total bases). AMERICAN MALACOLOGICAL BULLETIN 19+ 1/2 * 2004 populations of freshwater gas- tropods. Lydeard et al. (1998) compared 8 individual Gonio- basis (or Elimia) carinocostata (Lea, 1845) from five sites and obtained a maximum 16S se- quence divergence of 3.9%. A B Cl C2 Gis. Gc. The maximum CO1 divergence G. proxima A 528 6.1 7.4 Zit 11.2 12.5 among conspecific individuals G. proxima B 0/3(3) 525 6.2 16.4 9:3 12.9 of Tryonia from Death Valley a. proxima 1(2)/0 3(5)/0 530 14.5 9.8 12.8 was 5.2% (Hershler et al. G. proxima C2 3(5)/2(2) 4(6)/0 4(7)/2(6) 531 14.7 i) 1999b). Within subspecies of G. semicarinata 0/2(4) 1(1)/2(2) 0/3(6) 1(1)/4(8) 524 11.8 Oncomelania hupensis, the G. catenaria 2(7)/3(3) 3(8)/1(1) 4(8)/4(6) 8(12)/3(11) 3(8)/0 532 : ¢ ‘ maximum COl1 divergence seemed to average around 2.1% (Davis et al. 1999), while maxima between the subspe- cies reached 14.2-15.3% (Davis et al. 1998). In Gontobasis proxima, we have discovered maximum divergences of p = 14.7% or ML = 16.9% for the CO1 gene and p = 17.1% or ML = 18.7% for the 16S gene. These rank among the highest intraspecific value for sequence diver- gence yet reported for molluscs. Thomaz ef al. (1996) sug- ? gested four (overlapping) explanations for the high levels of f sequence divergence they observed among populations of iD | ‘ the land snail Cepaea nemoralis (Linné, 1758): a large effec- - ! cr tive population size, fragmentation of the range into isolated “| Figure 3. The six unique mitochondrial 16S sequence fragments diagramed by their maximum likelihood distances. The circles are Gontobasis proxima (A, B, Cl, C2), the square is Goniobasis semi- carinata (Gs), and the triangle is Goniobasis catenaria (Gc). Thick segments join haplotypes that are less than 10% different, thin segments connect haplotypes ranging from 10%-15% different, and dashed segments join haplotypes of greater than 15% maximum- likelihood distance. (Hayashi and Chiba 2000), 11.1% in Helix Linné, 1758 (Guiller et al. 2001), 12.9% in Cepaea (Thomaz et al. 1996), 13% in Arianta Leach in Turton, 1831 (Haase et al. 2003) and 18.7% in Mandarina (Chiba 1999). It is difficult to generalize regarding the levels of se- quence divergence previously reported among conspecific demes, disruptive selection, and a systemically higher rate of mitochondrial evolution. Of these four, the factor most con- spicuously shared by the land and freshwater snails (but not by marine molluscs or by bivalves generally) is population fragmentation. Our three populations of G. proxima shared no connection through water and may have been isolated for millions of years. Our sample of three Race C snails included a pair of strikingly divergent haplotypes for both the genes we exam- ined. The population from which these snails were drawn (“Phlp” of Dillon 1984a) is homogeneous both in morphol- ogy and in gene frequency at seven enzyme-encoding nuclear genes. Apparently, conspecific pleurocerids sampled from adjacent rocks may show more mitochondrial DNA sequence divergence than verifiably distinct species isolated by 400 km overland. Data addressing the possible existence of intermediate forms between these two diverse mitochon- drial haplotypes in the Phlp population would cast light on whether such great genetic diversity may have evolved in situ or might reflect the admixture of two previously isolated populations. The levels of sequence divergence we observed within and among populations of Goniobasis proxima exceeded their (interspecific) divergence with Goniobasis semicarinata and Goniobasis catenaria in many cases. For the CO1 gene, haplotypes A and B were more similar to G. semicarinata or MITOCHONDRIAL DIVERGENCE IN POPULATIONS OF GONIOBASIS SPP. 75 proxima semicarinata catenaria Figure 4. The single most parsimonious tree (CI = 0.816, HI = 0.184) returned by phylogenetic analysis where Goniobasis semi- carinata was specified as root. Bootstrap values (percent of 1,000 replicates) are indicated at the nodes. G. catenaria than to either Cl or C2, regardless of the metric employed (Table 1, Fig. 2). For the 16S gene, haplotype C2 was so strikingly distinct as to render all the other five hap- lotypes neighbors by comparison (Table 3, Fig. 3). Phylogenetic analysis of the combined data set under the parsimony criterion suggested that Goniobasis proxima is paraphyletic with respect to either of its congeners in the American southeast. This may be a consequence of “lineage sorting” as depicted in Fig. 4 (Takahata and Nei 1985, Rosenberg 2003). The bootstrap support for most of the branches in the phylogeny was not high, however, and the tree topology may reflect more noise than signal. There is some evidence that the sites available for varia- tion in these genes may be approaching saturation in this sample of populations of Goniobasis. Haplotypes A and B appeared to be the most similar by almost all measures, and displayed an exceptionally high Ts : Tv ratio (Table 2). Omitting the A/B comparison for CO1, the overall average Ts : Tv ratio across the six populations droped from 4.14 to 3.24. The relationship between B and C1 was also apparently close (judging from 16S data) and characterized by a high Ts : Tv ratio. Setting aside these two individual comparisons, however, the Ts : Tv ratios we obtained were generally less than 4: 1. It is interesting to note that the greatest difference in CO1 amino acid sequence (5.5%) was posted between Goniobasis proxima B and C1, which the maximum likeli- hood analysis of 16S sequence data suggested as the most similar pair of populations. Our observation that interspe- cific differences in amino acid sequence were strikingly lower than intraspecific differences in most cases (Table 1) further suggests that sequence divergence may be approaching satu- ration in these highly isolated populations of freshwater snails. Under such circumstances, systematic inference must be made with care. The existence of a high level of sequence divergence can apparently be interpreted as little evidence that a pair of freshwater snail populations is specifically dis- tinct. The only other pleurocerid populations for which data are available on both gene frequencies at nuclear loci and mitochondrial sequence divergence are the Leptoxis spp. of Alabama. Lydeard et al. (1997) reported up to 19.4% se- quence divergence for the 16S gene among single individuals of three nominal species: Leptoxis ampla (Anthony, 1855), Leptoxis taeniata (Conrad, 1834), and Leptoxis picta (Con- rad, 1834). These results appeared incompatible with a much larger data set on gene frequencies at nine enzyme-encoding loci in six populations (30 individuals per population), which suggested that the three nominal species might be conspecific (Dillon and Lydeard 1998). It now seems appar- ent that an uncorrected sequence difference as high as 19% for the 16S gene does not necessarily contradict the conspe- cific hypothesis for pleurocerid populations. An observation of low levels of divergence may consti- tute some evidence that a pair of populations is conspecific, however. In addition to their data on the three individuals of Leptoxis spp. noted above, Lydeard and his colleagues (Lydeard et al. 1997, 1998, Holznagel and Lydeard 2000) have reported sequence data from the 16S rDNA gene for over 30 species (representing five genera) of North American pleurocerid snails. Their sampling effort has focused on the Mobile Basin of Alabama because of its putatively high pleu- rocerid diversity, and has been directed toward elucidating higher-level evolutionary relationships. Like most of the American freshwater gastropod fauna, however, the tax- onomy of the Alabama Pleuroceridae predates the modern synthesis, being based almost entirely upon minor attributes of the shell. The maximum divergence among the individual snails representing seven nominal species of Alabama Go- niobasis sequenced by Lydeard et al. (1997) was only 7.89% (uncorrected), with many pairwise values less than 2%. A critical re-examination of the biological species of pleuro- cerid snails in the Mobile Basin, and throughout most of North America, is long overdue. ACKNOWLEDGMENTS Laboratory assistance was provided by Shela Patel, Jen- nifer Ivey, Zachery Evans, and Jessica Pease. We thank Allan 76 AMERICAN MALACOLOGICAL BULLETIN Strand, Chuck Lydeard, and especially Amy Wethington for technical advice and assistance. Allan Strand and Matthias Glaubrecht provided helpful comments on an earlier draft of the manuscript. This research was funded by grants from the Department of Biology and College of Charleston R&D Committees. LITERATURE CITED Baco, A. R., C. R. Smith, A. S. Peek, G. K. Roderick, and R. C. Vri- jenhoek. 1999. The phylogenetic relationships of whale-fall vesicomyid clams based on mitochondrial COl DNA se- quences. Marine Ecology Progress Series 182: 137-147. Chase, M. R., R. J. Etter, M. A. Rex, and J. M. Quattro. 1998. Bathy- metric patterns of genetic variation in a deep-sea protobranch bivalve, Deminucula atacellana. Marine Biology 131: 301-308. Chiba, S. 1999. Accelerated evolution of land snails Mandarina in the oceanic Bonin Islands: Evidence from mitochondrial DNA sequences. Evolution 53: 460-471. Collin, R. 2000. 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Abstract: Survey data from electronic databases and the literature were used to summarize knowledge of the composition and geographic distribution of Virginia’s freshwater gastropod fauna. After excluding records likely based on misidentifications, we concluded that 53 species of freshwater gastropods occur in Virginia now or historically. A map and/or narrative description of statewide distribution was produced for each species. Several species appeared to be restricted to a few sites and highly endangered, including the hydrobiids Fontigens bottimeri, Fontigens morrisoni, Holsingeria unthankensis, and Holsingeria sp. 1. Absence of recent records for the hydrobiid Somatogyrus virginicus, the pomatiopsid Pomatiopsis cincinnatiensis, the pleurocerids Elimia arachnoidea and Pleurocera gradata, and the lymnaeid Stagnicola neopalustris indicated these species might also be imperiled if not already extirpated from Virginia. Although we have a good understanding of distributions of Fontigens spp., Holsingeria spp., and of several river-dwelling pleurocerids in southwest Virginia, other species and geographic regions (e. g., eastern shore and Big Sandy River drainage) are undersurveyed. We provide data to assist in designing surveys to fill these knowledge gaps and to monitor temporal changes in species’ distributions. Comparisons of historic and future data from field surveys will facilitate protection and management of endangered species by providing evidence of restricted or shrinking geographic ranges. Key words: Macroinvertebrates, biogeography, endangered species, snails In 1817, Thomas Say published the first descriptions of species of freshwater gastropods in North America (Say 1817, Martin 1999). Although our understanding of this continent’s freshwater gastropod fauna has advanced since that time, large gaps remain. For example, taxonomic con- fusion precludes accurate estimates of numbers of species inhabiting North America and makes it difficult to deter- mine the geographic distribution, environmental require- ments, ecological importance, and conservation status for many taxa (Neves et al. 1997). Additionally, many species are thought to have experienced dramatic population declines, but quantitative evidence to confirm this is rarely available. It has been determined, however, that at least 42 species have become extinct following European settlement of North America, and living specimens of several other species have not been seen in nearly a century (Neves 1991, Neves et al. 1997, Bogan 1998). Clearly, the lack of attention to fresh- water gastropods has been costly. Field surveys provide critical evidence of changes in freshwater gastropod assemblages, including population de- clines and shrinking or restricted geographic ranges. Al- though large quantities of survey data exist for many North American species, many data are scattered among museum collections and unpublished and published literature that are difficult to obtain. These data must be summarized and disseminated to gain a complete understanding of the dis- tribution and conservation status of our freshwater gastro- pods and to identify geographic regions requiring additional surveys. We reviewed and summarized data from field surveys in electronic databases and the literature to describe the species composition and geographic distribution of freshwater gas- tropods in Virginia, U.S.A. Geographic information associ- ated with collection records was used to produce maps and narrative descriptions of distributions of species inhabiting Virginia now or historically. This is the first comprehensive review of Virginia’s freshwater gastropod fauna in 30 years. Beetle (1973a) used museum collections to construct a checklist of Virginian species with names of counties or cit- ies, but not specific locality data. Burch (1950) and Burch (1952) published regional species checklists for the James River Basin and Hanover County. Other surveys in Virginia concentrated on specific drainage basins or taxonomic groups (Clench and Boss 1967, Stansbery and Clench 1974a, Stansbery and Clench 1974b, Stansbery and Clench 1977, Dillon and Benfield 1982, Hershler et al. 1990). *From the symposium “The Biology and Conservation of Freshwater Gastropods” presented at the annual meeting of the American Malacological Society, held 3-7 August 2002 in Charleston, South Carolina, USA. 80 AMERICAN MALACOLOGICAL BULLETIN METHODS Study area Virginia extends from 36°30'N to 39°30'N in latitude, and from 75°13'W to 83°40'W in longitude. Approximately 102,830 km* is contained within state boundaries (Wood- ward and Hoffman 1991). Because of Virginia’s variable physiography and climate, the state supports a diverse as- semblage of freshwater species. Many boreal species reach the southern limit of their geographic distribution in north- ern Virginia and many austral species reach the northern limit of their ranges in southern Virginia (Woodward and Hoffman 1991). Additionally, Virginia’s major rivers flow in different directions, restricting gene flow between popula- tions and contributing to high species richness. Rivers east of the New River flow southeast to the Atlantic Ocean, the New River flows north to the Ohio River, the Levisa and Russel Rivers of the Big Sandy watershed flow northwest to the Ohio, and other rivers west of the New River flow southwest to the Tennessee River (Fig. 1, Woodward and Hoffman 1991). Study design Distributional information was obtained from (1) pub- lished literature, (2) unpublished literature authored by R. T. Dillon, (3) museum records, and (4) the Virginia Depart- ment of Game and Inland Fisheries. With exception of un- attainable publications, we reviewed all peer-reviewed litera- ture that might contain records of freshwater gastropods in Virginia. Information from one unpublished report and a 19° 1/2 + 2004 Ph.D. dissertation was also used (Dillon 1977, Dillon 1982). We also included all museum records available on the World Wide Web, specifically those in the Florida Museum of Natural History (FMNH 2002) and the Illinois Natural His- tory Survey Mollusk Collection (INHS 2003). The Virginia Museum of Natural History (VMNH) also provided records. Additional records were obtained from the Virginia Fish and Wildlife Information Service (VDGIF 1998). A map and/or narrative description of geographic dis- tribution was produced for each species occurring in Vir- ginia now or historically. Records likely resulting from misidentified species were excluded from maps, but are dis- cussed. Species’ names and their authorities were based on Turgeon et al. (1998). RESULTS AND DISCUSSION Our review suggests that 53 species of freshwater gas- tropods occur in Virginia now or historically. Distributions and ecological requirements for these taxa are discussed be- low. Our review also uncovered records of eight species that we consider questionable. Records for questionable species are provided with rationale for why we feel they never oc- curred in Virginia (Table 1). Family Valvatidae Valvata tricarinata (Say, 1817). This species reaches the southern extent of its geographic range in Virginia (Clarke | he James ) ¢ y ) | nen me | fe \ ae / > ()y, A { fb , \.. / Appomatto (| Je aes a & Me. ~ a Nottowayx “4 oar |! Me, ¥ ") - ‘ fo Meherrj vA \ \ / | Figure 1. Major rivers of Virginia. Arrows indicate direction of flow (map adapted from Woodward and Hoffman 1991). FRESHWATER GASTROPODS OF VIRGINIA 81 Table 1. Although these species were reported from Virginia, it is unlikely that they ever occurred there. Species Records (and reference) Rationale for conclusion Family Hydrobiidae Birgella subglobosus Pyrgulopsis lustrica Cincinnatia integra Family Pleuroceridae Elimia carinifera Norfolk (FMNH 2002) Tazewell County (Beetle 1973a) Leptoxis clipeata Roanoke River, Montgomery County (FMNH 2002) Augusta and Washington Counties (Burch 1950, FMNH 2002) Lithasia obovata Family Lymnaeidae Stagnicola caperata (Beetle 1973a) Stagnicola oronoensis Fairfax County (Thompson 1977, 1984) Fairfax County (Thompson 1977, 1984) Fairfax, Page, and Shenandoah Counties Gretna, Pittsylvania County (FMNH 2002) No other records occur near Virginia (Thompson 1977, 1984) See Birgella subglobosus Occurs in the midwestern United States (Burch 1989) Inhabits Alabama River drainage and southern Tennessee River drainage (Burch 1989) Now extinct, but occurred in the Coosa River drainage, Alabama (Palmer 1985, Burch 1989) Occurs in Ohio River drainage, including states adjacent to Virginia (Burch 1989) A northern species that occurs as far south as Maryland (Burch 1989) Inhabits Maine and Ontario (Burch 1989) 1981, Burch 1989, VDGIF 1998). Valvata tricarinata is re- stricted to calcium-rich, permanent, slow-moving waters in- cluding lakes and backwaters of large rivers (Baker 1928a, Jokinen 1983, Strayer 1987). Such habitats occur in western Virginia, where this snail has most often been encountered (Woodward and Hoffman 1991, VDGIF 1998). Valvata tri- carinata has been recorded from Fairfax County, from the Powell River near Back Valley in Lee County, and from the Clinch River near Honaker and Fort Blackmore in Russell and Scott counties (Figs. 1-2, Beetle 1973a, VDGIF 1998). Family Viviparidae Viviparus georgianus (Lea, 1834). This is one of four freshwater gastropods to invade Virginia. Presumably due to introductions by aquarists, this native of Florida, Georgia, and Alabama is now discontinuously distributed across the United States (Clench 1962, Clench and Fuller 1965, Mills et al. 1993). Populations occupy soft substrates in large, slow- moving bodies of water (Clench and Fuller 1965, Clarke 1981, Strayer 1987). Viviparus georgianus has been recorded from the Potomac River near Hunter’s Point, from Little Hunting Creek and Mount Vernon in Fairfax County, from Great Creek in Chesterfield County, and from the Wythe- ville Fish Hatchery in Wytheville, Wythe County (Clench 1962, FMNH 2002). Cipangopaludina chinensis (Reeve, 1863) (= Viviparus chinensis, Viviparus malleatus |Reeve, 1863]). This Asian snail has invaded much of North America (Jokinen 1982, Mills et al. 1993). It inhabits soft substrata in static or slow- moving waters (Clarke 1981). It is rare in Virginia, but has been reported from the Dyke Marsh in Fairfax County and from the Azalea Gardens in Norfolk (VMNH). Campeloma decisum (Say, 1817) (= Campeloma decisa, Campeloma integra Say, 1821, Campeloma rufa {Haldeman, 1841]). Taxonomic confusion surrounds the genus Cam- peloma (Baker, 1928a). Although we found records of Cam- peloma crassula (Rafinesque, 1819) and Campeloma limum (Anthony, 1860), these species appear to occur outside of Virginia (Clench and Boss 1967, Dillon 1977, Burch 1989). Thus, we assigned all records of Campeloma to C. decisum. Campeloma has been collected from rivers and streams within the Atlantic, New River, and Tennessee River drain- ages (Fig. 3A, Goodrich 1913, Burch 1950, Burch 1952, Clench and Boss 1967, Beetle 1973a, Dillon 1977, FMNH 2002, VDGIF 1998, VMNH). This burrowing snail is often abundant in low-gradient rivers and pools with soft sub- strates (Clench 1962, Clarke 1981, Jokinen 1983). Lioplax subcarinata (Say, 1816). This species occurs on soft substrates in the Atlantic drainage (Baker 1928a, Clench and Turner 1955, Burch 1989). It has been recorded from Swift Creek in Chesterfield County, the James River near Cartersville in Cumberland County, near Maidens in Powhatan County, Mount Vernon and the Potomac River near Great Falls in Fairfax County, and Fluvanna County (Clench and Turner 1955, Clench and Boss 1967, Beetle 1973a, FMNH 2002). Family Bithyniidae Bithynia tentaculata (Linnaeus, 1758) (= Bulimus ten- taculatus). Most authors feel that North American popula- tions of B. tentaculata descended from European snails in- troduced into the Great Lakes in the 19" century (Baker 1928b, Mills et al. 1993). This species is common in the Great Lakes, but uncommon in Virginia where it reaches the Counties Accomack Albemarle Alleghany Amelia Amherst Appomattox Arlington Augusta Bath Bedford . Bland . Botetourt Brunswick . Buchanan . Buckingham . Campbell . Caroline . Carroll . Charles City . Charlotte . Chesterfield . Clarke . Craig . Culpeper . Cumberland . Dickenson CONDON BWN s© ee NNN NY WY BRWN oO NN NN AMERICAN MALACOLOGICAL BULLETIN 19+ 1/2 * 2004 : i‘ 30 \ > 7 be on he ee Ge Y67 AD SW Y PO. 24 fee on N56) Sf 86 £ ie = 739. os a Sas 7 LL y 85 o\ } 9 I a Lisa a 228, Ss MA SO te 1 oo oe 50km 3; / — A, a Peat, 2 S19 4 ; a. gee Be I Pe Ae a 4. > 2 60 - a fe ‘ e Aes ae se Te A aac i pat } 5 ree 2 a Nee a ey ear mel L Oe a ra] 2 | Va] fa 28. Essex 55. Lunenburg 82. Shenandoah 29. Fairfax 56. Madison 83. Smyth 30. Fauquier 57. Mathews 84. Southampton 31. Floyd 58. Mecklenburg 85. Spotsylvania 32. Fluvanna 59. Middlesex 86. Stafford 33. Franklin 60. Montgomery 87. Surry 34. Frederick 61. Nelson 88. Sussex 35,. Giles 62. New Kent 89. Tazewell 36. Gloucester 63. Northampton 90. Warren 37. Goochland 64. Northumberland 91. Washington 38. Grayson 65. Nottoway 92. Westmoreland 39. Greene 66. Orange 93. Wise 40. Greensville 67. Page 94. Wythe 41. Halifax 68. Patrick 95. York 42. Hanover 69. Pittsylvania 43. Henrico 70. Powhatan Independent Cities 44. Henry 71. Prince Edward 96. | Chesapeake 45. Highland 72. Prince George 97. Hampton 46. Isle of Wight 73. Prince William 98. | Newport News 47. James City 74. Pulaski 99. Norfolk 48. King and Queen 75. Rappahannock 100. Poquoson 49. King George 76. Richmond 101. Portsmouth 50. King William 77. Roanoke 102. Suffolk 51. Lancaster 78. Rockbridge 103. Virginia Beach 32. Lee 79. Rockingham 53. Loudoun 80. Russell 54. Louisa 81. Scott i) —~ . Dinwiddie Figure 2. Counties and selected independent cities of Virginia (map adapted from Woodward and Hoffman 1991). FRESHWATER GASTROPODS OF VIRGINIA 83 A Campeloma decisum Amnicola limosus ie, i eee Figure 3. Distributions of (A) Campeloma decisum, (B) Gillia altilis, (C) Amnicola limosus, (D) Fontigens nickliniana, (E) Fontigens orolibas, (F) Elimia catenaria, (G) Elimia semui- carinata, and (H) Elimia clavaeformis in Virginia. Shading indicates counties and inde- pendent cities where the taxon has been found. Specific localities of occurrence, if known, are indicated by dots. Filled dots represent records collected during or after 1952. Unfilled circles indicate earlier records. See figures 1-2 for names of rivers, counties, and inde- pendent cities. southern extent of its North American range (Baker 1928a, Burch 1989). It has been recorded from Rockbridge County, and from the Potomac River near Alexandria and Mount Vernon in Fairfax County in the 1920s and 1930s (Pilsbry 1932, Marshall 1933, Beetle 1973a, Dundee 1974). Family Hydrobiidae Littoridinops tenuipes (Couper, 1844) (= Amnicola tenu- ipes, Bythinella tenuipes). This species inhabits the Atlantic drainage in streams that are brackish for part of the year (Pils- bry, 1952; Hershler and Thompson, 1992). It has been reported from Hampton, Newport News, and Norfolk cities, and from King George and Northampton counties (Beetle 1973a). Gillia altilis (Lea, 1841). This species is common in in- land rivers and streams (Fig. 3B, Clench and Boss 1967, Gillia altilis OA Q A TASES BEES ER i. ; ” [ay p k ie Elimia clavaeformis IS? Deak Beetle 1973a, Thompson 1984, FMNH 2002). Most authors consider G. altilis to be restricted to the Atlantic drainage, so Beetle’s (1973a) record from Lee County, Virginia (Tennessee drainage) should be examined further (Thompson 