Phylogenetic Relationships Within the North American Hydrobiid Snail Tgonia: Taxonomic and Biogeographic Implications

Robert Hershleri , Hsiu-Ping Liu2, and Margaret Mulvey3 'Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, D.C. 20560; 2Department of Biology, Olin Hall, Colgate University Hamilton, NY 13346; 3Savannah River Ecology Laboratory, P.O. Drawer E, Aiken, South Carolina 29802.

Introduction The aquatic biota of western North America is of interest from the standpoint of evolutionary biology and biogeography because elements often are profoundly isolated by inhospitable deserts and mountain ranges, live in extremely restricted and/or harsh environments, and present distributional and phylogenetic patterns which have been molded by the extremely complex and dynamic(Cenozoi physical history of the region. Biotic distributions and evolutionary relationships may also provide clues as to early regional drainage relationships for which geological evidence often/has been obscured by subsequent deposition, deformation and erosion. Whereas early regional biogeographic treatments of fishes (e.g., Hubbs and Miller 1948; Hubbs 1974) focused on dispersal opportunities afforded by a highly integrated late Pleistocene "pluvial" drainage, Minckley et al. (1986) accepted great antiquity (Oligocene- ) of this fauna and consequently emphasized the complex role of geological events in effecting vicariance, and the likelihood that fishes have been introduced to and/or transferred within the region by rafting on allochthonous terranes, intra-continental microplates, and other tectonically displaced or extended crustal fragments. The limited phylogenetic data then available for western American fishes demonstrated partial congruence with hypotheses for historical area relationships advanced by these authors, although they emphasized the need for additional Sich studies. While this work seemingly set the stage for a new era in biogeographic study of western American fishes and other aquatic biota, few pertinent phylogenetic hypotheses have since been generated, and these have focused almost exclusively on one group, fishes (e.g., Echelle and Dowling 1992; Smith 1992; but also see Hendrickson 1986). Freshwater mollusks have figured prominently in the development of provocative, albeit non-phylogenetic hypotheses for western American aquatic biogeography (e.g., Taylor 1985). Few elements of this biota have as much potential for evolutionary and biogeographic inquiry as the family , which is the most diverse group of freshwater snails in North America2' with about 35 genera and 285 described (Turgeon et al. In Press). Hydrobiids are ubiquitous and locally abundant in many freshwater ecosystems of the West. These small benthic snails are obligately aquatic and disperse but slowly, features which link them tightly with drainage history (Taylor and Bright 1987) and, together with their antiquity (the regional age of the family is minimally Paleogene; Taylor 1985), make them ideal tools Col evaluating biotic response to vicariance. \Howeveta paucity of rigorously proposed phylogenetic hypotheses has i:trevented use of hydrobiids in such studies. One of the most speciose groups of western hydrobiids is the genus Tryonia, which is composed of at least 22 Recent species (Hershler and Thompson 1992). Most of these snails are locally endemic in major drainages of the Southwest and sympatry of congeners is rare, suggesting that the phylogeny of Tryonia may be informative with respect to regional historical biogeography. The Great Basin contains eight unique congeners, while smaller numbers of species are endemic to the Pecos River-Rio Grande (six), Gila River (one), and Colorado River (one) drainages. The uniquely parthenogenetic T. protea (unpublished) lives in both the Great Basin and Colorado River drainage, while additional species live along the Pacific (T. imitator) and Gulf of California (T. quitobaquitae) coasts. Tryonia also is represented by two broadly disjunct species in peninsular Florida, and a poorly known fauna in northern Mexico, whose sole described representative, T. hertleini, went extinct several decades ago following drying of the springs at its type locality. The possibility that congeners early ranged into Central America and northern South America is suggested by regional Neogene fossils (Taylor 1966; also see Wesselingh 1996; Wesselingh et al. 1996), although allocations of these to Tryonia are controversial (Nuttall 1990). Tryonia typically lives in inland, thermal springs, and some species are salt tolerant: T. sauna, for instance, lives in Cottonball Marsh in , in which salinities range up to several times that of seawater (LaBounty and Deacon 1972). Floridian species live in lakes as well as springs (Thompson 1968) while T. imitator lives in coastal strand habitats and tolerates a

2 broad range of salinity regimes (Kellogg 1985). Tryonia also is of interest because it closely parallels patterns of distribution and endemism documented for one of the better studied groups of western fishes, the genus Cyprinodon (; Miller 1981). As with the overwhelming majority of the Hydrobiidae, scope and content of Tryonia have not been established within a phylogenetic context. In a recent review of the hydrobiid subfamily Cochliopinae (Hershler and Thompson 1992), Tryonia was placed in the informal " group" along with 12 other New World genera in which males have glandular papillae on the penis; Mexipyrgus, locally endemic in northern Mexico, was conjectured as its closest relative. Tryonia has been loosely defined on the basis of a few distinctive (but not demonstrably synapomorphic) shell and genitalic features (Taylor 1966; Hershler and Thompson 1992), and even these details have not been published for most of the species currently allocated to the genus. Specific hypotheses of congener relationships have been proposed on the basis of patterns of penial ornament (Taylor 1985, 1987; Hershler 1989). The cosmopolitan distribution of the Cochliopine suggests that this group arose during the late Mesozoic and main sub-groups diverged prior to late to early break-up of Laurasia (Hershler and Thompson 1992). The fossil record of Tryonia, although not overly useful given the weak phylogenetic signal provided by hydrobiid shells (Taylor 1987), nevertheless suggests a minimal Miocene age for the genus, based on fossils from the Mint Canyon Formation (southwest California) identified as T. imitator (Kew 1924; Oakenshott 1958). The possibility of an even more ancient origin is suggested by high-spired shells resembling Tryonia in early post-Laramide lake beds of the West (e.g., Paleocene-Eocene Flagstaff Formation; La Rocque 1960), although affinities of these are uncertain (Taylor 1975) and in need of more study. Taylor (1987) conjectured that extant species of Tryonia are generally of Miocene age. In order to provide a robust framework for biogeographical analysis of Tryonia, we are examining the monophyly and evolutionary structure of the genus using mitochondrial DNA seqUences. In a preliminary study, sequences from the cytochrome c oxidase subunit I (COI) gene were used to generate a robust phylogeny for species of the Death Valley system, (Hershler et al. submitted). Herein we analyze DNA sequence variation for this gene for the remaining congeners. We evaluate monophyly of Tryonia, propose a hypothesis of phylogenetic

3 relationships within the genus, and evaluate congruence of Tryonia biogeography with historical area relationships implied by geological history of the West.

Materials and Methods Specimens Sequence data for species from the Death Valley region are from Hershler et al. (submitted). We also analyzed all other species currently allocated to Tryonia, as well as three undescribed species (two from the Great Basin and on:-.) fiom the coastal plain of Alabama) conforming to the genus in general features. Effort to collect suitable material proved unsuccessful only in the case of T. brunei, which we were unable to find at its single known locality in wesei'exasA (Taylor 1987). In order to test monophyly of Tryonia, we also analyzed other members of the "Littoridina group" (Aphaostracon, , Mexipyrgus, Onobops, Pyrgophorus) as well as representatives of the presumably more distantly related "Heleobia group" (Heleobia, Heleobops). Australasian taxa (Ascorhis, Phrantela) considered among the more plesiomorphic hydrobiids (Ponder and Clark 1988; Ponder et al. 1993) were used as outgroups (trees were rooted with Phrantela). Multiple samples were analyzed for several species of Tryonia (including the more widespread congeners) to evaluate intra-specific variation. Taxa and localities are listed in Table 1 and sampling localities for Tryonia are in Figure 1. All analyzed specimens were live-collected in the field, and either placed directly into concentrated ethanol or flash-frozen in a portable liquid nitrogen cannister. Voucher material from these samples is reposited in the Recent mollusk collection of the National Museum of Natural History (USNM). Laboratory Methods Genomic DNA was extracted from entire frozen snails using the Chelex method of Walsh et al. (1991), while the CTAB extraction method (Bucklin 1992) was used for ethanol-preserved specimens. A 710 base pair segment of mitochondrial gene was amplified via polymerase chain reaCtion (PCR) using primers COIL 1490 and COIH 2198 (Folmer et al. 1994). Protocols for amplification, sequencing and alignment are of Hershler et al. (Submitted). Sequence Analysis

4 We plotted number of transitions (TS) and transversions (TV) versus genetic distance (uncorrected for multiple hits) for all pairwise comparisons in order to determine whether in- group comparisons were demonstrating TS saturation. Previous phylogenetic studies (***) have shown that TS increases linearly, then levels off and becomes saturated as genetic distances increase. Step matrices for the phylogenetic analysis were selected on the basis of the observed pattern of base substitution (***). Phylogenetic Analysis Cladistic analyses were performed using PAUP* (Swofford 1998). As a relatively large number of taxa was analyzed, heuristic searches for shortest trees were performed. Uninformative characters were ignored and zero length branches were collapsed. Bootstrapping (Felsenstein 1985) with 500 iterations was used to estimate the reliability of branches on the shortest trees. Maximum likelihood scores using several models (***) were calculated for equally parsimonious trees in order to help guide selection of a preferred topology. Results DNA sequence variation Alignments were unambiguous as COI is a protein coding gene. Direct sequencing of PCR products yielded an aligned matrix of 603 base pairs (bp). This sequence corresponds to positions 1566-2168 in the homologous Drosophila yakuba mtDNA sequence (Clary and Wolstenholme 1985). The complete sequences have been deposited in GenBank under accession numbers *****. Of the 601 bp sequence, 247 sites were variable and 212 of these were phylogenetically informative. Average base frequencies for the total data set were 25% A, 40% T, 17% C, and 18% G and base composition bias was not evident among taxa. A plot of the absolute number of TS and TV versus genetic distance (Figure 2) conformed to prior evolutionary fmdings for mitochondrial genes (***). The absolute number of both TS and TV increased linearly as genetic distance increased. TS outnumbered TV among closely related taxa and as genetic distances increased, TS leveled off while TV continued to increase, suggesting that saturation occurred in comparisons of outgroups (Ascorhis, Phrantela) versus cochliopine snails. We also plotted genetic distance versus the absolute number of TS and TV for the first, second, and third codon positions (genetic distance value corresponds to distances calculated for each codon position). For the first codon position (Figure 3), there was