1984, Burch 1989). Somatogyrus virginicus (Walker, 1904). This is one of the most endangered species of freshwater gastropods in North America. The species was described from a population in the Rapidan River at Bar- nard’s Ford, Culpeper County, Virginia (Walker 1904a). Neves (1991) recognized the rarity of records of S. virginicus for Virginia, but was unsure if the Rapidan had been sampled after 1904. Somatogy- rus virginicus has also been reported from North Carolina, but its status there is also uncertain (Neves et al. 1997). Somatogy- rus virginicus is a candidate for federal an endangered species status (Neves ef al. 1997). Amnicola limosus (Say, 1817) (= Am- nicola limosa). This species occurs in a variety of habitats of the Atlantic drain- age, including lakes, rivers, and perma- nent streams (Fig. 3C, Rehder 1949, Beetle 1973a, Hershler and Thompson 1988, FMNH 2002, VDGIP 1998). Lyogyrus granum (Say, 1822) (= Am- nicola grana). This species is also re- stricted to the Atlantic drainage, and is often associated with physical structure in eutrophic, slow-moving, permanent wa- ters (Clarke 1981, Burch 1989). It has been reported from Norfolk City, and from Fairfax, Hanover, Henrico, and New Kent counties (Beetle 1973a). Holsingeria unthankensis (Hershler, 1989). The “thank- less ghostsnail” was described as a tiny, pale-colored, stream- dwelling snail inhabiting undersides of stones in Unthanks Cave, Lee County, Virginia (Hershler 1989, Kabat and Hershler 1993). This species is listed as “endangered” by the Virginia Department of Conservation and Recreation, but has not been provided federal protection under the Endan- gered Species Act (Roble 2001). Before Hershler’s (1989) taxonomic revision, H. unthankensis and the yet unnamed Holsingeria sp. 1 were grouped under the name Fontigens holsingerit (Burch 1989, Kabat and Hershler 1993). Holsingeria sp. 1 sensu Hershler (1989). Populations of the “skyline caverns snail” have been collected only from 84 AMERICAN MALACOLOGICAL BULLETIN streamside pools in Skyline Caverns, Warren County, Vir- ginia (Hershler 1989, Batie 1991). Although its taxonomic status remains uncertain, this species is certainly imperiled due to limited geographic range (Roble 2001). Fontigens nickliniana (Lea, 1838). This species and its congeners occur in cool, calcium-rich waters of western Vir- ginia (Hershler et al. 1990). It inhabits caves, springs, streams, and small lakes (Fig. 3D, Haldeman 1842, Goodrich 1913, Baker 1928a, Burch 1950, Beetle 1973a, Holsinger and Culver 1988, Hershler et al. 1990, Richardson et al. 1991, FMNH 2002, VDGIF 1998, VMNH). Hershler et al. (1990) list synonyms for this species. Fontigens orolibas (Hubricht, 1957). This species inhab- its springs and cave streams in western Virginia (Fig. 3E, Haldeman 1840-1845, Hubricht 1957, Beetle 1973a, Hols- inger and Culver 1988, Hershler et al. 1990, Richardson et al. 1991, FMNH 2002, VMNH). Hershler et al. (1990) list syn- onyms for this species. Fontigens morrisoni (Hershler, Holsinger, and Hubricht, 1990). This is another endangered species that is not pro- tected by law, although it is found at only four sites: Blowing and Butler caves in Bath County, and two springs near Mus- toe in Highland County (Hubricht 1976, Holsinger and Cul- ver 1988, Hershler et al. 1990, Roble 2001). Environmental requirements and life history features are poorly known. Fontigens bottimeri (Walker, 1925) (= Paludestrina bot- timeri). This species is limited to a few caves and springs, but has not been granted legal protection (Hershler et al. 1990, Roble 2001). In Virginia, it is known only from Ogden’s Cave in Frederick County (Hershler et al. 1990). Family Pomatiopsidae Pomatiopsis cincinnatiensis (Lea, 1840). Limited historic distribution and absence of recent records suggest P. cin- cinnatiensis is imperiled in or extirpated from Virginia. Goodrich (1913) recorded this species from brooks near Cleveland, in Russell County, and Beetle (1973a) reported sightings from Lee and Scott counties. Pomatiopsis cincin- natiensis is described as a semiaquatic species (van der Schalie and Dundee 1955). Its habitat consists of a narrow, moist zone on riverbanks (Baker 1928a, van der Schalie and Getz 1962). Pomatiopsis lapidaria (Say, 1817). This species is also semiaquatic (Baker 1931, Berry 1943). It has more general- ized habitat requirements than Pomatiopsis cincinnatiensis, and occurs in swampy areas and wet pastures as well as stream edges (Baker 1931, van der Schalie and Dundee 1955). Beetle (1973a) recorded this species from Arlington, Buchanan, Fairfax, Giles, Grayson, Lee, Mecklenburg, Mont- gomery, New Kent, Patrick, Prince William, Scott, Smyth, Washington, and Wythe counties and from the city of New- port News. 19° 1/2 + 2004 Family Pleuroceridae Elimia arachnoidea (Anthony, 1854) (= Goniobasis arachnoidea, Goniobasis spinella Lea, 1862). This species is limited to small streams in Tennessee and southwestern Vir- ginia (Goodrich 1940, Burch 1989). Goodrich (1913) re- corded populations from Little and Big Moccasin Creeks near Gate City in Scott County, and Goodrich (1940) found it in Lee County. Lack of recent records for E. arachnoidea makes its present status unclear. Elimia catenaria (Say, 1822) (= Goniobasis catenaria). This species of the Atlantic Coastal Plain has been recorded from several streams and rivers in southcentral Virginia (Fig. 3F, Goodrich 1942, VMNH). Elimia semicarinata (Say, 1829) (= Goniobasis semicari- nata). This species occurs in the upper New River and sur- rounding tributaries and in Campbell and Greensville coun- ties (Fig 3G, Goodrich 1942, Dillon 1977, Dillon and Davis 1980, Dillon 1982, VDGIF 1998). The discontinuous distri- bution probably indicates an incomplete understanding of its geographic range. Elimia aterina (Lea 1863) (= Goniobasis aterina). This rare species is restricted to a few springs and small streams in Tennessee and southwest Virginia (Goodrich 1913, Burch 1989, FMNH 2002). It has been recorded from Beaver Creek near Bristol in Washington County, and from Stock Creek and a mountain brook near Gate City in Lee County (Good- rich 1913, FMNH 2002). The most recent record was from 1914, so the present status of E. aterina is uncertain. Elimia clavaeformis (Lea, 1841) (= Goniobasis clavaefor- mis). This species is restricted to streams and small rivers in the Tennessee River drainage (Goodrich 1940, Burch 1989). It occurs in tributaries of the Powell and Holston Rivers (Fig. 3H, Goodrich 1913, Beetle 1973a, Dillon 1989, FMNH 2002, VDGIF 1998). Elimia simplex (Say, 1825) (= Goniobasis simplex). This species occurs in the Tennessee and New River drainages (Fig. 4A, Goodrich 1940, Burch 1989). Populations in the New River drainage are restricted to a few small creeks (Fig. 4A, Dillon 1977, Dillon and Davis 1980, Dillon 1982). In the Tennessee drainage, however, E. simplex occurs in both small and large rivers, including the Clinch and Holston rivers (Fig. 4A, Say 1825, Tryon 1873, Goodrich 1913, Stans- bery and Clench 1974a, 1974b, 1977, Goudreau et al. 1993, FMNH 2002, VDGIF 1998, VMNH). This species has also been collected from cave streams (Holsinger 1964, Holsinger and Culver 1988). Elimia proxima (Say, 1825) (= Elimia symmetrica {Hal- deman, 1841], Goniobasis proxima, Goniobasis symmetrica). This species inhabits Atlantic and Tennessee drainages (Goodrich 1942, Goodrich 1950, Dillon and Keferl 2000). Populations inhabit small streams in addition to large rivers or FRESHWATER GASTROPODS OF VIRGINIA 85 A Elimia simplex Zn, «30k is Figure 4. Distributions of (A) Elimia simplex, (B) Elimia proxima, (C) Elimia virginica, (D) Io fluvialis, (E) Leptoxis praerosa, (F) Leptoxis carinata, (G) Leptoxis dilatata, and (H) Pleurocera uncialis in Virginia. Shading indicates counties and independent cities where the taxon has been found. Specific localities of occurrence, if known, are indicated by dots. Filled dots represent records collected during or after 1952. Unfilled circles indicate earlier records. The star in (B) indicates the location of a population resulting from an intro- duction by Dillon (1986). The star in (D) indicates the location of a population reestab- lished as a result of reintroduction efforts (Ahlstedt 1991). See figures 1-2 for names of rivers, counties, and independent cities. B aS Elimia proxima See, Io fluvialis (Say, 1825) (= Fusus flu- vialis, Io brevis Anthony in Reeve, 1860, Io clinchensis Adams, 1914, Io lyttonenesis Adams, 1914, Io paulensis Adams, 1914, Io powellensis Adams, 1914, Io spinosa Lea, 1837). The “spiny riversnail” occurs in some large tributaries of the Tennessee River, but only in flowing, well oxygen- ated habitats with abundant limestone (Fig. 4D, Say 1825, Tryon 1873, Adams 1900, Adams 1915, Goodrich 1913, Clench 1928, Lutz 1951, Dazo 1961, Beetle 1973a, Stansbery and Clench 1974a, Stansbery and Stein 1976, McLeod and Moore 1978, Ahlstedt 1979, Ahlstedt 1991, Neves ef al. 1997,. PMNEH 2002, INHS 2003, VDGIF 1998, VMNH). Dra- matic population declines of I. fluvialis from 1900 through the 1970s were docu- mented through field surveys (Adams 1915, Lutz 1951, Stansbery and Stein 1976, McLeod and Moore 1978). Habitat degradation caused by deforestation and industrial pollution destroyed several Virginian populations during this time, including all of those in the North Fork Holston River south of Saltville, Smyth County (Adams 1915, Ahlstedt 1979, Ahlstedt 1991). Due to its small numbers, specific habitat requirements, and con- tinuing threats to its survival, I. fluvialis is classified as “threatened” by the Virginia Department of Conservation and has been considered for protection under the federal Endangered Species Act (Neves et al. 1997, Roble 2001). Attention directed spray zones of falls or springs (Fig. 4B, Tryon 1873, Burch 1950, Goodrich 1950, Dillon 1977, 1982, 1988, Dillon and Davis 1980, Dillon and Keferl 2000, FMNH 2002, VMNH). A population in Coyner Springs, Augusta County, consists entirely of descendents from snails introduced from tribu- taries of the Dan and New Rivers (Fig. 4B, Dillon 1986). Elimia virginica (Say, 1817) (= Gontobasis virginica). This is the most abundant and widespread species of Elimia in large rivers of the Atlantic drainage (Fig. 4C, Burch 1950, Burch 1952, Clench and Boss 1967, Beetle 1973a, FMNH 2002, VDGIF 1998, VMNH). On rare occasions, it is re- corded from small streams (Fig. 4C, VMNH). Based on its established range, we concluded that a report of E. virginica from Lee County is in error (Goodrich 1942, Burch 1989, FMNH 2002). to I. fluvialis has had positive effects. Pol- lution abatement programs in the 1970s enabled successful reestablishment of [. fluvialis in part of its historic range. Ahlstedt (1979) contributed to this recovery in 1978 by re- introducing this species to two sites on North Fork Holston River where it had been absent for almost 100 years. By 1986, Ahlstedt (1991) saw evidence of reproduction and increased population densities at downstream and upstream sites, in- cluding one site in Scott County (Fig. 4D). Leptoxis praerosa (Say, 1821) (= Anculosa praerosa, An- culosa subglobosa Say, 1825, Leptoxis subglobosa, Melania subglobosa). This species also inhabits tributaries of the Ten- nessee River (Fig. 4E, Say 1825, Goodrich 1913, 1940, Stans- bery 1972, Beetle, 1973a, Stansbery and Clench 1974a, 1974b, 1977, Goudreau et al. 1993, Reed-Judkins et al. 1998, FMNH 2002, VDGIF 1998, VMNH). Leptoxis praerosa is 86 AMERICAN MALACOLOGICAL BULLETIN often associated with Jo fluvialis in large rivers, but L. praerosa also occurs in small tributaries that do not support Io fluvialis (Figs. 4D, E). Similar to I. fluvialis, L. praerosa suffered severe population declines during much of the 20" century (AhlIstedt 1979). However, populations of L. praerosa recovered more rapidly than I. fluvialis following water quality improvements. Leptoxis carinata (Bruguiere, 1792) (= Anculosa cari- nata, Leptoxis nickliniana Lea, 1839, Melania nickliniana, Mudalia carinata, Nitrocris carinata, Spirodon carinata). This is the most abundant and widespread pleurocerid in eastern and central Virginia; densities can reach 500 individuals/m° on rocky bottoms of rivers and small creeks (Fig. 4F, Tryon 1873, Pilsbry 1894, Goodrich 1942, Burch 1950, Burch 1989, Clench and Boss 1967, Beetle 1973a, Miller 1985, Dillon 1989, Stewart and Garcia 2002, FMNH 2002, VDGIF 1998, VMNH). Leptoxis carinata is restricted to the Atlantic drain- age, so we did not plot records from Buchanan, Montgom- ery, Pulaski, and Wythe counties (Goodrich 1942, Beetle 1973a, FMNH 2002). Leptoxis dilatata (Conrad, 1835) (= Nitrocris dilatatus, Spirodon dilatata). This species occurs in the New River drainage (Fig. 4G, Tryon 1873, Goodrich 1940, Beetle 1973a, Dillon 1977, Burch 1989, Farris et al. 1994, Reed-Judkins et al. 1998, FMNH 2002). Dillon (1977) found this species to be among the most common molluscs in the upper New River drainage and noted its occurrence in the main river and tributaries. We did not plot records from Alleghany, Amherst, Rockbridge, and Scott counties because these lo- calities are within Atlantic and Tennessee drainages, where L. dilatata is replaced by Leptoxis carinata and Leptoxis praerosa, respectively (Goodrich 1940, Goodrich 1942, Beetle 1973a, FMNH 2002). Pleurocera canaliculata (Say, 1821) (= Pleurocera cana- liculatum). This species is restricted to the Tennessee River drainage. Beetle (1973a) reported this species from Lee, Scott, Smyth, Washington, and Wise counties. Pleurocera gradata (Anthony, 1854) (= Pleurocera gra- datum). This species has not been seen in Virginia in over 100 years. The only record was from the Holston River, Washington County (Tryon 1873). Despite its rarity, the species has not been granted statewide or federal protection (Roble 2001). Pleurocera uncialis (Haldeman, 1841) (= Goniobasis un- cialis, Pleurocera “unciale”). This species occurs in upper tributaries of the Tennessee River (Fig. 4H, Goodrich 1913, 1937, 1940, Beetle 1973a, Stansbery and Clench 1974a, 1974b 1977, Burch 1989, Goudreau et al. 1993, Reed-Judkins et al. 1998, FMNH 2002, VDGIF 1998). It is the most com- mon species of Pleurocera in western Virginia, but pollution has caused declines in abundance (Goudreau et al. 1993). 19 * 1/2 * 2004 Family Lymnaeidae Fossaria spp. The taxonomy of the genus Fossaria is in a confused state, with species distinguished by minor differ- ences in shell attributes that might be ecophenotypic in ori- gin. Records occurred for the following Fossaria “species”: F. humilis (Say, 1822), F. dalli (Baker, 1907), F. galbana (Say, 1825), F. obrussa (Say, 1825), and F. parva (Lea, 1841) (Fig. 5A, Goodrich 1913, Burch 1950, Beetle 1973a, Dillon 1977, Dillon and Benfield 1982, FMNH 2002). Fossaria spp. occur in lakes, ponds, and streams, and can thrive in waters with low levels of dissolved oxygen (Baker 1911, Goodrich 1913, Dillon 1977, Dillon 2000). These snails are often semiaquat- ic, inhabiting moist areas above the water line (Haldeman 1840-1845, Baker 1911, Baker 1928a). Pseudosuccinea columella (Say, 1817) (= Lymnaea colu- mella). This species is found in ponds, lakes, and stream pools across Virginia (Fig. 5B, Rehder 1949, Burch 1950, Burch 1952, Burch and Wood 1955, Beetle 1973a, Dillon 1977, Dillon and Benfield 1982, FMNH 2002). It withstands oxygen fluctuations characteristic of eutrophic habitats, and individuals often occur above the water line on mud and other substrates (Baker 1928a, Jokinen 1983). Radix auricularia (Linnaeus, 1758). This Eurasian spe- cies invaded North America and now occurs at scattered locations (Burch 1989, Mills et al. 1993). Populations fre- quent eutrophic lentic habitats and can be found on mud or plants (Clarke 1979, 1981). In Virginia, R. auricularia has only been reported from Giles County (Beetle 1973a). Stagnicola neopalustris (Baker, 1911). Baker (1911) de- scribed a new species of lymnaeid from Orange township, Orange County, Virginia. This is the only known record for this species. Family Physidae Physella gyrina (Say, 1821) (= Physella ancillaria |Say, 1825], Physella aurea |Lea, 1838], Physella crocata (Lea, 1864], Physella elliptica {Lea, 1831}, Physellaa inflata {Lea, 1841], Physella microstoma {Haldeman, 1840]). Most popu- lations of Physella (= Physa) show little reproductive isola- tion (R. T. Dillon, Jr. pers. comm.). Here we synonymize six nominal species reported from Virginia under the oldest veritable name, P. gyrina. This species can be found in al- most any environment supporting freshwater snails (Clarke 1981, Dillon 2000). However, taxonomic uncertainties and lack of attention directed to pulmonates have resulted in few records for P. gyrina or its synonyms (Fig. 5C, Tryon 1865, Walker 1918, Baker 1928a, Burch 1950, Clench and Boss 1967, Beetle 1973a, Wethington et al. 2000, VMNH). Physella acuta (Draparnaud, 1805) (= Physella hender- sont [Clench, 1925], Physella heterostropha [Say, 1817], Phy- sella pomilia Conrad, 1833). After finding no evidence of reproductive isolation among three species of Physella (= FRESHWATER GASTROPODS OF VIRGINIA 87 B pendent cities. Pseudosuccinea columella H Laevapex fuscus $52. ERS ALB Pe Figure 5. Distributions of (A) Fossaria spp., (B) Pseudosuccinea columella, (C) Physella gyrina, (D) Physella acuta, (E) Gyraulus deflectus, (F) Helisoma anceps, (G) Ferrissia rivu- laris, and (h) Laevapex fuscus in Virginia. Shading indicates counties and independent cities where the taxon has been found. Specific localities of occurrence, if known, are indicated by dots. Filled dots represent records collected during or after 1952. Unfilled circles indicate earlier records. See figures 1-2 for names of rivers, counties, and inde- Aplexa elongata (Say, 1821) (= Ap- lexa hypnorum Linnaeus, 1758). This spe- cies is primarily an inhabitant of vernal freshwater habitats, including temporary woodland pools, but has been found in small streams (Baker 1928a, Clarke 1981). It is rare in Virginia (Beetle 1973a, Joki- nen 1983, Burch 1989). Records exist from Greene and Surry counties (Beetle 1973a, FMNH 2002). Family Planorbidae Gyraulus deflectus (Say, 1824) (= Gy- raulus hirsutus). This species reaches the southern limits of its geographic range in Virginia and adjacent states (Clarke 1981, Burch 1989). This small snail occurs in mesotrophic and eutrophic lakes, large ponds, and quiet areas of rivers (Fig. 5E, Baker 1928a, Burch 1950, Burch 1952, an Burch and Wood 1955, Beetle 1973a, Clarke 1979, Strayer 1987, VDGIF 1998). Gyraulus parvus (Say, 1817). This species 1s commonly found in heavily vegetated lakes and ponds, and occasion- ally lotic habitats (Baker 1928a, Strayer 1987). It has been recorded from Au- gusta, Frederick, Giles, Rockbridge, Shen- andoah, Wythe, and York counties and the city of Newport News (Burch 1950, Beetle 1973a). Helisoma anceps (Menke, 1830) (= Helisoma antrosa Conrad, 1834, Planorbis bicarinatus Say, 1819). This species is Physa) reported from Virginia, including P. acuta, P. hender- soni, and P. heterostropha, Dillon et al. (2002) assigned the name P. acuta to this entire group. Physella acuta could be the most abundant and cosmopolitan freshwater gastropod in the world (Dillon et al. 2002). The snail occurs in streams, rivers, brooks, ditches, permanent and temporary ponds and lakes and is found on hard and soft substrates (Clarke 1981, Jokinen 1983, Strayer 1987). It inhabits oligo-, meso-, and eutrophic waters (Clarke 1979). Taxonomic confusion and lack of attention directed to pulmonates resulted in few Vir- ginian records for P. acuta and its synonyms, although this species probably occurs throughout the entire state (Fig. 5D, Pilsbry 1894, Goodrich 1913, Baker 1928a, Rehder 1949, Burch 1950, Burch 1952, Burch and Wood 1955, Beetle 1973a, 1973b, Dundee 1974, Dillon, 1977, Dillon and Ben- field 1982, VDGIF 1998, FMNH 2002). common throughout Virginia (Fig. 5F, Pilsbry 1894, Walker 1909, Goodrich 1913, Baker 1945, Burch, 1950, Burch 1952, Burch and Wood 1955, Clench and Boss 1967, Beetle 1973a, Dillon 1977, Dillon and Benfield 1982, Burch 1989, FMNH 2002, VDGIF 1998, VMNH). Among planorbids, this species is unusual in that it is most commonly found in lotic habitats, although it also inhabits ponds and lakes (Jokinen 1983, Dillon 2000). Micromenetus brogniartianus (Lea, 1842) (= Menetus brogniartianus). Both global and Virginian distributions of this small planorbid are poorly known (Burch 1989). It was reported from Surrey County and the city of Newport News (Beetle 1973a). Micromenetus dilatatus (Gould, 1841) (= Menetus di- latatus). Baker (1945) recorded this species from the vicinity of Luray, Page County, Virginia. Additional records exist from Culpeper, Fairfax, New Kent, and Prince William 88 AMERICAN MALACOLOGICAL BULLETIN Counties and the cities of Hampton and Newport News (Beetle, 1973a). This species is encountered in vegetated len- tic habitats and also in upland streams (Jokinen 1983, Strayer 1987). Planorbella trivolvis (Say, 1817) (= Helisoma trivolvis). Although this large planorbid is distributed throughout the eastern and midwestern United States, we found few records (Baker 1928a, Burch 1989). It has been reported from the Holston River, near Marion in Smyth County, and from Fairfax County and the cities of Newport News and Norfolk (Beetle 1973a). Planorbella trivolvis occurs in lentic habitats and areas of slow flow in rivers and streams (Baker 1928a, Clarke 1981). Planorbella armigera (Say, 1821). This snail is usually associated with vegetation in perennial, lentic habitats (Baker 1928a, Clarke 1981, Burch 1989). It has been re- ported from Fairfax County and the cities of Hampton, Newport News, and Virginia Beach (Beetle 1973a). Promenetus exacuous (Say, 1821) (= Menetus exacuous). This species inhabits still areas of permanent and vernal freshwater habitats (Baker 1928a, Clarke 1979, Burch 1989). It has been recorded in Dinwiddie and Prince George coun- ties (Beetle 1973a). Family Ancylidae Ferrissia fragilis (Tryon, 1863) (= Ancylus pumilus Lea, 1845, Ferrissia californica |Rowell, 1863}, Ferrissia shimekii [Pilsbry, 1890], Gundlachia meekiana Stimpson, 1863). This tiny gastropod occurs in lentic, eutrophic waters (Basch 1963, Clarke 1979, Burch 1989). Walker (1904b) recorded this species from Fairfax County near Alexandria, Virginia. Additional records occur from Hanover and Rockbridge counties (Burch 1952, Beetle 1973a). Ferrissia parallela (Haldeman, 1841) (= Ancylus paralle- lus). This limpet reaches the southern extent of its range in northern Virginia (Basch 1963, Burch 1989). The only rec- ord we found was from Fairfax County (Beetle 1973a). Fer- rissia parallelus inhabits lentic habitats where it is often found clinging to vegetation (Baker 1928a, Clarke 1981). Ferrissia rivularis (Say, 1817) (= Ancylus depressus Hal- deman, 1844, Ancylus haldemani Bourguignat, 1853, Ancylus rivularis, Ancylus tardus Say, 1830). This is the most com- monly reported ancylid in Virginia (Fig. 5G, Haldeman 1840-1845, Walker 1904b, Basch 1963, Beetle 1973a, Dillon 1977, Reed-Judkins et al. 1998, VDGIF 1998). Ferrissia rivu- laris lives in lotic habitats, where it occupies cobbles or other hard substrates in riffles (Baker 1928a, Jokinen 1983). Laevapex fuscus (Adams, 1841) (= Ancylus fuscus, Fer- rissia fusca). This species typically inhabits lakes and slow- flowing areas of streams and rivers, with occasional records of collections from rivulets (Fig. 5H, Rehder 1949, Beetle 1973a, Dillon 1977). Laevapex fuscus can be found attached 19° 1/2 + 2004 to vegetation, rocks, and man-made objects (Baker 1928a, Basch 1963). CONCLUSIONS A diverse group of freshwater gastropods inhabits Vir- ginia, with more than 50 species occurring there historically or presently. However, the lack of recent records for several species is cause for concern, as is evidence that some species are found only at one or two locations. Because intensive survey efforts have been directed to them, it is clear that Fontigens bottimeri, Fontigens morrisoni, Holsingeria unthan- kensis, and Holsingeria sp. 1 are extremely rare and endan- gered. Other species with few records, including Somatogyrus virginicus, Pomatiopsis cincinnatiensis, Elimia arachnoidea, Pleurocera gradata, and Stagnicola neopalustris are also likely endangered or extirpated. However, field surveys are still needed to determine their statuses. Other taxa and specific geographic regions should also be surveyed. Specifically, pulmonates (families Lymnaeidae, Physidae, Planorbidae, and Ancylidae) have been undersur- veyed, thus their maps underestimate their distributions. Additionally, we found no records from the Big Sandy drain- age, including Buchanan and Dickenson counties and the Levisa and Russel Rivers, and only one record (Littoridinops tenuipes; Beetle 1973a) from the eastern shore (1. e., Acco- mack and Northampton Counties). Absence of records from these regions are in contrast to the Tennessee drainage in southwest Virginia, where detailed surveys of rivers revealed declines and subsequent recoveries of pleurocerid popula- tions (Adams 1915, Ahlstedt 1979, 1991). By summarizing survey data from different sources, we hope to stimulate research that will improve our under- standing of the freshwater gastropod fauna of Virginia. We identified species and geographic regions that have been well surveyed, as well as those requiring additional study. Fur- thermore, we provide critical baseline data for measuring temporal changes in gastropod abundance and distributions. Comparisons of data from historic and future field surveys will facilitate legal protection of endangered species by pro- viding evidence of restricted or shrinking geographic ranges. Consequently, effective management plans can be developed for species in need of assistance. ACKNOWLEDGMENTS We thank the American Malacological Society and staff at the College of Charleston for organizing the 2002 AMS meeting. Special thanks to Amy Martin (Virginia Depart- ment of Game and Inland Fisheries) for access to the Vir- FRESHWATER GASTROPODS OF VIRGINIA 89 ginia Fish and Wildlife Information Service electronic data- base, Elizabeth Moore (Virginia Museum of Natural History) for museum records, and Christopher Register (Longwood University) for cartographic assistance. Com- ments by Eileen Jokinen, Janice Voltzow, and an anonymous reviewer improved the manuscript. LITERATURE CITED Adams, C. C. 1900. Variation in Io. Proceedings of the American Association for the Advancement of Science 49: 208-225. Adams, C.C. 1915. The variations and ecological distribution of the snails of the genus Io. Memoirs of the National Academy of Sciences 12: 1-92. Ahlstedt, S. A. 1979. Recent mollusk transplants into the North Fork Holston River in southwestern Virginia. 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Malacological Publications, Hamburg, Michigan. Burch, P. R. 1950. Mollusks. In: James River Project Committee of the Virginia Academy of Science, ed., The James River Basin. Past, Present and Future., Virginia Academy of Science, Rich- mond, Virginia. Pp. 129-137. Burch, P. R. and J. T. Wood. 1955. The salamander Siren lacertina feeding on clams and snails. Copeia 3: 255-256. Clarke, A. H. 1979. Gastropods as indicators of trophic lake stages. Nautilus 93: 138-142. Clarke, A. H. 1981. The Freshwater Mollusks of Canada. National Museums of Canada, Ottawa, Ontario, Canada. Clench, W. J. 1928. Jo fluvialis turrita Anthony. Nautilus 42: 36. Clench, W.J. 1962. A catalogue of the Viviparidae of North America with notes on the distribution of Viviparus georgianus Lea. Occasional Papers on Mollusks 2: 261-287. Clench, W. J. and K. J. Boss. 1967. Freshwater Mollusca from James River, VA and a new name for Mudalia of authors. Nautilus 80: 99-102. Clench, W. J. and S. L. H. Fuller. 1965. 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Butler, eds., Proceedings of the First Freshwater Mollusk Conservation Society Symposium, 1999, Ohio Biological Survey, Columbus, Ohio. Pp. 245-251. Woodward, S. L. and R. L. Hoffman. 1991. The Nature of Virginia. In: K. Terwilliger, ed., Virginia’s Endangered Species, Proceed- ings of a Symposium, The McDonald and Woodward Publish- ing Company, Blacksburg, Virginia. Pp. 23-48. Accepted: 2 March 2004 AMERICAN MALACOLOGICAL BULLETIN 19 1/2 + 2004 Environmentally and genetically induced shell-shape variation in the freshwater pond snail Physa (Physella) virgata (Gould, 1855)* David K. Britton and Robert F. McMahon Department of Biology, Box 19498, The University of Texas at Arlington, Arlington, Texas 76109, U.S.A. Abstract: Species of North American freshwater snails within the genus Physa are distinguished primarily on differences in shell shape. However, shell shape in this genus is ecophenotypically influenced by environmental factors. The present study examined the degree to which genes and environment influenced spire angle in specimens of Physa virgata collected from a single population in Arlington, Texas. Five separate genetic lines were inbred for five generations, after which progeny were reared under one of three temperature regimes (20°, 25°, or 30°C) until reaching adult size (approximately 5 mm in shell length). The spire angle of each individual was then measured and comparisons made across thermal regimes and genetic lines. Genetic line and temperature both significantly influenced spire angle. In general, snails reared at warmer temperatures had wider spire angles than snails reared at cooler temperatures. Some genetic lines had wider mean spire angles than others, regardless of temperature. Individual spire angles differed by as much as 23.8° under controlled conditions. These results suggest that shell shape is neither a consistent character nor taxonomically diagnostic. Thus, the number of currently recognized species of Physa is problematic. Key words: Physa, shell shape, reaction norms, phenotypic plasticity Species with populations occupying heterogeneous en- vironments are exposed to different, often dynamic, envi- ronmental conditions. Thus, different populations of a given species may be acted upon by differing selective pressures, resulting in no exclusively optimal phenotype for the species in general (Via and Lande 1985). In response to naturally varying selective pressures, many such species have evolved the ability to modify an individual’s phenotype in response to environmental cues (Pigliucci 2001). This phenotypic plasticity may potentially allow populations to inhabit oth- erwise hostile environments by providing protection from local hazards (Via and Lande 1985). However, the ability to modify phenotype is almost certainly constrained by costs such as those associated with sensory and regulatory mecha- nisms related to plasticity, and limitations to benefits such as those imposed by unreliable environmental cues (reviewed by DeWitt et al. 1998). Life history patterns, in particular, are well studied in relation to environmental modification. For example, intra- specific competition affects growth rates in freshwater pul- monates. Brown (1979) demonstrated that increasing popu- lation densities of experimental populations of the freshwater snail Lymnaea stagnalis Linnaeus, 1758 led to re- duced growth rates and delayed maturity in this species. Similarly, individuals of Physa gyrina (Say, 1821) decreased shell growth rates as population density increased (Brown, 1982). Yet, growth rate in this species increased and fecun- dity decreased when individuals of P. gyrina were reared in competition with Stagnicola elodes (Say, 1821) (Brown 1982). Kawata and Ishigami (1992) similarly reported that individuals of Physa acuta (Draparnaud, 1805) exposed to water containing chemical cues from of a second (naturally sympatric) species (Lymnaea sp.) exhibited accelerated growth rates relative to individuals exposed to water condi- tioned by conspecifics and controls (without snail cues). The presence or absence of predators has also been demonstrated to affect phenotypic characters (Crowl 1990, Crowl and Covich 1990, DeWitt 1996). Crowl and Covich (1990) reported that when exposed to chemical cues created by crayfish feeding on conspecifics, experimental popula- tions of Physa (Physella) virgata (Gould, 1855) accelerated growth rates and decreased reproductive rates until reaching a larger size relative to populations not exposed to the cues. Crowl (1990) reported that the presence of predators (cray- fish) is at least as important as environmental instability (measured by stream permanence) in inducing life-history trait variation in P. virgata. Life-history traits, however, are not the only phenotypic characters in freshwater gastropods influenced by environ- mental cues. DeWitt (1996) showed that individuals of Physa (Physella) heterostropha (Say, 1817) exposed to a fish preda- tor produced rounder shells with slower growth rates com- “From the symposium “The Biology and Conservation of Freshwater Gastropods” presented at the annual meeting of the American Malacological Society, held 3-7 August 2002 in Charleston, South Carolina, USA. 94 AMERICAN MALACOLOGICAL BULLETIN pared to snails exposed to a crayfish predator, which pro- duced more elongate shells with faster growth rates. Burnside (1998) demonstrated that population density and experimentally manipulated growth rates were also associ- ated with differential shell shapes in P. virgata, with higher densities and faster growth rates leading to rounder apertures. Phenotypic plasticity in shell shape is under-appreciated and problematic with regard to freshwater pulmonate snails because many species are distinguished based on shell shape parameters. If morphological variation can be induced by environmental cues, then the validity of routine use of shell shape parameters as indicators for gastropod species identi- fication must be questioned. The freshwater snail genus Physa Draparnaud, 1801 (Pulmonata: Basommatophora) is a worthy choice for illus- trating how shell shape parameters might be problematic for the identification of species of freshwater pulmonates. Cur- rently, physid species are primarily or solely based on slight variations in shell shape (Te 1975, Burch 1989). Although anatomical differences exist between some currently ac- cepted species, they are often relatively minor, with several accepted species sharing similar anatomy (Te 1973, 1975). Te (1973) reported that species of Physa include only a handful of complexes that can be distinguished based on differences in penile anatomy. Nevertheless, approximately 40 species of Physa are currently recognized (Turgeon ef al. 1998), and are typically identified by comparing collected specimens to Burch’s (1982, 1989) illustrations of physid shells, which have little or no description of anatomical or other physical differences. Many gastropods, including physids, are known to have considerably plastic shell shapes influenced by both biotic and abiotic factors, including water velocity (Urabe 1998), presence or absence of predators (Appleton and Palmer 1988, DeWitt 1996, 1998), population density and growth rate (Burnside 1998). If characteristics of shell shape are assumed to be consistent within a species but are actually ecophenotypically plastic, then measured differences be- tween geographically distinct populations may lead to erro- neous species designations if shell shape is the primary or sole characteristic considered. Thus, more species of Physa could be described than actually exist. Many of the currently identified North American physid species may be ecophe- notypic variants of a much smaller number of reproductively distinct species. Carefully planned breeding experiments are one way to test whether species are distinct. Dillon ef al. (2002) were able to synonymize Physa integra (Haldeman, 1871) and Physa heterostropha with Physa acuta, showing a clear lack of reproductive isolation between these phenotypi- cally similar “species.” Consistency of any character, including shell shape, within a species is essential for it to be taxonomically useful. 19° 1/2 + 2004 Because both genetic and environmental conditions are likely to affect shell shape, it is important to examine the relative degree to which each exhibits influence. The objec- tive of this study was to investigate the degree to which the shape of the shell of Physa virgata can be influenced by controlled genetic and environmental conditions and to ad- dress the validity of shell shape as a taxonomically useful character for this genus. MATERIALS AND METHODS Thirty immature juveniles of Physa virgata less than 4.0 mm in shell length (SL) were collected from a single popu- lation in Trader Horse Creek on the University of Texas at Arlington Campus, Arlington (Tarrant County), Texas, USA (32.72739°N, 97.11274°W). Sampled snails were returned to the laboratory and isolated in 250-ml plastic containers filled with de-chlorinated tap water, maintained in an incubator at 25°C under a 12-hr light/dark cycle. Snails were fed Ward- ley® Total Tropical® flake fish food ad libitum. Each isolated individual was used to produce a separate, random genetic line of progeny by allowing it to mature in isolation and reproduce by self-fertilization, creating a genetic bottleneck for its subsequent line of progeny. Offspring were left in parental containers until they reached about 0.5 mm in shell length. At this time, the parent and all but three or four juveniles were removed. The remaining juveniles were maintained in the same manner as their parent, except that they were allowed to inbreed with siblings or self-fertilize after reaching sexual maturity. No attempt was made to determine if individual snails self- fertilized or outcrossed with siblings. A total of five genera- tions of progeny were produced in the laboratory in this manner. Because individuals of Physa virgata are diploid, a heterozygote for a hypothetical locus (Aa for example) could potentially produce offspring with three different genotypes (AA, Aa, or aa) even if the individual reproduced by self fertilization. However, restricting reproduction to selfing or outcrossing among siblings prevented new alleles from add- ing genetic variation to each line. Additionally, inbreeding within such small laboratory populations could potentially remove rare alleles over generations by way of genetic drift. Thus, inbreeding allowed maintenance or reduction of ge- netic variation within each genetic line over each generation. Growth and reproductive rates were highly variable among the 30 lines. Some lines produced mostly fast- growing progeny while others produced only a few, non- viable, or slowly growing offspring. Thus, 5 of the original 30 genetic lines were chosen for all subsequent experimentation based on their ability to consistently produce numerous, viable offspring. SHELL-SHAPE VARIATION IN PHYSA VIRGATA 95 After five generations of inbreeding at 25°C, approxi- mately 15 hatchlings (<0.5 mm SL) were collected from each genetic line. Each selected hatchling was isolated in a new 250-ml plastic container and randomly assigned to a thermal regime of 20°, 25°, or 30°C. Individual genetic lines did not produce equal numbers of viable offspring. Each of the three thermal regimes received as many snails as were available. A minimum of five snails (range = 5 to 12 snails) from each of the five genetic lines were assigned to each treatment. It was hypothesized that different thermal regimes would induce different growth rates and thereby indirectly influence shell shape. Snails remained in containers within incubators un- der a 12-hr light/dark cycle and were fed ad libitum. When each F2 snail approached about 5 mm in SL (range approxi- mately 3 to 7 mm), it was removed and stored in 70% ethanol. Snails that perished during the experiment were discarded. Shell growth rates were estimated by subtracting 0.5 mm (1e., the approximate hatchling shell length when initially isolated) from the final shell length and dividing by the number of weeks elapsed under treatment. Digital images of all snail shells were captured with shells held in a position corresponding to the images of Burch (1989). Images were captured with a Sony CCD-IRIS color video camera (Model DXC-107A) fitted with a Navitar 7000 TV zoom lens. The long axis (from apex of the spire to the most distant point along the edge of the aperture) and apertural plane were held parallel to the focal plane of the camera. Morphometric measurements were recorded using digital image analysis software (SigmaScan Pro 5.0 by SPSS), including shell length in mm and spire angle in degrees. Shell length was measured along the long axis as described above, and spire angle was measured from the penultimate whorl to the apex of the spire (Fig. 1). Because allometric shell growth is common in gastro- pods, it was necessary to assess whether shell length and/or growth rate should be included as covariates when testing whether spire angles differed between treatments. As a rough evaluation of whether shell length was the major factor in- fluencing spire angle (1.e., allometric growth had occurred), a reduced major axis (RMA) regression of shell length (in- dependent variable) on spire angle (dependent variable) was performed using data combined from all genetic lines. Five additional independent RMA regressions were also per- formed, each examining the relationship between shell length and spire angle for a specific genetic line. Similarly, an RMA regression was performed to test whether growth rate (independent variable) influenced spire angle (dependent variable) using data combined for all ge- netic lines. The relationship between growth rate and spire angle was also evaluated independently for each genetic line resulting in five additional RMA regressions. Type I] (RMA) regressions were necessary because the independent vari- Figure 1. Shell measurements made on specimens of Physa virgata. SA is spire angle measured in degrees. SL is shell length measured in mm. ables (SL and Growth Rate) were not measured without error, an assumption in type I regression. Mixed-model two-way ANCOVAs were performed to as- sess whether growth rates and spire angles differed among treatments. Shell length was included as covariate in the first ANCOVA (assessing growth rate means across treatments) and shell length and growth rate were included as covariates in the second (assessing spire angle means across treatments). Genetic line (arbitrarily assigned 1, 2, 3, 4, or 5) and thermal regime (20°, 25°, or 30°C) were independent variables (with genetic line treated as a random variable) in both ANCOVAs. RESULTS Growth rates for individuals of Physa virgata ranged from 0.049 to 0.405 mm/day, averaging 0.146 mm/day (sd = 96 AMERICAN MALACOLOGICAL BULLETIN 0.077) with data combined across all genetic lines. Shell spire angles ranged from 55.2° to 79.0° with a mean of 66.6° (sd = 5.5), including data from all lines (Fig. 2). Measurements were made on snails ranging in shell length (SL) from 3.0 to 6.9 mm with a mean of 4.9 mm (sd = 0.80). The reduced major axis (RMA) regression of shell length on spire angle that combined data for all genetic lines and thermal regimes, although statistically significant at a = 0.05 (1° = 0.046, p = 0.043), suggested that shell length was not a major factor influencing spire angle over the range of shell sizes studied (Fig. 3). Two of five independent RMA regressions of shell length on spire angle for individual ge- netic lines were statistically significant (Genetic Line 4, 7° = 0.273, p = 0.01; Genetic Line 5, r° = 0.198, p = 0.03) with both indicating a negative correlation between shell length and spire angle. Neither of these accounted for more than 27.3% of the variation revealed in spire angle (Fig. 3). The three independent RMA regressions for the remaining ge- netic lines did not reveal a significant (a = 0.05) relationship between shell length and spire angle. Although growth rates varied substantially in this study, they did not significantly influence shell shape measured as spire angle. The RMA regression of growth rate on spire angle, combining data from all genetic lines and thermal regimes, was not significant (7° = 0.0014, p = 0.73). More- over, none of the individual RMA regressions of growth rate on spire angle for individual genetic lines were significant (Fig. 4). ANCOVA revealed a significant effect of inbred line as well as an interaction between the effects of temperature and inbred line on growth rate (Table 1). The influence of tem- perature on growth rate was dependent on genetic line (Fig. 15 10 Frequency 45 50 55 60 65 70 75 80 8 Spire Angle in Degrees Figure 2. Histogram of the distribution of spire angles measured from all specimens of Physa virgata utilized in experiments. 19+ 1/2 * 2004 5), although some lines had consistently higher growth rates than others, regardless of temperature. Both genetic line and temperature significantly influ- enced spire angle (Table 2). Some genetic lines had wider spire angles than others regardless of temperature, and the snails in the 25° and 30°C regimes had wider spire angles than those in the 20°C regime (Fig. 6). The relationship between temperature and spire angle was fairly consistent and not confounded by an interaction with inbred line. By dividing the variation for each effect by the sum of the measured variation (sum of squares) for all effects (tempera- ture, genetic line, and interaction) one can estimate the rela- tive proportion that each effect contributed to the total mea- sured variation in spire angle. Genetic line, temperature regime, and interaction accounted for approximately 44%, 37%, and 19%, respectively, of the measured variation in spire angle in individuals of Physa virgata. DISCUSSION When data from all specific genetic lines were com- bined, the total sample reflected a reasonably normal distri- bution of spire angles with a mean of approximately 67°. In comparison, Burch’s (1989) illustration of Physa virgata had a spire angle of approximately 73°. The standard deviation of the studied sample was 5.5°, making the shape of Burch’s (1989) illustrated specimen consistent with the expected dis- tribution based on spire angles obtained in the present study. Indeed, assuming that spire angles are normally distributed and there is nothing special about the Trader Horse Creek population of P. virgata, one should expect 95% of adult individuals of P. virgata to have a spire angle somewhere between 55.6° and 77.6° (two standard deviations on either side of the mean). Remarkably, this range includes the spire angles of most of the physid species depicted in Burch (1989). Although it was anticipated that shell growth rate would influence shell shape in Physa virgata, this was not demon- strated. The relationship between shell growth rate and tem- perature was dependent on genetic line examined. Specifi- cally, temperature had little impact on the shell growth rates of Lines 1 and 5 (a change of about 0.04 mm/day from 20°-30°C), but had substantial influence on Line 2 (an in- crease of >0.25 mm/day over the same temperature range). Two of the three lines had maximal growth rates at 25°C while three had maximal rates at 30°C (Fig. 5). Although growth rate did not influence adult spire angle in this study as expected, temperature did. In general, higher tempera- tures (25° and 30°C) corresponded to wider spire angles in P. virgata. The relationship between spire angle and tempera- ture produced generally similar reaction-norm patterns for SHELL-SHAPE VARIATION IN PHYSA VIRGATA 97 mental variable (temperature), and the height of the lines can be attributed to the genotypic influence (Pigliucci 2001). The individuals of Physa virgata uti- lized in this study were all derived from a single population taken from one loca- tion during a single collection event. Nev- ertheless, shell shape and shell growth rates varied substantially among indi- Spire Angle (degrees) Spire Angle (degrees) viduals. Although most measured varia- tion in spire angle was attributable to genetic line (44%), a considerable pro- portion (37%) was attributable to envi- ronmentally induced differences (i.e., temperature regime). After isolating dis- tinct genetic lines and controlling rearing temperature, snails produced descen- dants with remarkably different shell shapes. Spire angles ranged from ap- proximately 52° to 79°, as much or more variation in spire angle than depicted in the illustrations of the shells of the over Spire Angle (degrees) Spire Angle (degrees) 40 identified North American species of Physa (Burch 1982, 1989). Physa virgata is an r-selected invasive species, as are most freshwater pulmo- 80 nates (McMahon 1983). It has evolved Ui © 6 tolerance of the varying conditions typi- 70 ; oe cal of the small, ephemeral ponds and e? streams where this species is often found eS. ed (McMahon 1983). The ability to self- 60 fertilize, capacity to produce numerous offspring, and ability to readily disperse 2 (aided, perhaps, by transport on the feet Genetic Line 3 A - ; Ph atce of waterfowl), allows single individuals to 2 BPA 8S SG OF quickly found new populations and/or repopulate ephemeral ponds and streams SL (mm) ae 50 Spire Angle(degrees) when water returns (Dillon and Wething- ton 1995, Dillon 2000). This study sug- gests that individuals (including those that found new populations) can be ge- Figure 3. Scatter plots of spire angle vs. shell length in specimens of Physa virgata. Data from each genetic line are indicated in separate panels. Shell length was not significantly correlated with spire angle when all data were combined. The relationship was also not significant for three genetic lines when examined separately. However, two genetic lines netically predisposed for wider (or nar- had significant regressions: both Genetic Line 4 (17 = 0.273, p = 0.01) and Genetic Line | rower) spires, and that this genetic pre- 5 (r° = 0.198, p = 0.03) indicated a negative correlation (depicted in the figure as solid disposition can be ecophenotypically lines) between shell length and spire angle but explained very little of the observed enhanced or diminished by non-genetic, variation. environmental influences such as tem- perature. Although the extensive genetic variation in shell shape revealed in this each genetic line, although each was vertically shifted with study could be the basis for the evolution of adaptive shell respect to the others, indicating a genetic influence on spire shapes within populations subjected to long-term, specific angle (Fig. 6). The slopes of these reaction norms can be selection pressures (e.g. crayfish predation or rapid water attributed to the ecophenotypic influence of the environ- velocity), a large proportion of variation in the spire angle of 98 AMERICAN MALACOLOGICAL BULLETIN 80 80 e a ® ° mn . ® 75 ee, ® 75 ®e a ro a 0 e © a e ae ® e ® Ve =) : D 2 ec 60 = 01» 30) were collected from the sides and undersides of rocks in depths less than 1 m. Specimens were generally restricted to rock surfaces embedded in sand/ pebble substrata. To avoid size bias, all specimens on a sampled rock were removed by sliding a scalpel blade gently under the edges of their shells, preventing shell or tissue damage. Rocks were sampled until an appropriate sample TEMPORAL VARIATION OF SHELL SHAPE IN A FRESHWATER LIMPET 103 size (n > 30) was attained. The population was sampled annually in the fall between September and October from 1973 to 1987 and in the spring between April and June from 1977-1988. All samples were immediately fixed in 10% neu- tralized formalin and returned to the laboratory for mea- surement of parameters of shell shape. The limpet population had an annual semelparous re- productive pattern. Adults oviposited in May/June to pro- duce a cohort that grew through summer and fall to oviposit the following spring. Adults died soon after spring oviposi- tion giving each generation a one-year life span. Individuals collected in the fall had experienced approximately six month’s growth from hatching, while those collected the following spring were from the same annual cohort after approximately a year’s growth. Spring samples were col- lected during the oviposition period just prior to hatching of juveniles of the next generation. All annual cohorts de- scribed herein are indicated by the year during which they were produced. The shell aperture length (AL, the greatest anterior- posterior distance across the aperture), aperture width (AW, the greatest distance across the aperture 90° to the anterior- posterior axis), and height (SH, the greatest vertical distance from the shell apex to the plane of the aperture) of each individual were measured to the nearest 0.1 mm with a binocular dissecting microscope at 10X using an ocular mi- crometer (McMahon and Whitehead 1987) (Fig. 1). AL and AW were measured for specimens submerged in a glass Petri dish. SH was measured using the tip of a fine brush to transfer the specimen from the bottom of the dish onto the side of a vertically mounted glass cover slip to which moist- ened specimens adhered by water surface tension. In this position the shell was viewed in lateral profile, allowing mea- surement of SH. Shell morphometric variables were mea- sured for a total of 1,787 specimens. Data on monthly rainfall and air temperature over the course of the experiment were obtained from annual sum- maries of climatological data recorded at Station 341168/ 99999 on Broken Bow Lake Dam (National Climatic Data Center, National Oceanic and Atmospheric Administration), approximately 3 km from the collection site. ANOVA/ANCOVA and post hoc Scheffe’s test were uti- lized to compare differences within spring and fall samples in mean AL, AL-adjusted sample means of AW and SH, AL-adjusted mean AW or SH of spring and fall samples within single generations, and sample shell AL growth versus mean air temperature and total precipitation over the prior six month period. ANCOVA was utilized to generate sample mean SH and AW values adjusted to eliminate the influence of the covariant AL. The relationship between individual AL and AW or SH for all individuals and between shell growth in AL and the AL-adjusted mean AW and SH of spring and fall samples were examined with reduced major axis regres- sion analysis. Least squares linear regression analysis was utilized to examine the correlation between cohort growth in shell AL and mean air temperature or total precipitation over the prior six month period. All statistical analyses were preformed with Statgraphics Plus® version 6.1 (Statistical Aperture Length (AL) Figure 1. Diagrams indicating shell mea- surements taken. Aperture Width (AW) Dorsal Aspect Lateral Aspect Shell Height (SH) 104 AMERICAN MALACOLOGICAL BULLETIN Graphics Corporation) with the exception of reduced major axis regression analyses preformed with a program devel- oped by Andrew J. Bohonak of San Diego State University. The «@ value for all statistical analyses was 0.05. RESULTS ANOVA revealed variation in mean aperture length be- tween annual fall (n = 15, F = 41.3, P < 0.00001) and spring samples (n = 11, F = 15.5, P < 0.00001) (Fig. 2). A Scheffé’s test allowing multiple pair-wise mean testing indicated that of 105 possible pair-wise comparisons between the sample mean AL values of 15 annual fall collections, 71 (67.6%) were significantly different (P < 0.05). Similarly, of 55 pos- sible pair-wise comparisons of mean AL values between the 11 spring samples, 35 (63.6%) were significantly different. Reduced major axis regressions of either AW or SH against AL for all sampled individuals were significant (AW, a = 0.078, b= 0,741,.n = 1787, r = 0.956, P <.0.0001; SH, a = —0.139, b = 0.395, n = 1787, r = 0.864, P < 0.0001). Regres- sion slope values were significantly less than unity, indicating that relative shell AW and SH decreased slightly with in- creasing AL (Fig. 3). When subjected to ANCOVA with individual AL as a covariant to account for size allometry, cohort year had a significant impact on mean cohort SH in both the fall (n = 15, F = 26.29, P < 0.00001) and spring samples (n = 11, F = 35.90, P < 0.00001). A Scheffe’s test indicated that 36 (34.3%) of 105 and 28 (50.9%) of 55 possible pair-wise comparisons of AL adjusted mean cohort SH were signifi- 19 * 1/2 + 2004 cantly different in the fall (Fig. 4A) and spring samples (Fig. 4B), respectively. AL-adjusted annual mean SH ranged from 1.07 mm (s.e. = +0.014, n = 54) for the fall 1987 cohort to 1.29 mm (s.e. = 0.016, n = 37) for the fall 1975 cohort (Fig. 4A) and from 1.25 mm (s.e. = 0.035, n = 11) for the spring 1983 cohort to 1.56 mm (s.e. +.0.015, n = 62) for the spring 1979 cohort (Fig. 4B). Similarly, ANCOVA and a Scheffé’s test indicated that 49 (46.7%) of 105 and 26 (47.3%) of 55 possible pair-wise comparisons of AL-adjusted mean cohort AW were significantly different in the fall (n = 15, F = 48.22, P < 0.00001) and spring samples (n = 11, F = 17.80, P < 0.00001), respectively. AL-adjusted annual mean AW ranged from 2.48 mm (s.e. = +0.008, n = 132) for the fall 1981 cohort to 2.77 mm (s.e. 0.015, n = 54) for the fall 1987 cohort (Fig. 4C) and from 2.94 mm (s.e. = +0.031, n =17) for the spring 1977 cohort to 3.18 mm (s.e. = +0.022, n = 35) for the spring 1986 cohort (Fig. 4D). In all cases, mean spring sample AL increased over that of the fall sample AL within any one annual cohort (1977- 1987) (Fig. 2), allowing cohort shell growth rate to be esti- mated as the increase in mean sample AL between the fall and spring cohort samples. Because all individuals in a fall sample of a cohort were produced in May/June, cohort growth after hatching could be estimated as the mean cohort AL of the fall samples. When subjected to reduced major axis regression analysis, increases in mean AL from cohort spring hatching to fall sampling and from fall sampling to subse- quent spring sampling versus AL-adjusted mean cohort sample AW or SH as dependent variables proved insignificant (fall AW: n = 15, P = 0.664; spring AW: n = 11, P = 0.824, fall SH: n = 15, P = 0.340; spring SH: n = 11, P = 0.824). 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Aperture Length (mm) ‘73 ‘74 ‘75 ‘76 ‘77 ‘78 ‘79 ‘80 ‘81 Annual Generation *82 ‘83 *84 ‘85 ‘86 ‘87 Figure 2. Variation in mean aperture length (AL, vertical axis) of samples of annual generations (horizontal axis) in a population of freshwater limpets. Inner bars about means represent standard de- viations of the mean and outer bars the range of AL recorded in the sample. Samples collected during the fall are in- dicated by an “F” at the upper end of the error bar and those collected in the spring by an “S.” Paired fall and spring samples of the same annual cohort are connected by solid lines (cohorts ’78 through ’87). Mean fall cohort values reflect the in- crease in AL in the first six months of life, while the difference in mean AL between fall and subsequent spring samples of co- horts represents the mean increase in co- hort AL in the subsequent six months of life in this semelparous, spring- ovipositing species. Aperture Width (AW) or Shell Height (SH) in mm 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 TEMPORAL VARIATION OF SHELL SHAPE IN A FRESHWATER LIMPET 105 10 #15 20 2.5 3.0 3. AW = 0.078 + 0.741(AL in mm) n= 1787, r= 0.956, P <0.000 -0.139 + 0.395(AL in mm) 7, r=0.864, P <0.0001 5 40 45 50 5.5 Aperture Length (AL) in mm Figure 3. Relations of shell aperture width (AW, circles) and shell height (SH, triangles) (vertical axis) to aperture length (AL, horizontal axis) in all sampled individuals (n = 1787) of a population of freshwater limpets col- lected from 1973-1988. Data points for all 1787 measured individuals do not appear in this figure due to extensive point over- lap. Solid lines are best fits of significant (P < 0.0001) reduced major axis regres- sions. Regression equations relating AW and SH to AL and associated statistical parameters are indicated above the AW data and below the SH data, respectively. Mean Shell Height (SH) in mm Mean Aperture Width (AW) in mm 87 82 85 81 83 74 73 78 76 77 84 80 86 7975 58 81 73 82 75 80 76 79 78 77 74 86 85 84 83 87 ghee! Fall Samples Annual Generation t § Fall Samples Annual Generation Mean Shell Height (SH) in mm Mean Aperture Width (AW) in mm 3.20 3.15 3.10 3.05 3.00 2.95 2.90 Spring Samples 83 81 85 87 78 84 77 86 82 80 79 Annual Generation Spring Samples pit 77 78 80 79 84 85 81 87 82 83 86 Annual Generation Figure 4. Aperture length-adjusted mean shell height (SH) and aperture width (AW) (vertical axis) of samples of annual cohorts (year of cohort formation indi- cated on the horizontal axis) of a popu- lation of freshwater limpets. A. Adjusted mean SH of cohort samples collected during the fall. B. Adjusted mean SH of cohort samples collected during the spring. C. Adjusted mean AW of cohort samples collected during the fall. D. Ad- justed mean AW of cohort samples col- lected during the spring. Vertical bars about the means represent standard er- rors of the means. Horizontal bars in the upper portion of the figures encompass means that were not significantly different. 106 AMERICAN MALACOLOGICAL BULLETIN 150 B © Fall Samples 77 78 79 80 81 82 83 84 85 86 87 Annual Generation The difference between AL-adjusted mean AW and SH of fall and spring samples within a specific annual cohort was tested by ANCOVA with fall and spring sample periods as the treatment and AL as the covariant. Application of a Bonferroni correction for sequential tests to a = 0.05 indi- cated that the mean AW of the fall sample was significantly different from that of the spring sample in 4 (36.4%) of 11 paired annual cohort fall and spring samples (i.e., the 1977, 1981, 1982, and 1986 cohorts, Fig. 5A). Similarly, the mean SH of the fall sample was significantly different from that of the spring sample in 6 (54.5%) of 11 paired annual cohort fall and spring samples (1e., the 1978, 1980, 1981, 1982, 1983, and 1987 cohorts, Fig. 5B). Variation in mean AW and SH between fall and spring cohort samples was non- directional. Mean AW was significantly lower in spring com- pared to fall samples in the 1977 cohort and significantly higher in the 1981, 1982, and 1986 cohorts (Fig. 5A). The mean SH of the spring sample was significantly lower in the 1978, 1981, and 1983 cohorts and significantly higher in the 1980, 1982, and 1987 cohorts (Fig. 5B) ANOVA of cohort increase in mean AL between fall and spring samples (1.e., overwinter) indicated that neither av- erage air temperature (n = 11, P = 0.081) or total precipi- tation (n = 11, P = 0.459) significantly impacted shell growth rate measured as increase in AL. In contrast, ANOVA indi- cated that growth of annual cohorts between time of hatch- ing and fall sampling (i.e., over summer) was positively cor- related with both average air temperature (n = 15, P = 0.035) and total precipitation (n = 15, P = 0.05). Because air tem- perature and precipitation levels potentially affected shell size, the relationship between either mean air temperature or total precipitation during the preceding six month growth period and AL-adjusted mean cohort sample AW or SH was investigated with least squares linear regression analysis. Ap- T S 4 Spri pring Samples & 1.40 é a c = = = s ‘ & 1.30 <= : —) s ”Q 120 x?) ~ = = 2 1.10 ( Subspecies = incipient differentiation pos Se ea ett ag clinal variation Monotypic species Figure 1. Classifying borderline cases of the microtaxonomic differentiation between subspecies and “good” species. The terminological framework of components in the speciation process is given (under the BSC) as discussed in the text. Stages 1-6 are intermediates in the continuous differentiation of groups of populations. Roman numerals mark the different levels of species limits according to the relevant species concepts, with I-II indicating “cladistic” species categories: I—phylogenetic species, II—evolutionary species, II—biological (“multi-dimensional”) species, [V—species under the recognition concept, V—zoogeographical species [Adopted and combined from Haffer (1985: 53, fig. 1) and Haffer (1992: 116, tab. 1)]. TREATING SPECIES AS DYNAMIC ENTITIES 119 Criticism Even in view of the fact that interbreeding in nature is the finest possible evidence for evolutionary units, the BSC was often held to be non-universal, non-operational and non-applicable (for example Cracraft 1983, Endler 1989, Hull 1997, Mayden 1997, see also more recent discussions in Eck 1998, Wheeler and Meier 2000, Hey, 2001a, 2001b). The BSC was also accused of confusing pattern and process with a bias towards a particular type of speciation. Accordingly, Mallet (1997) criticized that “by postulating an ideal species. rather than a practical approach to sorting actual taxa, Mayr opened a Pandora’s box.” It was even claimed that the BSC althogether “is not very useful” (Schilthuizen 2001: 19). However, difficulties in the application of the BSC (for ex- ample those arising from allopatry, lack of information or cases of incomplete reproductive isolation and hybridiza- tion) in themselves do not detract from the validity of the concept. Another cause for misunderstanding the value of the BSC is the lack of distinction between species concept and species taxon (or species category), as discussed above. Admittedly, there are limitations and a genuine inap- plicability of the BSC in the cases of asexuality (uniparental reproduction) and allopatry. Indeed, the BSC is only appli- cable to organisms reproducing bisexually. For those organ- isms, however, that reproduce non-bisexually, either com- pletely unisexually (parthenogenesis) or asexually, the agamospecies becomes available, rendering the claim of only a single universal species concept inappropriate. For a dis- cussion of the species concept in parthenogenetic taxa see Maslin (1968), Sudhaus (1984), and Hauser (1987). The ex- istence of reproductive isolation in nature can only be de- termined with certainty when taxa are sympatric. Given the gradual process of speciation, non-continuous populations may or may not have reached the level of biological species. The status of such taxa, including subspecies belonging to the same species or paraspecies, allospecies, and semispecies (Fig. 1), can be determined by inference only (Mayr 1997, 2001). These inferences have to be made within a taxon- specific framework using the degree of morphological and other differences or, with recent advances in molecular methods, by comparison of genetic distance. Making those inferences is advocated here as necessary and logical proce- dure that follows from the theory of the BSC. Although McKitrick and Zink (1988: 3) rightly called the inapplicabil- ity of the BSC to allopatric forms “an underemphasized problem because there are thousands of allopatric popula- tions,” the inability to treat allopatric populations objectively and provide a foolproof system for the correct assignment of isolated populations or other cases of evolutionary interme- diacy is inherent in all other species concepts as well. The BSC is most often criticized as being non- dimensional. However, by viewing a species as existing and extending its populations in a geographical framework, the BSC actually is two-dimensional. With this horizontal no- tion a biological species appears as a real unit in nature. Although the BSC is admittedly not vertical, that is not primarily a historical concept, if we add the time dimension, three-dimensionality is gained, as suggested by the chrono- species concept. This latter concept (artificial delimiting of portions of phylogenetic species lineages), however, is essen- tially nothing more than a morphospecies concept applied to fossils. Accordingly, the chronospecies concept uses the cri- teria of the BSC combined with the morphological approach in geological time (Reif 1984, Willmann 1985, see also pa- pers in Eck 1998). Expanding the concept Emphasizing again the distinction between species con- cepts and species taxa (as discussed above), we find that the species concept is based on the non-dimensional situation, while the species taxon is multi-dimensional. Adding the dimensions of geography and time permits a way to treat populations taxonomically. Applying the ideas of the BSC and, in addition, subsequently testing how many popula- tions and presumed subspecies (due to their distinctness) actually deserve species status even in allopatry, this proce- dure became a valuable and more heuristic endeavor, in ornithology, for example, than the often meaningless dispute over naming “species” or “subspecies” in allopatric and/or parapatric situations. The last decades have seen the devel- opment of the methodological tools to treat the various in- stances of evolutionary intermediacy. To accommodate taxonomically these stages of the microgeographical differ- entiation process in concert with the BSC, a terminological framework has been developed, in particular in ornithology (Fig. 1). Terms such as para-, allo-, semi-, and superspecies proved most valuable in the context of zoogeographic and phylogenetic studies, and entire faunas have been studied utilizing this approach (see Haffer 1985, 1992, Amadon and Short 1992, Sibley and Monroe 1990, see also literature cited therein for examples from ornithology). Adequate system- atic inferences and the application of allo-, semi-, and syn- species of a superspecies is advocated, for example, by Mayr (1942, 1963), Amadon (1966), Sudhaus (1984), Haffer (1986), Sibley and Monroe (1990), Mayr and Ashlock (1991), Glaubrecht (1993, 1996, 2000), and Helbig (2000). THE DEBATE CONTINUES How to delimit species in a cladist’s world? The last two decades have seen a particularly active phase of debate about how to define and delineate species in 120 AMERICAN MALACOLOGICAL BULLETIN a cladist’s world. Phylogenetic systematics, as proposed originally by Hennig (1950, English translation 1966, see also Ax 1984 and Wagele 2000), and particularly its application and recent computation utilizing advanced methods in bio- informatics and technology by cladists, produced a revolu- tion in systematics. Unquestionably, the rigorous application of cladistic analysis had a major impact also on many other aspects of systematic biology, resulting in “tree thinking” (O’Hara 1994). Because the BSC was considered insufficient for this purpose, phylogenetic thinking, or the cladistic ap- proach to nature, necessitated the re-evaluation of concepts in systematics, with cladistics recognizing the importance of a species concept that serves their methodology of branching patterns and clades defined by synapomorphies, leading to “(re) inventional word games,” as Avise (2000b: 1831) rightly noted in his elegant and eloquent book review on “the speciational wonderland.” Currently, there are 22 ways to view and perceive what a species is, according to a review by Mayden (1997); Hey (2001b) lists 24 concepts. While the battle over the best cladistic species concept continues among cladists, proponents of the BSC, especially Mayr (2001), denied that any of the new phylogenetic concepts is legitimate, since “none of the authors of these new concepts has understood the difference between a species concept and a species taxon. Instead of new concepts, they have proposed new operational criteria of how to delimit species taxa.” Following a suggestion by Haffer (1992, 1998) and Git- tenberger (1972), who both tried a systematization among the multitude of species definitions instead of a mere com- pilation, I will distinguish here between “horizontal” and “historical” species concepts. Although a species as defined by the BSC can be viewed as a horizontal cross-section of a phyletic lineage at any given time, a historical species con- cept results in the vertical delimitation of species as sug- gested, for example, under the Hennigian Species Concept (HSC) (Meier and Willmann 2000), the Evolutionary Spe- cies Concept (ESC), and (implicitly) under the Phylogenetic Species Concept (PSC). Proponents of the HSC, ESC, and PSC consider species to be parts of a “vertical” evolutionary lineage between two consecutive cladogenetic events (that is speciation and/or termination through extinction). The fol- lowing brief review will restrict itself to the vertical concepts ESC and PSC, other concepts are considered of minor im- portance, because they reflect only special aspects with slightly changed emphases. The Evolutionary Species Concept Simpson (1961: 153) suggested defining species as “a lineage (an ancestral-descendent sequence of populations) evolving separately from others and with its own unitary evolutionary role and tendencies.” Later, Wiley (1978: 18) proposed in a slightly revised version that “a species is a 19° 1/2 + 2004 single lineage of ancestral descendent populations of organ- isms which maintains its identities from other such lineages and which has its own evolutionary tendencies and historical fate.” More recently, Wiley and Mayden (2000) have re- defined and defended the evolutionary species as “an entity composed of organisms which maintains its identity from other such entities through time and over space and which has its own independent evolutionary fate and _ historical tendencies.” Although considered relevant for both living and extinct groups and to sexual and asexual organism, the ESC is non- operational and subjective (that is, containing undefinable criteria rendering them useless in practice), and even Simp- son himself has abandoned his own concept as being rather nebulous for systematic purposes (Reif 1984, O'Hara 1993, Mayr 1992, 2001). How, for example, is one to describe and determine “evolutionary tendencies” or “historical fate” of a population or taxon? Nevertheless, the ESC is still supported and frequently recommended (e.g. Maslin 1968, Wiley 1980, 1981, Ax 1984, Willmann 1985, Otte and Endler 1989, Frost and Hillis 1990, Mayden 1997, Peters 1998). However, the ESC failed to provide its main objective, namely a clear delimitation of a species in the time dimension that turned out to be illusory in all cases of gradual species transforma- tion, as is illustrated in particular by the classical formen- reihen of freshwater gastropods (see Willmann 1981, Wil- liamson 1981, further discussions in Reif 1984, Eck 1998). In essence, the ESC is a typological morphospecies concept that is not operational; it can only assume that characteristic features are consistent and thus diagnosable throughout the entire historical existence of the evolutionary lineage. The Phylogenetic Species Concept Cladistic methodology views the world as branching patterns. According to this philosophy, every lineage starts and ends with a branching event (speciation) or its extinc- tion and is characterized by at least one autapomorphy. This view led to the need for a species concept consistent with phylogenetic principles. The reproductive criterion (i.e. breeding compatibility) is considered inappropriate among cladists to group organisms into species. Instead, the exis- tence of unique patterns of shared and diagnosable charac- ters is proposed and defended as a sufficient criterion. Espousing PSC as alternative to the BSC, for example, Cracraft (1983: 170) defined species “as the smallest diag- nosable cluster of individual organisms within which there is a parent pattern of ancestry and descent.” He later defined species under the PSC as “an irreducible (basal) cluster of organisms diagnosably distinct from other such clusters, and within which there is a parental pattern of ancestry and descent” (Cracraft 1989: 34-35). For a historical review and application of the many versions of the PSC see Mishler and TREATING SPECIES AS DYNAMIC ENTITIES 121 Brandon (1987), McKitrick and Zink (1988), Mayden (1997) and Mishler and Theriot (2000). One of the most serious problems with the PSC is that there are too many versions, which leads to confusion about the specific definition. Regrettably, irrespective of the two decades of debate, cladists have failed to set or agree on any standards for what to consider a “phylogenetic species.” This lack of consenus is both a problem for communication and acceptance, and is a source of much confusion and many misconceptions, which hampers scientific progress. Mayden (1997) sorted out two main approaches, the diagnosable version and the monophyly version of the PSC, the latter stemming from the debate of cladists whether the concept of monophyly should be extended from higher categories to the species level (de Quieroz and Donoghue 1988, Nixon and Wheeler 1990). The diagnosable version In the definition of Nixon and Wheeler (1990: 218) a species is “the smallest aggregation of populations (sexual) or lineages (asexual), diagnosable by a unique combination of character states in comparable individuals (semapho- ronts).” Because this definition is character-based it renders a phylogenetic species analogous to a morphospecies, even if now allowing to do so on an additional level, such as the molecular features. Accordingly, using the increasingly im- proved methods of molecular genetics, it currently becomes more and more standard procedure to characterize each and all geographically separated populations by apomorphic fea- tures (i.e. sequence differences), with smaller populations more easy to track than large polymorphic ones. In conclu- sion, those populations which turn out to be consistently diagnosable by characters or character combinations recog- nizable by ordinary (= arbitrary) morphological means and/ or molecular means (single fixed nucleotide base pairs) are considered as species under the PSC. The monophyly version Rosen (1978) stated that “a geographically constrained group of individuals with some unique apomorphous char- acters, is the unit of evolutionary significance.” More re- cently, Mishler and Theriot (2000) defined species as “the least inclusive taxon recognized in a formal phylogenetic classification.” Accordingly, “taxa are ranked as species. be- cause they are the smallest monophyletic groups deemed worthy of formal recognition.” This concept equates species with monophyletic units and speciation with character transformation. There are a few problems with monophyly in this context, for example, the question of whether species are, must, or can be monophyletic (Wiley 1981, Willmann 1983, McKitrick and Zink 1988, Wheeler and Nixon 1990). Establishing “monophyly” for species has proven difficult in practice, as Nelson (1989) admitted. Generally, PSCs have been criticized for three different reasons. In addition to being (1) typological and to diagnose evolutionary units on the basis of (partly trivial) characters, they are (2) arbitrary and reductionistic in the sense that they not include important biological criteria, and are (3) leading to the recognition of too many species. Apart from the question of what actually is “phylogenetic” about a spe- cies, for example, Mayr (2001: 167) criticized the various phylogenetic species concepts as “simply typological pre- scriptions of how to delimit species taxa.” Starting from the conviction that without clear-cut definitions no progress in the clarification of concepts and theories is possible, it is not helpful that in the last two decades so many cladists have come up with several more or less divergent definitions of the phylogenetic species concept. Often even the same au- thors have different formulations and wordings at different times, without reaching any compelling synthetic consenus. This results in confusion of what an author means when referring to a species under the PSC and it certainly defeats the cladists’ claim to provide with the PSC a viable alterna- tive to the BSC. Given the purported demise of the BSC, the recent debate, illustrated in Wheeler and Meier (2000), re- veals that the “revolutionaries,” as Avise (2000b) stated, have not come up with something much better. In addition to the often rather nebulous and vague us- age of words in the PSC definitions, any focus on diagnos- able differences between phylogenetic lineages, not necessar- ily representing populations, renders it reductionistic and non-biological, because it ignores the relationship to other populations or taxa within a geographical and historical con- text. The operational orientation of the PSC (serving better diagnosability) not only leads to subjectivity, it also renders the PSC a clearly typological concept close to essentialism, which had been overcome with the modern synthetic theory of evolution. As a result, the PSCs will eventually lead to the same ballooning of species numbers as observed in the 19"? century. In combination with the arbitrariness of changing de- limitation of species under the different versions currently available, the PSCs render comparative studies of faunas and speciation processes hazardous. Only very disputably is the PSC truely a species concept, i.e. in the sense of having any relevance to a species as a natural entity in nature. Defined in this way, a phylogenetic species does not play any role in the ecosystem nor seem to have any interaction with other populations of the same species or with other species, but only serves as a description of a taxon on a cladogram. In addition, the various stages of differentiation in geographi- cally vicariant populations or taxa are not distinguished taxonomically. Because of the subjectivity of delineating populations in the patchy, allopatric situations of continental areas, the ap- plication of the PSC under the diagnosability version endan- gers consensus among systematists and, therefore, taxo- nomic stability (see critique in Eck 1998, also Snow 1997, Haffer 1995b, 1998, Wheeler and Meier 2000). Irrespective of these problems, the PSC has been widely stated as pro- viding an objective species concept. It was strongly advo- cated, first by ornithologists (for example see Cracraft 1983, 1989, McKitrick and Zink 1988), but later also by other practical zoologists (see Kottelat 1997 and Lydeard et al. 2000 for two examples). Implications and consequences Proponents have failed to develop a single useful, stan- dard definition for a “phylogenetic species” that secures con- gruent use. In addition, the PSC is an attempt to combine two widely disparate concepts, namely monophyly and di- agnosibility, into one species definition, which increases the number of practical and theoretical problems (see for ex- ample Sluys 1991). Thus, with respect to the alternative defi- nitions under the PSC, the criteria given do not achieve the demanded degree of objectivity. Ultimately, the immanent subjectivity of the definition will result in arbitrary species delimitations. In contrast, the criterion of reproductive isolation under the BSC provides an objective means of separating sympatric species. This criterion also represents the causal factor that produces and maintains discrete entities. Avoiding an infla- tion of “species” by naming even slightly differentiated forms or populations with unbalanced degrees of differen- tiation, which can happen using the PSCs, the BSC makes an attempt to delineate taxa as species with respect to the same degree of differentiation. When systematists apply a narrow morphological spe- cies concept, they arrive at higher numbers of species. For an example from birds see Figure 2, for other examples from fishes and insects that apply the PSC see Kottelat (1997) and Packer and Taylor (1997), respectively. Thus, so-called “wide” versus “narrow” approaches of delineating species have direct consequences for the assessment of biodiversity and conservation. As a consequence of the perception of species as diagnosable units, the application of PSCs will result in a great proliferation of species and inflate the bio- logical diversity on the lowest taxonomical level, even if one accepts only phenotypic differences and not molecular ge- netic differences. In conclusion, the PSC is certainly not an improvement. Although not perfect, the BSC is still the most useful and meaningful concept, while PSC lacks objectivity and is a step backward to the days of typological thought. Not by acci- dent, the BSC became the “working definition” of species 2 AMERICAN MALACOLOGICAL BULLETIN 19 * 1/2 + 2004 among most population and evolutionary biologists for most of the last century, although it was formulated not for convenience but for its correspondence to natural phenom- ena, as Coyne ef al. (1988) pointed out. Taken together, the problems with the BSC are fewer than those faced by other species concepts, particularly those based on morphology. Therefore, the difficulties of applying the BSC are not such as to justify its rejection in favor of other, logically and biologically worse concepts. The BSC is to be favored be- cause it is the only definition that is based primarily on the biological significance of a species. TWO EXAMPLES FROM FRESHWATER GASTROPODS Initially in malacology (when it was perceived essentially as conchology), a plethora of nominal species were de- scribed, followed by only a slight tendency to reduce the numbers of species after population thinking was imple- mented in some areas of the study of molluscs. Generally, it was concluded that much of the observed conchological variation actually represented population-level phenomena. However, in the absence of studies explicitly focussing on reproductive isolation in sympatry, most taxonomic deci- sions are still largely subjective and primarily based on morphology. Because more freshwater biotopes occur in isolated ar- eas than do marine or terrestrial ones, populations of fresh- water gastropods tend to be isolated (Rensch 1929, Huben- dick 1954, Meier-Brook 1993, Glaubrecht 1996). This discontinuous distribution not only leads to morphological variations in isolated populations and microgeographic races, but consequently also results in the naming of nearly each of these populations as distinct species under typologi- cal-morphological species concepts. This will also be the case under the PSC, because these concepts do not take into account biological phenomena such as geographical distri- bution and genetic cohesiveness, even of temporarily sepa- rated populations. Two case studies of freshwater gastropods of the former “melaniid species basket” (“Melaniidae” = Thiaridae sensu lato of the superfamily Cerithioidea, see Glaubrecht 1996, 1999) illustrate this point, using taxa of Lavigeria Bourguig- nat, 1888 from Lake Tanganyika (which should be grouped as belonging to the Paludomidae instead of Thiaridae s. str., see Glaubrecht 1999, Strong and Glaubrecht 2002), and the North American Pleuroceridae. Case 1: “Le Bourguignatisme”—an example from Lake Tanganyika One of the most unhappy episodes in the history of malacology is the French school of the so-called “Nouvelle TREATING SPECIES AS DYNAMIC ENTITIES 1 Number of forms Species and subspecies i) ies) Mayr 30,000 28,500 © Subspecies Sharpe 20,000 : 18,939 9 species / and subspecies / Z G.R. Gray A 11,162 10,000 7 eo oo G.R. Gray Pi 6,000 : Latham 70 Species 2,951 (9°77 Brisson) = Lo ~ 3,779 Illiger 1,500 Oo” 2000 A.D. 1800 1750 1850 1900 1950 Figure 2. Implication of applying different species concepts. Marked by the works of ornithologists during the last 250 years the increase of numbers of species and subspecies of birds is shown, culminating in the recognition of 18.939 species around the turn to the 20" century. Applying the multidimensional species concept (under the theory of the BSC and influenced by the “Berlin circle” and Ernst Mayr, see text), eventually halted this process after 1900. Reversing the situation during the 1920s and 1930, many morphospecies were reinterpreted as subspecies and combined in more widely conceived biological species taxa. Immediately, this resulted in a precipitous decline in the number of species recognized. It was the emphasis of the existence of closely related allopatric and parapatric species (together forming a superspecies) that led to a moderate stability regarding species numbers during the 1930s and 1940s, and to an estimated total number of known birds of today around 9700 species. This process, starting during the late 1920s, when geographically representative biospecies were discovered, can be aptly called a “quiet revolution” in systematics, that is still missing in malacology [after Haffer 1992: 147, fig. 4]. Ecole,” with Jules-René Bourguignat [1829-1892] as its main proponent. Bourguignat considered as distinct species (and merited a name to) taxa that could be distinguished on the grounds of three or more constant characters. Called the “bete noir of European malacology” (Dance 1970) and the “great species manufacturer” (Kobelt 1881), Bourguignat was the most radical among conchological splitters. Unable to comprehend or accept the concept of species as a bio- logical entity, he regarded species as abstractions that did not exist outside his imagination. One of Bourguignat’s special interests was the so-called thalassoid (marine-like) molluscan fauna of Lake Tanga- nyika. Someone who could conjure up dozens of novelties from an average European lake or make 20 species out of a well-known species of European freshwater mussel, could work miracles with these thalassoid gastropods. Based on collections from various French naturalists, Bourguignat de- scribed 75 new “species” and proposed 9 new genera in his final masterpiece, the “Histoire malacologique du Lac Tan- ganika” (Bourguignat, 1890). For example, he split Lavigeria as we know it today into five genera and compiled 51 named species for it, introducing 46 new species between 1885 and 1890 (another 7 species were described in subsequent de- cades). In contrast, only one or two species, Lavigeria nassa (Woodward, 1859) and Lavigeria grandis (Smith, 1881), re- spectively, were accepted by Leloup (1953) and Brown (1980, 1994) under the (implicit) application of the BSC. For an illustration of the taxonomic history of Lavigera see Table 1. Bourguignat and his colleagues can be excused by the fact that they lived when there was little agreement over the species problem. However, this situation is quite different from today with some systematists only being dissatisfied 124 AMERICAN MALACOLOGICAL BULLETIN Table 1. The taxonomic history of Lavigera, a thalassoid gastropod endemic to Lake Tanganyika, East Africa, illustrates the changing number of species (and generic) names applied under different taxonomic concepts. Author No. of species No. of genera Bourguignat (1890) 51 species 5 genera Pilsbry and Bequaert (1927) 22 species 1 genus Martens (1897) 10 species 1 genus Leloup (1953) 2 species 1 genus Brown (1980, 1994) 2 species 1 genus Michel (2000) 20+ species 1 genus Todd and Michel (2001) 30+ species 2 genera with the answers already available to the species question. It is interesting to note that this dissatisfaction has again re- sulted in an increase of the number of named species in Lavigeria. While only two species were accepted since Le- loup’s (1953) treatment, more recently the existing morpho- logical disparity in Lake Tanganyika has been approached by naming the smallest diagnosible units (as suggested, for ex- ample, under the PSC) and implying a local radiation within this genus (e.g. Michel 2000, Todd and Michel 2001). Re- grettably, not only is Bourguignat’s typological approach re- peated this way, but also a general weakness of his treatment, i.e. proposing high numbers of species in the absence of providing a modern systematic revision. Thus, it is currently difficult to understand the biology and systematics of the genus Lavigeria. For example, it has been proposed that viviparity in Lake Tanganyika gastropods in general and in Lavigeria in particular was a major factor in the causation of the radiation of species flocks and species richness, respectively (Cohen and Johnston 1987, Michel 1994). This claim has been discussed and rejected by Glau- brecht (1996, 2001, see also Strong and Glaubrecht 2002). First, most thalassoid gastropods are actually not viviparous but oviparous, and second, those genera that are viviparous, other than Lavigeria, in particular Tanganyicia Crosse, 1881 and Tiphobia Smith 1880, are monotypic. What is special about Lavigeria? This taxon has a unique morphological di- versity, tempting Todd and Michel (2001) to, “delimit work- ing species-concepts using these shell characters, indepen- dent of geographical considerations to prevent occurrence information biasing our identifications and then assign the nominal species to our concepts.” Apart from the authors’ unconventional idea of what a “concept” of a species is and how to “assign” the latter to the former (see discussion above), this procedure resulted in their conclusion that their “systematic framework for the genus currently consists of over 30 species,” and that “many more species remain to be discovered as sampling improves” (Todd and Michel 2001: 355). The question remains unanswered what natural (spe- 19+ 1/2 * 2004 ciation) mechanism causes this enormous species flock to evolve and whether there is any ecological and/or geographi- cal correlation indicative of, for example, habitat specificity and fragmentation and/or intralacustrine allopatry. In con- trast to the procedure chosen by these authors, who do not want to be “biased” by information on occurrences, evalu- ating the actually morphological disparity and purported taxonomic diversity in Lavigeria within a microgeographical framework that includes the ecological context would be a most promising research program to address the species question. Case 2: The species question in North American Pleuroceridae The Pleuroceridae has long been recognized “as one of the most difficult families of American mollusks,” (Pilsbry and Rhoads, 1896: 495). For more than a century, high degrees of shell variation caused authors to describe a plethora of species and subspecies. The bewildering variety of shell phenotypes, interpreted as the result of an extensive endemic radiation particularly in streams and rivers of the southeastern USA posed tremendous problems to the sys- tematics of this group. Accordingly, Dillon (1984: 70) noted that “pleurocerid taxonomy is currently in a confused state.” The outstanding (and still most comprehensive) sys- tematic monograph of this family by George W. Tryon (1873) listed a total of 464 species for North America. In his treatment of the genus Elimia H. and A. Adams, 1854 (= Goniobasis Lea, 1862) alone, Tryon recognized 255 species. He later clearly saw that a reduction of pleurocerid species must be made, coming to the belief in 1888 that “there were not more than a tenth as many good species as names” (see Pilsbry and Rhoads 1896: 496). In the introduction to his monograph, Tyron (1873: li) had remarked concerning the morphological variation found in pleurocerids: “We thus find that no one character (with very few exceptions) can be relied on in species discrimination, but rather a combination of characters, with a general idea of the necessary allowance for variation pervading other species of the same general type, or contiguous locality.” Like many of his contemporaries, Tryon was aware of the species problem but not of the solution to it. In adding to his monograph the correspondence with another contem- porary malacologist, James Lewis, Tryon gave some insight into the debate. For example, in discussing the most variable species from the creeks in Tennessee with “a perfect series of differentiations of carinated apices,” Lewis (cited in Tryon 1873: 424-426) remarked that “one cannot tell where to assign limits. Limits are apparently obliterated and species have no existence. We are very largely at the mercy of opin- ion, some of which, no doubt, are but the reflex of the idiosyncrasies of the persons with whom they originate.” TREATING SPECIES AS DYNAMIC ENTITIES 125 What Lewis very aptly called the “key to the origin of many of our species” provides an explanation for the typological species-making in freshwater gastropods. Because it was common practice of local collectors to send only single shells for identifications to experts of the group, it appears as no “wonder then, that the descriptive naturalist should unwit- tingly fall into a very natural mistake and describe these shells as new species” (see Tryon 1873: 426) Pilsbry (in Pilsbry and Rhoads 1896: 496) was aware that the same species often occurs in some localities with the shell sculptured throughout, in others with sculpture only on the upper portion, and in still other localities only with the characteristic sculpture on the earlier whorls. Antici- pating a research program finally taken up much later, he concluded that “these shells must be collected and studied by river-systems.” Goodrich (1940, 1942), for example, studied members of the Pleuroceridae in the Ohio river system and the Atlantic coastal plain, compiling data for 81 species from these drainage complexes (leaving others unmentioned, however). Thus, he first tried to sort out named shells from real biological entities and to clarify some of the confusion over the various names erected for this over-described family of aquatic snails. In his compilation of North American freshwater snails, Burch (1982) provided the most and only recent overview, listing a total of 212 pleurocerid taxa (including subspecies and “morphs”), of which 152 were attributed species status. For the diverse and morphologically disparate genus Elimia alone he reduced the number given by Tryon (1873) by two-thirds, recognizing 83 species. Although it is generally realized now that there are problems with the delineation of species based solely on shell morphology in snails that ex- hibit clinal variation, Burch’s compilation still provides the only attempt so far to comprehensively treat the entire group. Nevertheless, any attempt to revise the Pleuroceridae and provide a formal systematic monograph is lacking, probably due to the enormous problems caused by the chaotic taxonomy resulting from former typological approaches. Several studies comparing the amount of phenotypic and genotypic variability in pleurocerid species from various river drainages, using measurements of genetic divergence/ similarity based on allozymes (for example Chambers 1980, Dillon and Davis 1980, Dillon 1984, Dillon and Lydeard 1998) or mitochondrial sequence data (Lydeard et al. 1997, 1998, Holznagel and Lydeard 2000, Mihalcik and Thompson 2002), reveal conflicting evidence as to the morphological and genetic/molecular concordance within and among spe- cies and genera of pleurocerids. From the results on pleu- rocerids it was concluded, (1) that morphological variability is correlated with environmental differences, (2) that species identification using shell morphology alone is often unreli- able, (3) that because intrapopulation genetic variation is low and interpopulation divergence is high gene flow even among conspecific populations connected through water can be quite low, and (4) that there are different views of species relationships and taxonomy based on electrophoretic studies and molecular genetic data compared to previous work based on shell morphology. For example, for taxa of the Elimia (= Goniobasis) floridensis (Reeve, 1860) species complex in Florida, Chambers (1980) reported that the di- vergence in shell sculpture was accompanied by little or no genetic divergence, which has been greatly facilitated by the low frequency of dispersal between drainage systems. Given that the geographic distribution of these freshwater gastro- pods are subdivided by the discontinuities of their habitat, Chambers (1980) favored an allopatric model of speciation when concluding that geographic barriers between popula- tions have probably played a major role in promoting the complex pattern of speciation observed in the evolution of Pleuroceridae. Studying species of Elimia occurring from Virginia to Georgia, Dillon (1984) emphasized a strong correlate of geo- graphic distance with genetic divergence between popula- tions. Thus, although the range of a species is fragmented into a large number of isolated populations separated from one another by mountains between drainages and by stretches of large, apparently uninhabited river (Dillon and Reed 2002), genetic cohesion is maintained even with neg- ligible gene flow. Geographically isolated populations not sharing alleles at many studied allozyme loci did not dem- onstrate reproductive isolation, as Dillon and Lydeard (1998) noted. Similarly, in a study of the species of the pleurocerid genus Leptoxis inhabiting the Mobile River basin of Alabama, Dillon and Lydeard (1998) found some of their data to be more consistent with a hypothesis of geographic isolation rather than reproductive isolation (see also Dillon and Reed 2002). Nevertheless, they strongly advocate special attention and conservation status for those pleurocerid populations to which species status would be attributable on the basis of high genetic divergence. With respect to the number of species as well as how and where to delineate species-level taxa in Pleuroceridae, many contradicting arguments have been put forward, at least in part based on the considerable mismatch between morphologically distinguishable taxa and those found either by electrophoretic studies or by molecular genetic analysis (mtDNA). For example, stating that certain shell characters (for example, sculpture) can give a misleading view of in- terspecific boundaries and relationships, Chambers (1990) recognized only four species of Elimia in Florida river drain- ages, namely Elimia floridensis (Reeve, 1860), Elimia dickin- 126 AMERICAN MALACOLOGICAL BULLETIN sont (Clench and Turner, 1956), Elimia boykiniana (Lea, 1840), and Elimia curvicostata (Reeve, 1861), where earlier treatments had considered 10 species. In contrast, Thomp- son and Mihalcik (2002) and Mihalcik and Thompson (2002) identified the previously recognized Elimia curvico- stata from rivers in western Florida to Georgia, for which Chambers has listed 10 junior synonyms, as a complex of 14 morphologically distinct species, describing five new species and two new subspecies. These authors propose that because of convergence in adult shells, the juvenile shells are of pri- mary importance in distinguishing species. In their parallel molecular analysis they found five distinct species clusters that correlate geographically to different river drainages. Earlier, Thompson (2000) described, based on morphologi- cal evidence only, four additional species of Elimia from the Coosa River drainage in Alabama. A similar association of clades in a molecular phylogeny with drainage basin rather than with traditional morpho- logical groupings of the currently recognized taxa was found in studies of the pleurocerids from the Mobile Basin (Sides 2002). Minton (2002) found, in a cladistic analysis of the genus Lithasia from the Cumberland, Ohio, and Tennessee River drainages that morphological characters (shells and radulae) alone neither recover currently or historically rec- ognized groups at the species level nor do they match with those taxa delineated based on molecular phylogenetic analysis (see also Lydeard et al. 1997). In addition, Minton and Savarese (2002) found evidence for the existence of an undescribed phylogenetic species in the Harpeth River, Ten- nessee, this time explicitly applying the concept of phyloge- netic species in their study. Based on studies on genetic variation at allozyme loci among populations of two species of Elimia, Elimia proxima (Say, 1825) and Elimia catenaria (Say, 1822) from the At- lantic drainages of the Carolinas, Dillon and Reed (2002) called into question the species’ identifications and status of some nominal species and subspecies and their relationship in neighboring Atlantic drainages. For example, they sug- gested that E. catenaria might occur also in Georgia (and maybe even further south), instead of applying different names to populations with slightly distinct morphological (shell) characters whenever found in different drainages of an adjacent state. Although the century-old suggestion to study pleuro- cerid systematics by river systems instead of typological naming of individual shells has finally been taken, a general disagreement on how to apply species concepts to these highly polymorphic freshwater gastropods in light of new biochemical methods has not yet greatly improved the situ- ation. Currently, the systematics of Pleuroceridae are con- strained between the Scylla of a relatively wide approach of 19 * 1/2 + 2004 molecular phylogenetics that chiefly resolves intergeneric re- lationships in an effort to understand the evolution of the entire family (Holznagel and Lydeard 2000) and the Charyb- dis of a narrow focus on populations within individual rivers or drainages and a restriction to only few species-level taxa (Dillon 1984, Lydeard et al. 1997, Dillon and Reed 2002, Minton 2002, Sides 2002). Taking the geographical context into consideration on a larger scale, such as comparing con- generic taxa like Elimia or Leptoxis across their entire distri- butional ranges and all inhabited drainage systems, in con- cert with a cladistic analysis of morphological and molecular data would greatly enhance our understanding of the nature of species in these North American gastropods. CONCLUSION Nature, in some respects, comes to us as continua, not as discrete objects with clear boundaries... But since nature has built a continuum, we must en- counter ambiguity at the center. Some cases will be impossible to call—as a property of nature, not an imperfection of knowledge (Gould 1985) Species as dynamic entities Species are, and therefore should be conceived of as, dynamic entities that need to be placed in historical as well as geographic contexts. Biological discontinuities such as re- productive isolation by which the species are characterized in nature should be utilized to define them. Among the plethora of species concepts suggested in the past, the BSC and the PSC(s) confront us with the twin dangers of either “overlumping” obviously distinct specific variation (on phe- notypic and on genetic grounds) via strict application of the BSC, or oversubdividing biodiversity on lowest taxonomic levels. Because things in nature that seem distinct may rep- resent the extremes of a continuum, I have emphasized (1) the historic dimension of the species debate and (2) the horizontal dimensionality of the species concept, that is the geographical factor in the discussion on the nature of spe- cies. In order to recognize biological species as evolutionary and ecological units we need to combine data on geographic variation with information on dispersal and environmental history (i.e. the biogeographical patterns). To this purpose, the BSC provides the only non-arbitrary criterion available, namely the presence or absence of interbreeding between two populations coexisting temporally and spatially. In con- trast, the PSC determines species status based on the sub- jective and arbitrary criterion of diagnosability (that is, spe- cies as the smallest diagnosable units). Biologists should be more aware and, consequently, ex- plicit in applying different conceptual approaches to the spe- cies problem. If not using the concept of a biological species TREATING SPECIES AS DYNAMIC ENTITIES 127 as reproductive community but focussing on diagnosability only (either at the morphological or molecular level), au- thors should explain their line of argument as to their per- ception of species in nature. For the most interesting and spectacular case studies of enlarged species diversity as re- cently discovered, for example, in limnic hybrobiids in North America and Australia, or some thalassoid molluscs in ancient lakes such as Lake Tanganyika and the central lakes on Sulawesi, the taxonomic descriptions of the many new taxa should be supplemented by addressing the general problems of species discrimination with a non-essentialistic species concept. Within the framework of evolutionary knowledge and population thinking the discussion and analysis of speciation in those cases would certainly enrich the century-old debate on the orgin of species diversity. There are many approaches in malacology today to over- come the purely descriptive tradition that resulted from the often uncritical multiplication of taxa names during the ty- pological times of the 19'" century. Modern taxonomy is in- creasingly aware of the uniqueness of individuals on the one hand and the wide range of variation within any population of individuals on the other hand. While malacology is often too narrowly focussed on accumulating data, other disciplines, such as ornithology, led the way in testing general evolutionary theories, including the predictions from species concepts and speciation hypotheses. What is needed is the integrated syn- thesis between the malacologist compiling observations from the field and laboratory and the malacologist evaluating theories within the framework of historical achievements. Rather than a lack of definitions, the real neglect is the absence of a clear statement of why and on which grounds decisions on species status have been made. Too often in systematic revisions and other taxonomic accounts, any ref- erence to the species concept is either lacking or the defini- tions given and/or used are unconventional, incorrect, or misleading. As long as this situation continues, the progress in systematic science is hampered as much as during Dar- win’s days, when “different naturalists made different deci- sions on different grounds, with the result that the deci- sions—and the entities dealt with—certainly did appear purely arbitrary” (Kottler 1978: 296). In this context, and in turning around the traditional tendency to look and describe “specific” differences, we should start with a single species as null hypothesis. In examining any set of morphological and genetic data we should only accept the more complex hy- pothesis of two or more species if a better fit with the data available necessitates this. Towards a phylogeographical synthesis There is a long research tradition in zoology of geo- graphical variation and the characterization of geographic varieties. We need to re-vitalize this tradition and at the same time employ newly available molecular and other tech- niques, as exemplified recently in phylogeography. This field of study is concerned with the principles and processes gov- erning the geographical distribution of genealogical lineages, especially those within and among closely related species (for review see Avise 2000a). A primary requirement of the ex- pansion of empirical studies of comparative phylogeography is the acquisition of biogeographic information on a regional scale. In many cases in invertebrate zoology, however, those basic biogeographic data are not available, thus hampering the integration of genealogical data. Concerning the ques- tion of how to delineate species, we are not suffering from a lack of definitions, but rather from incomplete biological information. The recent molecular revolution of phyloge- netics with the now widely-used methods of PCR and se- quencing has provided powerful tools for species-level stud- ies based on the reconstruction of past events and geographic modes. For example, within limnic molluscs with confusing taxonomy and poorly understood biogeography, this is most recently exemplified by the mudsnail species of the hydrobiid genus Hydrobia (Wilke et al. 2000) and by the limnic bivalves of the genus Corbicula (Pfenniger et al. 2002). There is a great need for the integration of more data, not only on morphological and molecular variation but also on geographic distribution. The fact that the range of intra- specific variation over a given region is often insufficiently known renders any evaluation of gene flow among popula- tions hazardous. The importance of knowing the structure of the population genetics of a species or species complex as a prerequisite for determining the genetic units has been illustrated recently for the epidemiologically important vec- tor of malaria Anopheles gambiae s. str. (della Torre et al. 2002) and for some snails that are vectors for schistosomiasis (reviewed recently by Blair et al. 2001). Future challenges Any in-depth debate of the species question in malacol- ogy faces two major challenges: first, to get more of the relevant data for as many taxa and case studies as possible and second, because natural processes are constrained by a three-dimensional space, to make inferences in the appro- priate spatial and temporal context. To gain the data for these inferences, the various stages of differentiation, par- ticularly in contact zones and nearby areas, should be fo- cussed on, and molecular and morphological variation tested in allopatry, parapatry, and sympatry, with the aim of attributing the status of allospecies, paraspecies, or semispe- cies to local populations (Fig. 1). Attempts to make these inferences are led by the conviction of Stebbins (1969), albeit in another context, that “the best system for any group is one synthesized from data of all kind.” Scrutinizing our ideas on the nature of species thus demands the integration of mor- 128 AMERICAN MALACOLOGICAL BULLETIN phology (from diagnostic biometry to anatomy and histol- ogy), molecular genetics, and biogeographical analyses supplemented by data from ecology, ethology, and other sources. Avise (2000a, 2000b) suggested that wedding the better elements of the traditional BSC and PSC will eventu- ally produce a synthetic conceptual framework for species recognition. In this ongoing phylogeographic synthesis, population-demographic and population-genetic principles should be supplemented by historical geographic consider- ations. Instead of trying to find another species definition or concept, we should make use of the heuristic properties of the existing biological and phylogenetic ones. A phylogeographic approach that combines the repro- ductive criterion of the BSC (such as barriers, isolates and geography) with the phylogenetic criterion of the PSC (namely historical and demographic aspects) will eventually lead to a most fruitful synthesis. The increasingly better and more detailed documentation of morphological and mo- lecular genetic differentiation of molluscs in their spatiotem- poral context will result in a taxonomically improved clas- sification based on insights from biology and phylogeny. Freeing malacology from the typological naming of whatever was previously diagnosed as “species” will then truly become Leopold von Buch’s legacy. ACKNOWLEDGMENTS Writing this account was triggered by many intensive discussions with colleagues over the past years, proponents of the concepts of bio- and phylogenetic species or not, for which I thank in particular George Davis, Rob Dillon, Jurgen Haffer, Chuck Lydeard, Michael Ohl, Winston Ponder, Ellen Strong, and John Wise. Special thanks go to the “thiarid tea circle” in Berlin, particularly Thomas von Rintelen, Frank Kohler, and Alexei Korniushin, for inspiring discussions. I am indebted, as always, to Mrs. Ingeborg Kilias for her in- dispensable help with finding even the most remote litera- ture, and to Nora Brinkmann for technical help. Special thanks to Jiirgen Haffer, Rob Dillon, and an anonymous reviewer who read an earlier manuscript for their many con- structive suggestions helping to shape this abbreviated ver- sion. 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Wethington' and Robert Guralnick* ' Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, U.S.A., awething@purdue.edu * University of Colorado Museum and Department of Ecology and Evolutionary Biology, University of Colorado at Boulder, Boulder, Colorado 80309, U.S.A., robert.guralnick@colorado.edu Abstract: A number of physid species, including Physa johnsoni the Banff Springs snail, were first described from hot water environments. Physid populations have a remarkable ability to cope with warm to hot temperatures. We present a comparative genetic analysis of the physid species living in hot springs in the context of a larger preliminary phylogeny of the Physidae. The molecular phylogeny, which was based on partial DNA sequences of the mitochondrial cytochrome oxidase c subunit | and 16S rRNA genes, place P. johnsoni in the Physa gyrina group, indistinguishable from other members of the Western United States within this group. Included in this P. gyrina group are the following putative species: P. johnsoni, Physa aurea (actually of the Eastern United States), Physa wrighti, and an individual of Physa gyrina (possibly Physa wolfiana) found at Hot Sulphur Springs, Colorado. Within the Physa acuta-like hot spring physids, Physa cupreonitens and Physa spelunca form genetically distinct and distant groups, which is indicative of a potentially effective barrier to gene flow between these hot springs and nearby populations of Physa acuta. Colonization of hot spring environments does not typically entail much sequence divergence from colder-water populations. Key words: hot springs; cave; life history evolution; population; systematics Species of the genus Physa Draparnaud, 1801 are more commonly found in heated waters than any other molluscan groups in North America and Europe (Clench 1926), so it is not surprising that there are a number of physids that were originally described from hot springs. Hot springs are local- ized, unique environments, many with a high sulphur con- tent, providing their inhabitants with stable temperatures throughout the year. Springs, and in particular, cave springs, can have a significant impact on gene flow. For example, Gooch and Hetrick (1979) found that ecophenotypic gam- marids in subterranean cave springs (and to some extent other spring populations of gammarids) have low heterozygosities compared to above-ground populations in first and second order streams. Low heterozygosities could be indicative of restricted or limited gene flow into these spring habitats. Given their constancy and persistence over long periods of geological time, it is possible that hot springs could be homes to relict species that have become endemic due to major climatic perturbations such as glaciation (Te and Clarke 1985). Members of the Physa acuta group as well as members of the Physa gyrina group were first described from hot spring environments. Both groups of physids are common and widespread in North America. The P. acuta group is diagnosed by having penial morphology “c” (Te 1978), one non-glandular sheath and a penial gland, and has a world- wide distribution. There is evidence from laboratory breed- ing (Dillon et al. 2002) and molecular studies (A. R. Weth- ington, pers. obs.) that Physa heterostropha (Say, 1817) and Physa integra (Haldeman, 1842) should be synonymized un- der the name Physa acuta (Draparnaud, 1801). The P. gyrina group has penial morphology “b” (Te 1978), which includes a penial gland and two distinct sheaths, one glandular and one non-glandular. They are more constrained by geography and are excellent at persisting in harsh environments such as temporary ponds (Clampitt 1970). Physids of both groups are able to withstand high tem- peratures as well as widely fluctuating temperatures. Indi- viduals of Physa gyrina (Say, 1821) are well suited to live in the elevated temperatures of shallow waters (Clampitt 1970) as well as along a reach artificially heated to 35°C by effluent from a local manufacturing plant (Agersborg 1929). Indi- viduals of Physa anatina (Lea, 1864) of the Physa acuta group can withstand temperatures as high as 40°C (Beames and Lindeborg 1968). Individuals of Physa virgata (Gould, 1855) of the P. acuta group did not alter their metabolic rate after living in artificially heated water for 54 generations (McMahon 1985), lending support to the hypothesis of San- durathri and Holmes (1976) that some species of physids are pre-adapted to living in heated waters, which would allow them to invade hot spring habitats. We focused on mitochondrial gene divergence between physids found in hot spring environments and nearby cold *From the symposium “The Biology and Conservation of Freshwater Gastropods” presented at the annual meeting of the American Malacological Society, held 3-7 August 2002 in Charleston, South Carolina, USA. spring environments to compare the evolutionary histories of hot spring physids in open environments as well as in a cave. This study included six named and one unnamed spe- cies from hot or warm springs: Physa aurea (Lea, 1838), Physa wrighti (Te and Clarke, 1985), Physa johnsoni (Clench, 1926), and Physa wolfiana (Lea, 1869) of the Physa gyrina group; Physa spelunca (Turner and Clench, 1974), an unnamed physid from Mt. Princeton Hot Springs Colorado; and Physa cupreo- nitens (Cockerell, 1889) of the Physa acuta group (Table 1). Most descriptions of physid species rely heavily on shell morphology, including those first described from hot springs. Exceptions include Physa spelunca and Physa wrighti, in which detailed anatomical descriptions are avail- able. Reliance on shell characters makes it hard to identify each putative species, especially outside its type locality, given the large degree of plasticity in shell characters. Shell shape can be influenced by the presence of predators (De- Witt 1995, DeWitt 1998, DeWitt et al. 1999, DeWitt et al. 2000, Langerhans and DeWitt 2002) as well as by environ- mental factors including temperature (Burnside 1998, Brit- ton 2004). According to the original descriptions, Physa wrighti and Physa spelunca have unusual anatomies that result from living in their environments. Individuals of P. wrighti share anatomical characters with three distinct groups (the Physa acuta group, the Physa pomilia group, and the Physa gyrina group) (Te and Clarke 1985). Physa spelunca was described as having no pigment (Turner and Clench 1974) but live specimens exhibit a wide range of color polymorphism that AMERICAN MALACOLOGICAL BULLETIN 19° 1/2 + 2004 includes the albino form (Megan Porter, personal commu- nication). Turner and Clench (1974) also describe P. spe- lunca as having reduced eyes and an increase in radular teeth size and protoconch size compared to other physids. These modifications could be adaptations to living in a cave with a constant, yet nutrient-poor environment, as Turner and Clench (1974) suggest. Our main goal was to use sequence divergence as an estimate of how long a particular form of physid has been in a hot spring environment and to use inferences based on experiments on reproductive isolation with other physids to explore the validity of hot spring taxa. We argue that for physids first identified from hot springs to be considered good species, they should show amounts of mitochondrial sequence divergence similar to other species-level diver- gences in other pulmonates. METHODS Due to the difficulties that can arise from matching newly collected snails to original descriptions of the shell, we collected snails from the type locality of each species. This allowed us to compare sequence identity/divergence between them and other physids collected from a broad geographic area near each hot spring environment. Given the isolated nature of hot springs from colder water environments, physids originally described from a hot spring and subse- quently collected from that spring should be the same spe- cies (see Table 1 for habitat descriptions). Table 1. Shell lengths, shell widths, reported water temperatures, and current environmental information for each species of physid from hot springs. Species Physa aurea Physa wolfiana Physa cupreonitens Physa johnsoni Physa spelunca Physella wrighti Site of original desciption Hot spring at Bath, VA Hot Springs, CO Hot springs at Wellsville, CO Middle spring of the Hot Sulphur Springs in Banff National Park, Alberta, Canada Cave in Lower Kane Cave between 800-900 feet above Big Horn River, near Kane, WY Alpha Stream, Liard Hot Springs Provincial Park, Alberta, Canada Shell length Shell width Reported water Number of of holotype of holotype temperature springs (mm) (mm) (°C) mentioned Human interference 12.7 7.62 21.1-26.7 one tourists and industry 7.62 4.8 37.7-43.3 one man-made chlorinated pools from hot spring 7.5 4.5 21.1-26.7 more than minimal, privately owned one 7.5 5.2 ~33.3 many historically great, but now in park protected by Canadian law 9.0 4.5 25.6 one minimal, gate is locked and cave has toxic sulphuric gas 5.3 2.8 ~35 one minimal, Provincial Park PHYSID POPULATIONS FROM HOT SPRINGS 137 Because Physa wrighti has been described as a basal member of the Physinae lineage, we have also included sam- pling from other major lineages within the group. If P. wrighti is indeed a relict left over from pre-glaciation events, then it should be distinctly different and basal to other physids, as predicted by Te and Clarke (1985). On the other hand, if P. wrighti is not genetically differentiated from sur- rounding physids, then it could have arisen from one or many recent migration events into the hot water habitat. In addition to representatives from each hot spring and cave population, specimens from populations located near each hot spring and cave environment were included, as well as two representatives from the Physa fontinalis group (Physa fontinalis [Linnaeus, 1758] and Physa jennessi [Dall, 1919]) and the following physids from type locales: Physa acuta and Physa virgata of the P. acuta group, Physa gyrina of the P. gyrina group, Physa zionis (Pilsbry, 1926) which Te (1978) placed in its own genus (Petrophysa Te, 1978), and Physa hendersoni (Conrad, 1834) of the Physa pomilia group. For rooting purposes in the parsimony run, two planorbid spe- cies, Biomphalaria obstructa (Morelet, 1849) and Planorbella trivolvis (Say, 1817), and one lymnaid species, Psuedosuc- cinea columella (Say, 1817), were included as representatives of two freshwater basommatophoran families that are closely related to Physidae. Shell and penial morphology of each individual was examined to aid in the initial placement of that individual into taxonomic groups. Specimens were preserved in 95% ethanol for DNA extraction. See Appendix 1 for taxonomic labels and locality data for each individual physid included in the present study. DNA was extracted from one to three individuals per population using standard phenol chloroform procedures (Sambrook et al. 1989). Pieces of mtDNA from genomic DNA were copied and augmented via the Polymerase Chain Reaction using 16S primers (L2510 and H3080 = 16Sar-L and 16Sbr-H, Palumbi et al. 1991) for a 550 base pair seg- ment and CO! primers (LCO1490 and HCO2198, Folmer et al. 1994) for a 650 base pair segment, cleaned using standard procedures and then cycle-sequenced. The double-stranded PCR products were generated using 50-500 ng of template genomic DNA in 25 ul volumes (10 mM Tris, 50 mM KCL, 2.5 mM MgCl,, 1 uM of each primer, 0.1 mM of each dNTP, 1.5 units Taq DNA polymerase; Fisher Scientific). The am- plification regime began with a denaturation at 92°C for two minutes followed by 35 cycles of the following: denaturation at 92°C for 40 seconds, annealing at 52°C for 60 seconds (16S) or 50°C for 60 seconds (CO1), and extension at 68°C for 90 seconds. The amplified DNA was then concentrated using Millipore Ultrafree MC filters and provided the tem- plate for cycle sequencing using the ABI BigDye kit following manufacturer’s instructions. The reactions were purified us- ing Quiagen DyeEx spin columns and sequenced on an ABI3100 genetic analyzer. The following physid sequences were obtained from gen- bank: Physa johnsoni (CApjomi, CApjolo, CApjoba, Capjoup, and CApjoca), Physa gyrina (CApgycb, CApgyml, CApgyfm, CApgycl and CApgyfly), Physa wrighti (CApwr323 and CApwr745), and Physa sp. (CApsp, probably Physa jennessi of the Physa fontinalis group) from Remigio et al. (2001). The resulting sequences were aligned by eye directly for COI and by using the LSU rDNA secondary structure for 16S (Lydeard et al. 2000). Physids have a large number of base pairs in the coding loop portion of the three dimen- sional 16S rDNA subunit as compared to other pulmonate taxa. Within physids it was impossible to line up one taxon with another, so the loops were excluded from analysis of the 16S data set. The truncated 16S sequences were combined with the CO1 sequences for analysis. Identical sequences were identified and removed from the phylogenetic analysis and the resulting data were ana- lyzed using PAUP* (Swofford 2001). A parsimony heuristic search was performed with 50 random addition replicates to test relationships within and between the various physid groups using Biomphalaria obstructa, Planorbella trivolvis, and Psuedosuccinea columella to root the analysis. One hun- dred bootstrap replicates were performed with 1078 charac- ters resampled at each replicate, optimality was set to par- simony, starting trees were obtained via stepwise addition, addition sequence was random each with ten replicates, and the branch swapping analysis was tree-bisection-reconnec- tion (TBA). To obtain genetic distance measurements for the haplotype network, Modeltest (Posada and Crandall 1998) was employed to discover the best base pair substitution model for the physids only. For the combined analysis, the General Times Reversible model was modified with a gamma distribution parameter (G) and the estimated num- ber of invariable sites (1) by hLRT in Modeltest version 3.06. The alpha level was set to 0.01. Base frequencies were as follows: A = 0.30180, C = 0.13850, G = 0.16990, and T = 0.38980. The assumed proportion of invariable sites was 0.3594. The Gamma distribution shape parameter was 0.7764. These settings were employed for 10,000 bootstrap replicates of a Neighbor-joining search using BioNJ method and maximum-likelihood distance measures. The data were also run solely with CO1 to add Physa wolfiana. For the CO] analysis, the General Times Reversible model was modified with a gamma distribution parameter (G) selected by hLRT in Modeltest version 3.06. The alpha level was set to 0.01. Base frequencies were as follows: A = 0.2808, C = 0.1389, G = 0.1454, and T = 0.4349. The pro- portion of invariable site (I) was none. The Gamma distri- bution shape parameter was 0.3612. This model was used in a neighbor-joining analysis with 10,000 replicates. 138 AMERICAN MALACOLOGICAL BULLETIN RESULTS The best score for the heuristic search was a tree length of 1330, held by 80 trees, for the combined analysis (Fig. 1). The combined data set yielded 1078 total characters; 544 characters were constant, 145 characters were variable but uninformative, and 389 characters were variable and infor- mative. The separate CO1 analysis retained 2122 trees, all of tree length 892, under the heuristic search criteria. The CO1 analysis yielded 658 total characters, 359 characters were constant, 67 characters were variable but uninformative, and 252 characters were variable and informative. Penial morphology correlated with the resulting mo- lecular phylogeny for Physidae in both analyses with four distinct groups represented: the Physa acuta group with bootstrap support of 99 (which included Physa zionis), the Physa pomilia group with bootstrap support of 100, the Physa gyrina group with bootstrap support of 100, and the Physa fontinalis group with bootstrap support of 97 for the combined analysis. Physids from hot springs either fell in the P. acuta group or the P. gyrina group and as such did not form a monophyletic group. In the combined analysis, each population from a hot spring for which more than one member was sampled represented a genetically distinct taxon within the P. gyrina group or the P. acuta group. The only exception was Physa johnsoni (Fig. 1), which was para- phyletic and included P. gyrina from Boyer River, Iowa (type locale of P. gyrina). None of the physids from hot springs within the Physa gyrina group (Physa johnsoni, Physa wrighti, Physa wolfiana, and Physa aurea) were especially distant genetically from other geographically nearby P. gyrina taxa (ranging from 0-1.6% different). The largest genetic distances between a hot-spring physid and nearby P. gyrina was 0.012 substitu- tions per site for P. aurea of Virginia in the combined analy- sis (Fig. 2). This same pattern was seen in the CO1 analysis. Physa cupreonitens was approximately 4.4% different from nearby coa021 (from Mount Princeton Hot Springs) and was 17.52% different from co43936 (also in Colorado but of another water drainage from the Arkansas River). Physa spelunca was about 10.45% different from wybhr2 collected just outside Kane cave in the Big Horn River (in the combined analysis). This gives support to there being an effective barrier to gene flow between these hot spring en- vironments and nearby Physa acuta populations (Fig. 2). The same result was uncovered in the separate CO1 analysis. DISCUSSION Physids of the Physa acuta and Physa gyrina groups can apparently invade hot water environments; there is not a monophyletic hot spring physid group. This is not surpris- 19° 1/2 + 2004 ing considering that P. acuta and P. gyrina cannot success- fully outcross (Wethington et al. 2000, Dillon et al. 2004). Physids living in hot springs of the P. gyrina group (Physa johnsont, Physa wrighti, Physa wolfiana, and Physa aurea) are much more similar to each other genetically than are physids of the P. acuta group living in hot springs (Physa cupreo- nitens, Physa spelunca, and a physid from Mt. Princeton Hot Springs) (Figs. | and 2). Physa wrighti does not fall basal to the P. gyrina plus P. acuta plus P. pomilia groups as predicted by Te and Clarke (1985). Within the Physa acuta group, Physa cupreonitens and Physa spelunca form monophyletic groups with 100% boot- strap support (Fig. 1). Although P. cupreonitens is most likely synonymous with P. acuta, P. spelunca is phylogenetically distinct and genetically distant enough that it could be a separate species, endemic to Lower Kane Cave. Its basal po- sition within the P. acuta group indicates that P. spelunca has been separated from other P. acuta for nearly as long ago as the cave formed. According to A. S. Engel (pers. comm.), the cave probably formed by sulfuric acid speleogenesis, in which sulphuric acid cut into limestone, less than 10,000 years ago (post-glacial). This time estimate is based on the terrace development on the Bighorn River (A. S. Engel, pers. comm.). Given that physids growing in hot waters can re- produce continuously (Agersborg 1929), this could be suf- ficient time for the Lower Kane Cave population to have diverged significantly from other members of the P. acuta group. The physids in Lower Kane Cave are well protected and seem to thrive in their unique environment so the chance of losing P. spelunca seems minimal. Reproductive isolation has been discovered between P. acuta and one basal member of the P. acuta group, similarly distant genetically, uncovered in a recent molecular phylogeny (R. T. Dillon, pers. comm.). Within the Physa gyrina group, Physa wrighti, Physa aurea, and the Physa gyrina of Coyner Springs, Virginia, USA, form distinct monophyletic groups (bootstrap support of 100, 86, and 67 respectively), but Physa johnsoni does not (Fig. 1). This same pattern was found in the COI analysis. This could, in part, be due to sampling, as there were only 2 individuals of P. wrighti, P. aurea, and P. gyrina from Coyner Springs represented in the analysis while there were 7 indi- viduals of P. johnsoni represented. Physa johnsoni, with its small size and proportionately wide shell, is morphologically distinct from the local P. gyrina. Therefore, the identification of the cave spring population as P. johnsoni should be correct. Hot sulphur water may cause physids to grow a smaller and more globose shell. This would be easy to test in the laboratory. Lepitzki et al. (2002) mention that they are rear- ing Physa johnsoni in the laboratory to re-introduce the snail to its former habitat as part of their recovery effort. The morphologies of the shells of P. johnsoni and Physa cupreo- nitens are very similar despite having long-separated ances- PHYSID POPULATIONS FROM HOT SPRINGS 139 physids from hot 65 arory | s springs co43686 86 eee z CL) ophysids from cave paP9 : wybhr2 : mo40408 65 paPlo Fy co43006 | 6atciéat group F253 = 29 co42241 99 co439l4 =F coa021 FY 100 eli coCUPl & c b " : Q = 5 —wySPE) utznp2 - Physa gions ol scystl, sc¥S powalia group scysr3 : c CApjon! : iobre | CApgycl gyring group mopoyfly Physidae oes 34 oo ut3 7088 107 CApwr3 23 m1 il CApwr? 45 49 3 vahsvl vahsv2 : vacsal vacsa2 Cee : all Ninf : # Lymnaeidae Nnf2 as midmpcl Planorbidae oor sched 16PB 2h jonanalis group Figure 1. Parsimony tree of mtDNA 16S + COI with one hundred bootstrap replicates. The numbers above the nodes indicate bootstrap values. The shaded individuals are from hot spring habitats (Physa spelunca is from a cave) and are labeled as follows: CApjon = Physa johnsoni; CApwr = Physa wrighti; vahsv = Physa aurea; coa021 = Mt. Princeton Hot Springs, Colorado; coCUP = Physa cupreonitens, and wySPE = P. spelunca. See appendix for additional taxa and location information for each label. 140 AMERICAN MALACOLOGICAL BULLETIN M onll Nnf 1 0.053 CApsp?46~ an49 i utznp2 co43914 con 2agage 001s 0043888 argyy | 0.010 “0.010 mo40408—paP1o Pa? _F23 “8.010 Fj 057 0.041 wybhr2 1obrg hose CApjon1 Doe ~coCUP2 i wy SPE1 wy SPE2 19+ 1/2 + 2004 CAnjon 0012 oat say e coaQ030 CARE L303 opgytly, CApeyel ut37088 Apgycb CApgym CApg coal07 plorsids from hot springs physids from cave a haplotypes that are similar or identical — 0.01 substitutions/site eee Figure 2. Haplotype network showing the number of substitutions per site between each taxon or group of taxa. The values for nodes having a substitution per site greater than 0.01 are given. The individuals shaded are from hot spring habitats (Physa spelunca is from a cave) and are labeled as follows: CApjon = Physa johnsoni; CApwr = Physa wrighti; vahsv = Physa aurea; coa021 = Mt. Princeton Hot Springs, Colorado; coCUP = Physa cupreonitens, and wySPE = P. spelunca. See appendix for additional taxa and location information for each label. tors. Britton (2004) found that Physa virgata from cold- water populations that were raised in warm waters (30- 35°C) for five generations manifested differences in shell shape. The generation 5 individuals had larger spire angles and hence more globose shells compared to the source population living in cold water. Britton (2004) concludes that there is a genetic and environmental temperature-based component to shell shape in this species. We assert that individuals of Physa gyrina invaded or were accidentally introduced into different hot springs en- vironments and over a short period of time (as few as five generations) these populations evolved distinctive shells. The genetic distance between members of this P. gyrina group are generally not greater than 6% and as such are probably all representatives of the same species, P. gyrina. This is very low compared to the genetic distance found between pulmonate species in general (Thomaz et al. 1996, Ross 1999, Wade et al. 2000, Davison 2000, DeJong ef al. 2001, A. R. Wethington, personal observation). Also, Dillon and Wethington (In press) have shown that Physa aurea can successfully outcross with P. gyrina to the F, generation. It is likely that all taxa of the P. gyrina group included in this study represent one biological species. In conclusion, colonization of physids into hot spring environments does not seem to entail much sequence diver- gence, especially in members of the Physa gyrina group. The sequence divergence uncovered within the Physa acuta group could be due to sampling error or could reflect un- derlying ecological or physiological differences between the physids living in hot springs (particularly Physa spelunca) and the rest. ACKNOWLEDGMENTS We thank Megan Porter of BYU who collected Physa splelunca for us from its type locale and Steve Finch of Salick, PHYSID POPULATIONS FROM HOT SPRINGS 141 Colorado, who graciously let A. R. Wethington and her sister see his hot spring and allowed us to collect Physa cupreo- nitens on his property, a type locality. We also thank Rob Dillon and Tom Smith of the College of Charleston who collected Physa aurea and Physa virgata, respectively, from their type locales. Rob Dillon also provided this study with Physa gyrina from Coyner Springs, Virginia. This study ben- efited from correspondence with Charles Pacas (Aquatic Specialist of Banff National Park), David Prescott (Endan- gered Species Specialist, Parkland Region Alberta Environ- ment), and Dwayne Lepitzki (Parks Canada), who agreed to send four specimens of Physa johnsoni from Banff National Park in ethanol for this study. Ellinor Michel and Philippe Jarne collected physids from some of the populations used in this study. Rob Dillon, Bryan Dillon, Christopher Rogers, Elliot Rogers, Katy Metzner-Roop, Andrew Roop, Susan Wethington, and Zelda Wethington all provided assistance in the field. George Oliver of the Utah Natural Heritage Program helped us to procure permission to collect Physa zionis from Zion National Park. Charles Lydeard, Rob Dil- lon, Dwayne Lepitzki, an anonymous reviewer, and Kathryn Perez offered some helpful comments and advice in the writ- ing of this manuscript. Funding sources include: NSF (to Charles Lydeard and Rob Dillon), Conchologists of America, Western Society of Malacologists, University of Alabama graduate research and travel grants, and DBI-0070351 NSF equipment grant (to Charles Lydeard), which provided the University of Alabama with an automated sequencer. This study was completed as partial fulfillment for A. R. Weth- ington’s dissertation at the University of Alabama. LITERATURE CITED Agersborg, H. P. K. 1929. The relation of temperature to continu- ous reproduction in the pulmonate snail, Physa gyrina Say. Nautilus 43: 45-49, Beames, C. G., Jr. and R. G. Lindeborg. 1968. Temperature adap- tation in the snail Physa anatina. Proceedings of the Oklahoma Academy of Science 48: 12-14. Britton, D. K. 2004. Environmental and genetically induced shell shape variation in the freshwater pond snail Physa virgata. American Malacological Bulletin 19: 93-100. Burnside, C. 1998. Ecophenotypic Variation in Shell Morphology within the Freshwater Pond Snail Genus Physella (Pulmonata: Basommatophora) and its Taxonomic Implications. Ph.D. Dis- sertation, University of Texas, Arlington. Clampitt, P. T. 1970. Comparative ecology of the snails Physa gy- rina and Physa integra (Basommatophora: Physidae). Malaco- logia 10: 113-151. Clench, W. J. 1926. Three new species of Physa. Occasional Papers of the Museum of Zoology 168: 1-8. Cockerell, T. D. A. 1889. Preliminary remarks on the molluscan fauna of Colorado. Journal of Conchology 6: 60-65. Davison, A. 2000. An east-west distribution of divergent mitochon- drial haplotypes in British populations of the land snail, Ce- paea nemoralis (Pulmonata). Biological Journal of the Linnean Society 70: 697-706. DeJong, R.J., J. A. T. Morgan, W.L. Paraense, J. P. Pointier, M. Amarista, P. F. K. Ayehkumi, A. Babiker, C.S. Barbosa, P. Brémond, A. P. Canese, C. P. de Souza, C. Dominguez, S. File, A. Gutierrez, R. N. Incani, T. Kawano, F. Kazibwe, J. Kpikpi, N. J. S. Lwambo, R. Mimpfoundi, F. Njiokou, J. N. Poda, M. Sene, L. E. Velasquez, M. Yong, C. M. Adema, B. V. Hofkin, G. M. Mkoji, and E. S. Loker. 2001. Evolutionary relationships and biogeography of Biomphalaria (Gastropoda: Planorbidae) with implications regarding its role as host of the human bloodfluke, Schistosoma mansoni. Molecular Biology and Evo- lution 18: 2225-2239. DeWitt, T. J. 1995. Functional Tradeoffs and Phenotypic Plasticity in the Freshwater Snail Physa. Ph.D. Dissertation, State Univer- sity of New York at Binghamton. DeWitt, T. J. 1998. Costs and limits of phenotypic plasticity: Tests with predator-induced morphology and life history in a fresh- water snail. Journal of Evolutionary Biology 11: 465-480. DeWitt, T.J., A. Sih, J. A. Hucko. 1999, Trait compensation and cospecialization: Size, shape, and antipredator behaviour. Ani- mal Behaviour 58: 397-407. DeWitt, T. J., B. W. Robinson, and D. S. Wilson. 2000. Functional diversity among predators of a freshwater snail imposes an adaptive trade-off for shell morphology. Evolutionary Ecology Research 2: 129-148. Dillon, R. T., Jr., C. Earnhardt, and T. Smith. 2004. Reproductive isolation between Physa acuta and Physa gyrina in joint cul- ture. American Malacological Bulletin 19: 63-68. Dillon, R. T., Jr. and A. R. Wethington. In press. No-choice mating experiments among six nominal taxa of the subgenus Physella (Basommatophora: Physidae). Heldia. Dillon, R. T., Jr., A. R. Wethington, J. M. Rhett, and T. P. Smith. 2002. Populations of the European freshwater pulmonate Physa acuta are not reproductively isolated from American Physa heterostropha or Physa integra. Invertebrate Biology 121: 226-234. Folmer, O., M. Black, W. Hoeh, R. Lutz, and R. Vrjenhoek. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mo- lecular Marine Biology and Biotechnology 3: 294-299. Gooch, J. L. and S. W. Hetrick. 1979. The relation of genetic struc- ture to environmental structure: Gammarus minus in a Karst Area. Evolution 13: 192-206. Langerhans, R. B. and T. J. DeWitt. 2002. Plasticity constrained: Overgeneralized environmental cues induce phenotype errors in a freshwater snail. Evolutionary Ecology Research 4: 857-870. Leptizki, D. A. W., C. Pacas, and M. Dalman. 2002. Resource Man- agement Plan for the Recovery of the Banff Springs Snail (Phy- sella johnsoni) in Banff National Park, Alberta. Wildlife Sys- tems Research and Parks Canada, Banff Alta, Alberta. Lydeard, C., W. E. Holznagel, M.N. Schnare, and R. R. Gutell. 2000. Phylogenetic analysis of molluscan mitochondrial LSU 142 AMERICAN MALACOLOGICAL BULLETIN 19° 1/2 * 2004 rDNA sequences and secondary structures. Molecular Phylo- genetic Evolution 15: 83-102. McMahon, R. F. 1985. Seasonal respiratory variation and metabolic compensation for temperature and hypoxia in the freshwater snail, Physella virgata (Gould), with special reference to the effects of heated effluents. American Malacological Bulletin 3: 243-265. Palumbi, S., A. Martin, S. Romano. W. O. McMillan, L. Stice, and G. Grabowski. 1991. The Simple Fool’s Guide to PCR. Privately distributed, Honolulu, Hawaii. Posada, D. and K. A. Crandall. 1998. MODELTEST: Testing the model of DNA substitution. Bioinformatics 14: 817-818. Remigio, E. A., D. A. W. Lepitzki, J.S. Lee, and P. D. N. Hebert. 2001. Molecular systematic relationships and evidence for a recent origin of the thermal spring endemic snails Physella johnsoni and Physella wrighti (Pulmonata: Physidae). Cana- dian Journal of Zoology 79: 1941-1950. Ross, T. K. 1999, Phylogeography and conservation genetics of the Iowa Pleistocene snail. Molecular Ecology 8: 1363-1373. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Commonly used techniques. In: J. Sambrook and D. W. Russell eds., Molecular Cloning: A Laboratory Manual Volume 3, 2" Edition, Cold Spring Harbor Laboratory Press, New York. Pp. E.3-E.4. Sandurathri, C.S. and J.C. Holmes. 1976. Effects of thermal effluents on the population dynamics of Physa gyrina Say (Mollusca: Gastropoda) at Lake Wabamun, Alberta. Canadian Journal of Zoology 54: 582-590. Swofford, D. L. 2001. PAUP*: Phylogenetic Analysis Using Parsi- mony (*and Other Methods), Version 4.065. Sinauer, Sunder- land, Maryland. Te, G. A. 1978. The Systematics of the Family Physidae (Basommato- phora: Pulmonata). Ph.D. Dissertation, University of Michi- gan, Ann Arbor. Te, G. A. and A. H. Clarke. 1985. Physella (Physella) wrighti (Gas- tropoda: Physidae), a new species of tadpole snail from Laird Hot Springs, British Columbia. Canadian Field-Naturalist 99: 295-299. Thomaz, D., A. Guiller, and B. Clarke. 1996. Extreme divergence of mitochondrial DNA within species of pulmonate land snails. Proceedings of the Royal Society of London (B) 263: 363-368. Turner, R.D. and W. J. Clench. 1974. A new blind Physa from Wyoming with notes on its adaptation to the cave environ- ment. Nautilus 88: 80-85. Wade, C. M., P. B. Mordan, and B. Clarke. 2000. A phylogeny of land snails (Gastropoda: Pulmonata). Proceedings of the Royal Society of London (B) 268: 413-422. Wethington, A.R., E.R. Eastman, and R. T. Dillon, Jr. 2000. No premating reproductive isolation among populations of a si- multaneous hermaphrodite, the freshwater snail Physa. In: P. D. Johnson and R. S. Butler, eds., Freshwater Mollusk Pro- ceedings—Part II: Proceedings of the First Freshwater Mollusk Conservation Society Symposium, March 1999. Ohio Biological Society, Columbus, Ohio. Pp. 245-251. Accepted: 22 March 2004 PHYSID POPULATIONS FROM HOT SPRINGS 143 Appendix 1. Identification label of each individual included in this study along with its accession numbers (where applicable), its classification, and where it was collected. Acc. # Acc. # State/ County/ Identity 16S Col Genus Subgenus Species Country Territory Region Locality Data argrvl AY651170 = AY651209_ — Physa Physella virgata USA Arizona Gila River argrv2 AY651171 AY651210 — Physa Physella virgata USA Arizona Gila River Capjonl AY651172.— AY651211 Physa Physella johnson Canada Alberta Banff Middle Spring (type) CApjon2 AY651173 AY651212 ~——~Physa Physella johnsoni Canada Alberta Banff Middle Spring (type) CApgycb AF346752. — AF346740 ~— Physa Physella gyrina Canada Alberta Banff C&B Marsh CApgyml AF346753—- AF346741 Physa Physella gyrina Canada Alberta Banff Muleshoe Lake CApgyfm AF346754 = AF346742 — Physa Physella gyrina Canada Alberta Banft Five Mile Pond CApgycl AF346755 = AF346743.—— Physa Physella gyrina Canada Alberta Banft Clear Spring mtpgyfly AF346756 AF346744 Physa Physella gyrina USA Montana Flathead Flathead Lake; Yellow Bay Lake CApjomi AF346747 AF346735 — Physa Physella johnsont Canada Alberta Banff Middle Spring (type) CApjolo AF346748 == AF346736 — Physa Physella johnson Canada Alberta Banff Lower Spring, Cave and Basin National Historic Site CApjoba AF346749 = AF346737 ~~ Physa Physella johnsoni Canada Alberta Banff Basin Spring, Cave and Basin National Historic Site CApjoup AF346750 = AF346738 — Physa Physella johnsoni Canada Alberta Bantt Upper Spring, Cave and Basin National Historic Site CApjoca AF346751 AF346739 —- Physa Physella johnsoni Canada Alberta Banff Cave Spring, Cave and Basin National Historic Site CApsp AF346758 AF346746 Physa Physa sp? Canada Alberta CApwr323 AF419322 AF419323 Physa Physella wright Canada Alberta British Laird Hot Springs Columbia CApwr745 — AF346757 AF346745 Physa Physella wrighti Canada Alberta British Laird Hot Springs Columbia c042241 AY651174 = AY651213.— Physa Physella acuta USA Colorado Mesa creek south of Vega Reservoir, near campground c043888 AY651175 AY651214 Physa Physella anatina USA Colorado Yuma Landsman Creek 043906 AY651176 AY651215 Physa Physella anatina USA Colorado Garfield Elk Creek c043914 AY651177 AY651216 Physa Physella anatina USA Colorado Rio Blanco Yellow Creek 6043936 AY651178 = AY651217 ~~ Physa Physella gyrina USA Colorado Garfield Garfield Creek coa006 AY651179 — Physa Physella wolfiana USA Colorado Grand Colorado River near Hot Sulphur Springs coa007 AY651180 = AY651218 — Physa Physella gyrina USA Colorado Grand western Tributary off Colorado River near Hot Sulphur Springs coa021 AY651181 AY651219 Physa Physella acuta USA Colorado Chaffe Farmers Ditch, trib of Boulder Creek (Mt. Princeton Hot Springs) coa030 AY651182 AY651220 Physa Physella gyrina USA Colorado coCUP1 AY651183 = AY651221 Physa Physella cupreonitens USA Colorado Fremont Hot Springs in Wellesville, Colorado coCUP2 AY651184 = AY651222 — Physa Physella cupreonitens USA Colorado Fremont Hot Springs in Wellesville, Colorado F23 AY651185 AY651223 Physa Physella acuta France Saint-Martin Rieutort Wadi, 25 km north of de Londres Montpellier, (near type) F7 AY651186 = AY651224 Physa Physella acuta France Saint-Martin Rieutort Wadi, 25 km north of de Londres Montpellier, (near type) iobrgl AY651187 = AY651225_— Physa Physella gyrina USA lowa Boyer Rver, North of Council Bluffs (type) mo40408 AY651188 AY651226 Physa Physella acuta USA Missouri Reynolds Hunter Hollow (off Black River) Nnfl AY651189 = AY651227 — Physa Physa fontinalis Netherlands Nooredemeek (Rykel de Bruyne’s site: 133/481) Nnf2 AY651190 = AY651228 — Physa Physa fontinalis Netherlands Nooredemeek (Rykel de Bruyne’s site: 133/481) 0k42234 AY651191 = AY651229 — Physa Physella gyrina USA Colorado Mesa creek north Sunset Lake paP10 AY651192 = AY651230 — Physa Physella heterostropha USA Pennsylvania Philadelphia Philadelphia; Schuylkill River at Fairmont Park paP9 AY651193 AY651231 Physa Physella heterostropha USA Pennsylvania Philadelphia Philadelphia, Schuykill River at Fairmont Park scysrl AY651194. — AY651232 Physa Physella hendersoni USA South Carolina Hampton South Carolina, Yamassee; Salkehatchie River off 17A, near 21 scysr2 AY651195 — AY651233—- Physa Physella hendersont USA South Carolina |= Hampton South Carolina, Yamassee; Salkehatchie River off 17A, near 21 scysr3 AY651196 = AY651234 — Physa Physella hendersoni USA South Carolina Hampton South Carolina, Yemassee; Salkehatchie River off 17A, near 21 ut37088 AY651197 = AY651235._—— Physa Physella gyrina USA Utah Box Elder (salt?) spring that empties into North Mud Flat of the Great Salt Lake 144 Appendix 1. (continued) Identity utznp2 vacsg | vacsg2 vahsv1 vahsv2 wybhr2 wySPE1 wySPE2 mi4mpcl scbctl] scpb2 Acc. # 16S AY651198 AY651199 AY651200 AY651201 AY651202 AY651203 AY651204 AY651205 AY651206 AY651207 AY651208 Acc. # Col AY651236 AY651237 AY651238 AY651239 AY651240 AY651241 AY651242 AY651243 AY651244 AY651245 AY651246 AMERICAN MALACOLOGICAL BULLETIN — 19° 1/2 + 2004 Genus Physa Physa Physa Physa Physa Physa Physa Physa Psuedosuccinea Biomphalaria Planorbella Subgenus Petrophysa Physella Physella Physella Physella Physella Physella Physella Species ZIONS gyrina gyrina aurea aurea acuta spelunca spelunca columella obstructa trivolvis Country State/ County/ Territory Region Utah Zion National Park Virginia Virginia Virginia Virginia Wyoming Wyoming Wyoming Michigan South Charleston Carolina South Charleston Carolina Locality Data Along rock face where seeping, The Narrows Coyner Springs Coyner Springs Hot springs in Bath, Virginia Hot springs in Bath, Virginia Big Horn River near Lower Kane Cave Lower Kane Cave near Kane, Wyoming Lower Kane Cave near Kane, Wyoming Four Mile Lake, Michigan Charles Towne Landing (see Dillon and Dutra-Clarke 1992) Malacological Review 25: 129-130 off Bees Ferry Road AMERICAN MALACOLOGICAL BULLETIN 19+ 1/2 * 2004 RESEARCH NOTE Method for mounting radulae for SEM using an adhesive tape desiccation chamber Fabio Moretzsohn* Department of Zoology, University of Hawaii, 2538 The Mall, Honolulu, Hawaii, 96822, U.S.A. Abstract: A method to mount radulae for low-magnification SEM study is described. The wet radula was arranged over the adhesive side of a piece of adhesive tape. The addition of a few drops of liquid delayed desiccation, permitting the radula to be positioned. A piece of clear plastic film was added to form a desiccation chamber. Pinpricks around the radula allowed it to desiccate slowly. The desiccation chamber was then cut near the radula, and the radula and its adhesive base were mounted on an SEM stub. The method is simple and prepares radulae with little deformation. Key words: radula, SEM, mounting, method, desiccation The radula is often studied using a scanning electron microscope (SEM). Several methods are available to prepare and mount radulae. Among them, Solem (1972) and Brad- ner and Kay (1995) suggest mounting the radula directly on the SEM stub using a thin layer of adhesive (double-sided tape or contact cement). There are problems with this ap- proach, however; it is difficult to reposition the radula once it touches the adhesive and the radular ribbon may curl and distort during desiccation, changing position or breaking. During the study of the Cribrarula cribraria (Linnaeus, 1758) species complex (Cypraeidae) (Moretzsohn 2003), I devel- oped a simpler alternative that allowed ample time to ma- nipulate the radula, minimized deformation due to desicca- tion, and prepared radulae suitable for SEM study at low magnification. THE METHOD I prepared a strip of adhesive tape with both ends looped backwards (adhesive side up) and attached it to a glass slide or piece of cardboard. I placed the radula flat with teeth up and radular ribbon down on the adhesive side of the adhesive tape (Fig. 1) in a drop or two of 70% ethanol. The radular ribbon could then be positioned. More liquid (25- 70% ethanol) was added with a Pasteur pipette to delay desiccation while the radula was being arranged on the ad- hesive tape. The method worked best when the radula was wet but not excessively wet (in which case the radula would * Current address: Texas A&M University, Center for Costal Stud- ies, 6300 Ocean Dr., Corpus Christi, Texas 78412, U.S.A., fmoretzsohn@hotmail.com curl; Fig. 3). When the alcohol began to evaporate, the ad- hesive tape became sticky and the radula could be attached to the tape. It could be re-arranged if more alcohol was added. I then covered the radula with non-adhesive clear plastic film (e.g. polyethylene or acetate) and pressed it against the adhesive tape all around the radula, thus forming a desicca- tion chamber with a wet radula inside. I used an insect pin to prick a few small holes around the radula, about 5-10 mm away from it (Fig. 2). The alcohol in the desiccation chamber evaporated slowly, depending on the temperature, humidity outside the chamber, and size of the holes. The clear film allowed me to monitor the radula as it desiccated. The radula was dry enough to be mounted on an aluminum SEM stub after 48 hours at room temperature. If the radula still looked wet or if there was condensation in the plastic cham- ber, I allowed it to dry longer. Extra pinpricks were necessary if desiccation was slow. I prepared a clean aluminum SEM stub (Solem 1972), coating it with nail polish, double-sided adhesive tape, or other adhesive to bond the desiccation chamber to the stub. I cut the adhesive tape chamber close to the radula. Using a pair of fine tweezers, | removed the non-adhesive cover film and placed the adhesive base with the radula on the SEM stub (Figs. 4-5). I pressed the sides of the adhesive base close to the radula against the stub to ensure a good bond. It was important that the radula and its tape base were oriented so that the radula was uppermost. Use of colored adhesive tape helped prevent misalignment. I placed the SEM stub in an airtight container with fresh desiccant (silica gel or similar) as soon as the radulae were mounted to prevent the settling of dust particles (Hickman, pers. comm.) and to ensure complete desiccation before sputter-coating the specimen 146 AMERICAN MALACOLOGICAL BULLETIN 4 radula (teeth up) 2 pin pricks 19 + 1/2 + 2004 Figures 1-6. 1, Diagram of a wet radula arranged on the adhesive surface of adhesive tape. The radula can be positioned with insect pins or tweezers prior to covering it with a clear plastic film (Fig. 2). 2, Dia- gram of a desiccation chamber adhesive tape (sticky side up) glass slide with gold-palladium (or equivalent) for observation with an SEM. ACKNOWLEDGEMENTS I thank Alison Kay, Carole Hickman, Janice Voltzow and two reviewers for suggestions on the manuscript. Some materials were donated by Kay and Hugh Bradner or were loaned from several museums. I also thank the Charles Ed- mondson Research Fund (University of Hawaii) and the Hawaiian Malacological Society for financial support; Tina Weatherby for help with SEM at the University of Hawaii Biological Electron Microscope Facility (BEMF); a grant to BEMF (#RR/AIO3061) partially subsidized SEM costs; and James Parrish and Lucy Kida made possible the use of a made with adhesive tape and clear non-adhesive polyethylene film for slow desiccation of radulae for SEM. Ethanol evaporates slowly through pinpricks made around the radula. 3, Photograph of a radula curling with excessive ethanol prior to being positioned in the desiccation cham- ber. Note the beads of adhesive on the surface of adhesive tape; scale = 10 mm. 4, Photograph of small radulae of cowries in the Cribrarula cribraria (Linnaeus, 1758) complex mounted on SEM stubs and coated with gold-palladium for SEM obser- vation; scale = 10 mm. 5, Scanning electron micrograph showing parts of two radulae of Cribrarula gaskoi- ni (Reeve, 1846) prepared as de- scribed in text. Note the plastic base cut close to the radulae and mounted on SEM stubs; scale = 1500 mm. 6, Scanning electron mi- crograph of the radula of Cribrarula gaskointi desiccated as described in the text, showing no significant dis- tortion of the radula and individual teeth. digital camera and dissecting microscope used to produce the figures shown here. LITERATURE CITED Bradner, H. and E. A. Kay. 1995. Techniques in preparing and photographing Cypraea radulae. The Festivus 27: 96-103. Moretzsohn, F. 2003. Exploring novel taxonomic character sets in the Mollusca: The Cribrarula cribraria complex (Gastropoda: Cypraeidae) as a case study. Ph.D. Dissertation, Department of Zoology, University of Hawaii. Solem, A. 1972. Malacological applications of scanning electron microscopy II. Radular structure and functioning. The Veliger 14: 327-336 + 6 pl. Accepted: 17 November 2003 THANKS TO OUR REVIEWERS We gratefully acknowledge the assistance of the following individuals who reviewed manuscripts in 2002-2003. Shirley Baker M. C. Barnhart Arny Blanchard Arthur E. Bogan Jayne Brim Box Gilianne D. Brodie Ken Brown Melbourne R. Carriker Rachel Collin W. Gregory Cope Kevin Cummings Marta deMaintenon Robert T. Dillon, Jr. Gorges Dussart Ken Emberton Ned Fetcher Susan Ford Bernard Fried Matthias Glaubrecht Daniel L. Graf Robert Guralnick Carole Hickman W. R. Hoeh Michael A. Hoggarth Daniel J. Hornbach Cristian Ituarte Erika V. Iyengar Eileen Jokinen Helen Elise Kitchel Annette Klussmann-Kolb David R. Lindberg Gerald L. Mackie Henry Madsen Maria Cristina Dreher Mansur André L. Martel Thomas M. McCarthy Robert F. McMahon Rachel Ann Merz Paula Mikkelsen Edna Naranjo Garcia Richard Neves Susan J. Nichols Dianna Padilla Timothy Pearce Melita Peharda David Richards c. A. Richardson Keven J. Roe Kent Rylander Sonia Barbosa dos Santos Allen E. Strand Richard Strathmann David Strayer Timothy W. Stewart James L. Theler Fred G. Thompson Andrew M. Turner Janet Voight Takashi Wada J. Evan Ward G. Thomas Watters Heike Wagele Amy R. Wethington Beaty, B. B. 19: 15 Britton, D. K. 19: 93 Brown, K. M. 19: 57 Cazier Shinn, D. 19: 33 Dillon, R. T. Jr. 19: 31, 63, 69, 79 Earnhardt, C. E. 19: 63 Frankis, R. C. Jr. 19: 69 INDEX TO VOLUME 19 AUTHOR INDEX Glaubrecht, M. 19: 111 Guralnick, R. 19: 25, 135 Johnson, P. D. 19: 57 Lee, T. 19: 1 McCarthy, T. M. 19: 47 McMahon, R. F. 19: 93, 101 Moretzsohn, F. 19: 145 Mower, C. M. 19: 39 Neves, R. J. 19: 15 Richards, D. C. 19: 33 Smith, T. P. 19: 63 Stewart, T. W. 19: 79 Turner, A. M. 19: 39 Wethington, A. R. 19: 135 PRIMARY MOLLUSCAN TAXA INDEX acuta, Physa 19: 40, 63, 93, 114, 135 acuta, Physella 19: 86, 106 acuta, Pleurocera 19: 31 adamsi, Pisidium 19: 5 Afropisidium 19: 2 altilis, Gillia 19: 83 Amblema 19: 69 Ambloplites 19: 16 Amnicola 19: 83 ampla, Leptoxis 19: 75 anatina, Physa 19: 135 anceps, Heliosoma 19: 87 ancillaria, Physella 19: 86 Anculosa 19: 85 Ancylus 19: 88, 102 Anodonta 19: 69 antipodarum, Potamopyrgus 19: 33 antrosa, Heliosoma 19: 87 Aplexa 19: 87 arachnoidea, Elimia 19: 84 Arianta 19: 74 armigera, Planorbella 19: 88 Astarte 19: 4 aterina, Elimia 19: 84 Athearnia 19: 59 aurea, Physa 19: 64, 136 aurea, Physella 19: 86 auricularia, Radix 19: 86 Balea 19: 64 Bembicium 19: 102 bicarinatus, Planorbis 19: 87 Biomphalaria 19: 64, 137 [first occurrence in each paper recorded] Bithynella 19: 83 Bithynia 19: 43, 81 bottimeri, Fontigens 19: 84 boykiniana, Elimia 19: 126 brevis, Io 19: 85 brogniartianus, Micromenetus 19: 87 Brotia 19: 113 Bulimus 19: 81 Byssanodonta 19: | californica, Ferrissia 19: 88 Campeloma 19: 43, 81 canaliculata, Pleurocera 19: 86 Candidula 19: 69, 73 carinata, Leptoxis 19: 86 casertanum, Pisidium 19: 5 catenaria, Elimia 19: 84, 126 catenaria, Goniobasis 19: 70 Cepaea 19: 74 chinensis, Cipangopaludina 19: 81 cincinnatiensis, Pomatiopsis 19: 84 Cipangopaludina 19: 81 clavaeformis, Elimia 19: 84 clinchensis, Io 19: 85 columella, Pseudosuccinea 19: 86, 137 compressum, Pisidium 19: 5 conventus, Neopisidium 19: 5 Corbicula 19: 4, 21, 69, 114 corneum, Sphaerium 19: 5 Costatella 19: 63 crassula, Campeloma 19: 81 Crepidula 19: 69 Cribaria 19: 145 148 cribaria, Cribaria 19: 145 crocata, Physella 19: 86 cubensis, Eupera 19: 2 cuperonites, Physa 19: 136 curta, Pleurocera 19: 31 curvicostata, Elimia 19: 126 Cyclocalyx 19: 2 dalli, Fossaria 19: 86 decisum, Campeloma 19: 43, 81 deflectus, Gyraulus 19: 87 depressus, Ancylus 19: 88 dickinsoni, Elimia 19: 125 dilatatus, Micromenetus 19: 87 Discus 19: 69 dislocata, Goniobasis catenaria 19: 70 downiei, Leptoxis 19: 58 Dreissena 19: 69 dubium, Pisidium 19: 5 Elimia 19: 43, 59, 70, 84, 124 elliptica, Physella 19: 86 elodes, Stagnicola 19: 40, 93 elongata, Aplexa 19: 87 Euhadra 19: 73 Eupera 19: 1 exacuous, Planorbella 19: 88 excentricus, Hebetancylus 19: 102 fabale, Sphaerium 19: 5 Ferrissia 19: 88, 107 floridensis, Elimia 19: 125 flumea, Corbicula 19: 21 fluvialis, Io 19: 60, 85 fluviatilis, Ancylus 19: 102 Fontigens 19: 83 fontinalis, Physa 19: 137 Fossaria 19: 86 fragilis, Ferrissia 19: 88 Fusconata 19: 113 fuscus, Laevapex 19: 88 Fusus 19: 85 galbana, Fossaria 19: 86 Gammatricula 19: 70 gaskointi, Cribaria 19: 145 georgianus, Viviparus 19: 81 Gillia 19: 83 glabrata, Biomphalaria 19: 64 Goniobasis 19: 69, 84, 124 gradata, Pleurocera 19: 86 grandis, Lavigera 19: 123 granum, Lyogyrus 19: 83 Gundlachia 19: 88 Gyraulus 19: 87 gyrina, Physa 19: 41, 47, 63, 93, 135 gyrina, Physella 19: 86 Gyrotoma 19: 59 haldemani, Ancylus 19: 88 Hebetancylus 19: 102 Heliosoma 19: 43, 59, 87 Helix 19: 74 hendersoni, Physella 19: 86 heterostropha, Physa 19: 40, 63, 93, 114, 135 heterostropha, Physella 19: 86, 106 hirsutus, Gyraulus 19: 87 holsingeri, Fontigens 19: 83 Holsingeria 19: 83 humilis, Fossaria 19: 86 hupensis, Oncomelania 19: 70 Hydrobia 19: 69, 127 hypnorum, Aplexa 19: 87 inflata, Physella 19: 86 insubrica, Marstoniopsis 19: 114 integra, Campeloma 19: 81 integra, Physa 19: 40, 63, 94, 114, 135 integra, Physella 19: 106 Io 19: 59, 85 iris, Villosa 19: 15 jennessi, Physa 19: 137 johnsoni, Physa 19: 136 Juga 19: 59 lacustre, Musculium 19: 5 Laevapex 19: 88, 102 Lampsilis 19: 15 lapidaria, Pomatiopsis 19: 84 Lasmigona 19: 69 Lavigera 19: 122 Lepaea 19: 69 Leptoxis 19: 58, 75, 85, 125 limosa, Neocorbicula 19: 4 limosus, Amnicola 19: 83 limum, Campeloma 19: 81 Lioplax 19: 81 Lithasia 19: 59, 126 littorea, Littorina 19: 107 Littoridinops 19: 83 Littorina 19: 107, 113 livescens, Elimia 19: 43 luteola, Lampsilis radiata 19: 15 Lymnaea 19: 86, 93 Lyogyrus 19: 83 lyttonenesis, Io 19: 85 magnifica, Tulotoma 19: 58 malleatus, Viviparus 19: 81 Mandarina 19: 70 Margaritifera 19: 15 margaritifera, Margaritifera 19: 15 Marstoniopsis 19: 114 meekiana, Gundlachia 19: 88 Melania 19: 85 Melanopsis 19: 113 Menetus 19: 87 Mercenaria 19: 69 Micromenetus 19: 87 microstoma, Physella 19: 86 morrisoni, Fontigens 19: 84 Mudalia 19: 86 Musculium 19: 1 Mytilus 19: 69 nassa, Lavigera 19: 123 nemoralis, Cepaea 19: 74 Neocorbicula 19: 4 neopalustris, Stagnicola 19: 86 Neopisidium 19: 2 nickliniana, Fontigens 19: 84 Nitrocis 19: 86 Notoacmaea 19: 69 Obovaria 19: 113 obrussa, Fossaria 19: 86 obstructa, Biomphalaria 19: 137 occidentale, Sphaerium 19: 5 Odhneripisidium 19: 2 Oncomelania 19: 70 orolibas, Fontigens 19: 84 149 Ostrea 19: 69 Paludestrina 19: 84 palustris, Stagnicola 19: 40 paralella, Ferrissia 19: 88 Partulina 19: 73 partumeium, Musculium 19: 5 parva, Fossaria 19: 86 paulensis, Io 19: 85 perversa, Balea 19: 64 Petrophysa 19: 137 Physa 19: 40, 47, 59, 86, 93, 1 Physella 19: 93, 102, 106 picta, Leptoxis 19: 75 Pisidium 19: | Planorbella 19: 88, 137 Planorbis 19: 87 Pleurocera 19: 31, 59 plicata, Leptoxis 19: 61 Pomatiopsis 19: 84 pomilia, Physa 19: 136 pomilia, Physella 19: 86 Potamilus 19: 113 Potamopyrgus 19: 33 powellensis, Io 19: 85 praerosa, Leptoxis 19: 85 proxima, Elimia 19: 84, 126 proxima, Goniobasis 19: 70 Pseudosuccinea 19: 86, 137 pumilus, Ancylus 19: 88 Quincuncina 19: 114 Radix 19: 86 14, 135 reintana, Samiculscospira 19: 107 revularis, Ferrissia 19: 107 rhomboideum, Sphaerium 19: rivularis, Ferrissia 19: 88 rufa, Campeloma 19: 81 rupestris, Ambloplites 19: 16 scholtzi, Marstoniopsis 19: 114 securis, Musculium 19: 5 5 semicarinata, Elimia 19: 59, 84 semicarinata, Goniobasis 19: 70 Semisulcospira 19: 107 serpenticola, Taylorconcha 19: shimeki, Ferrissia 19: 88 simile, Sphaerium 19: 5 simplex, Elimia 19: 84 Somatogyrus 19: 83 spelunca, Physa 19: 136 Sphaerium 19: 1 spinella, Gontobasis 19: 84 33 spinosa, Io 19: 85 Spirodon 19: 86 stagnalis, Lymnaea 19: 93 Stagnicola 19: 40, 86 Strephobasis 19: 31 striatinum, Sphaerium 19: 5 subcarinata, Lioplax 19: 81 subglobosa, Leptoxis 19: 85 sulcata, Astarte 19: 4 symmetrica, Elimia 19: 84 taeniata, Leptoxis 19: 75 Tanganyicia 19: 124 tardus, Ancylus 19: 88 Taylorconcha 19: 33 tentaculata, Bithynia 19: 43, 81 tenuipes, Littoridinops 19: 83 Tiphobia 19: 124 transversum, Musculium 19: 5 tricarinata, Valvata 19: 80 Tricula 19: 70 trivolvis, Helisoma 19: 43 trivolvis, Planorbella 19: 88, 137 Tryonia 19: 70 Tulotoma 19: 58 uncialis, Pleurocera 19: 86 unthankensis, Holsingeria 19: 83 150 Valvata 19: 80 variabile, Pisidium 19: 5 variegata, Tryonia 19: 70 Villosa 19: 15 virgata, Physa 19: 93 virgata, Physella 19: 102 virginica, Elimia 19: 85 virginicus, Somatogyrus 19: 83 vittatum, Bembicium 19: 102 Viviparus 19: 81 wolfiana, Physa 19: 136 wrighti, Physa 19: 136 zionis, Physa 19: 137 THE AMERICAN MALACOLOGICAL SOCIETY http://erato.acnatsci.org/ams Dr. Susan B. 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Report on the Mollusca, Pt. 2: Gastropoda and Scaphopoda. Bulle- tin of the Museum of Comparative Zoélogy 18: 1-492, pls. 10-40. Orbigny, A, d’. 1835-46. Voyage dans l’Ameérique Meridionale (le Brésil, la République Orientale de PUruguay, la République Argentine, la Patagonie, la Re- publique du Chili, la Republique de Bolivia, la République du Pérou), exécuté pendant les années 1826, 1827, 1828, 1829, 1830, 1831, 1832 et 1833, Vol. 5, Part 3 (Mol- lusques). Bertrand, Paris. Dates of publication: pp. 1-48, [1835], pp. 49-184 [1836], pp. 185-376 [1837], pp. 377- 408 [1840], pp. 409-488 [1841], pp. 489-758 + pls. 1-85 [1846]. Hurd, J. C. 1974. Systematics and Zoogeography of the Union- acean Mollusks of the Coosa River Drainage of Alabama, Georgia and Tennessee. Ph.D. Dissertation, Auburn Uni- versity, Alabama. U.S. Environmental Protection Agency. 1990. Forest riparian habitat survey. Available at: http://www.epa.gov/waterat- las/geo/iil6_usmap.html 25 January 2003. 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Institutional subscriptions are available at a cost of $48.00 per volume ($65.00 beginning with Volume 20). Membership in the American Malacological Society, which includes personal subscriptions to the Bulletin, is available for $60.00 ($20.00 for students, $60.00 for affiliated clubs). Outside the U.S. postal zones, add $5.00 surface and $10.00 airmail per volume. All prices quoted are in U.S. funds. For membership information and institutional subscriptions contact Dr. Susan Cook, Treasurer, American Malacological Society, 4201 Wilson Blvd., STE 110-455, Arlington, VA 22230. For other information, including avail- ability of back issues, contact Dr. Janice Voltzow, Department of Biology, University of Scranton, Scranton, PA 18510- 4625, USA. Complete information also available at the AMS website: http://erato.acnatsci.org/ams. WL 6 ll Reproductive isolation between Physa acuta and Physa gyrina in joint culture. ROBERT T. DILLON, Jr., CHARLES E. EARNHARDT, and THOMAS P. SMITH ............ 63 High levels of mitochondrial DNA sequence divergence in isolated populations of freshwater snails of the genus Goniobasis Lea, 1862. ROBERT T. DILLON, Jr. and ROBERT C. FRANKIS, Jr. 2.0.0... ec cee eee eee 69 Species composition and geographic distribution of Virginia’s freshwater gastropod fauna: A review using historical records. TIMOTHY W. STEWART and ROBERT T. DILLON, Jr. ..... 79 Environmentally and genetically induced shell-shape variation in the freshwater pond snail Physa (Physella) virgata (Gould, 1855). DAVID K. BRITTON and ROBERT F. MCMAHON ............0.. 000 cece eee tenes 93: A 15-year study of interannual shell-shape variation in a population of freshwater limpets (Pulmonata: Basommatophcra: Ancylidae). ROBERT F. MCMAHON .................0005- 101 Leopold von Buch’s legacy: Treating species as dynamic natural entities, or why Seourapiy, matters. MATTHIAS GLAUBRECHT ........... 0... cc cece cece ene ee eee een 111 Are populations of physids from different hot springs distinctive lineages? AMY R. WETHINGTON and ROBERT GURALNICK ....... 2.0.0.0... cece ees 135 Rcseqge Che hc eee eee eee eee ne en eee teens tnee eben 145 Re 147