5 clear separation between TS and TV frequencies, and TS and TV increased linearly with genetic distance. For the second codon position (not shown), very few substitutions occurred and there was no apparent pattern. The third codon position (Figure 4) showed a pattern similar to that of the first position. Up to about 16% sequence divergence, there is a decline in the rate of increase of TS and an increase in the rate of TV. This analysis suggests that the first and second codon positions are phylogenetically informative, especially among more distant taxa, while the third codon is more informative among more closely related taxa. For three species of Tryonia, intra-specific sequence differences (uncorrected for multiple hits) ranged from 0% - 0.2% (T. e.heatumi, n =2; T. circumstriata, n =2; T. imitator; n =2; T. protea; n = 6). For inter-specific comparisons among ingroup taxa, sequences differed by 1.3 - 14.8% (Table 2). The two samples of T. variegata (from widely spaced populations within the drainage) differed by 5%, suggesting that this species is in need of revision. Genetic distances among pairs of ingroup and outgroup taxa ranged from 16.1 to 22.5%.

Molecular phylogenetic analysis All characters were weighed equally as there was no evidence of base pair composition bias and saturation among in-groups. Eight trees of 863 steps were obtained (CI = 0.45) using maximum parsimony. Our preferred tree, that having the consistently lowest maximum likelihood score under the different models used (Table *), is shown in Figure 5. Australasian outgroups consistently occupied a basal position, supporting monophyly of the Cochliopinae. An undescribed species from the central Great Basin resembling Tryonia occupied the most basal in-group position. Four main clades having high bootstrap support were consistently resolved. Clade I is composed of four species currently assigned to Tryonia (two from the Pecos River-Rio Grande basin and two from the southeast United States). Clade II is composed of several other members of the "Littoridina group" (Littoridinops and an undescribed species referable to Pyrgophorus). Clade III is composed of two members of the "Heleobia" group. Clade IV is composed of the type species of Tryonia (T. clathrata), 13 other congeners, and the sister to this group, Mexipyrgus. Note that the sub-clade consisting of "true" Tryonia also had high bootstrap support. Phylogenetic structure within Tryonia will be discussed below.

6 All analyses indicated that Tryonia, as currently constituted, is polyphyletic, as these snails are distributed among distinct clades and also include a basally positioned, highly divergent species. The subgenus Pauperhyonia, which Taylor (1987) erected for five species from the Rio Grande basin (T. adamantina, T. alamosae, T. brunei, 7'. cheatumi, 7'. kosteri) which lack basal papillae on the penis, also is revealed as polyphyletic. Tree topology varied in placement of the clade composed of T. aequicostata and T. quitobaquitae, which was positioned as in Figure 5 or alternatively as sister to the clade composed of 7'. cetztumi, T. circumstriata, T. sauna, 7'. n. sp. 1, T. margae, and 7'. imitator. In addition, trees varied in relative positions of species within the sub-clades composed of T. ericae, 7'. elata, and T. variegata; and , L. monroensis, and Pyrgophorus sp.

Discussion Systematics Placement of cochliopine snails having penial glandular papillae ("Littoridina group") into distinct clades and the associated polyphyly of Tryonia (as well as its subgenus Paupertlyotlia) obviously are at odds with existing , but are more reflective of incomplete study of these taxa than indicative of a conflict between morphological and molecular data. Penial morphology alone, or in conjunction with shell characters, does not appear to be a good indicator of phylogenetic relationships. As mentioned above, most of the species allocated to Tryonia have not been thoroughly studied, particularly with respect to the female genitalia, which have been shown to be highly informative of relationships in hydrobiid and related snails (Ponder 1988b). Ongoing studies by one of us (RH, unpublished) have revealed a fundamental difference in female groundplan supporting recognition of the two major clades in congruence with the molecular-based cladogram: "true" Tryonia and Mexipyrgus (forming clade IV) share a short sperm tube (into which sperm is deposited by a copulating male) whereas all other members of the "Littoridina group" have an elongate sperm tube that is distally fused with the capsule gland with the exception of "Tryonia" n. sp. 2 and "T." robusta, which share the alternative condition. However, these two latter species have distinct types of penial glands that superficially resemble, but are not homologous with the glandular papillae shared by other snails

7 assigned to the "Littoridina group," suggesting a morphological basis for their highly divergent placements. The majority of hydrobiid genera currently in use similarly were not erected on the basis of well-studied synapomorphic characters, but instead represent "carry overs" from traditional taxonomy, and we predict that many will be identified as non-monophyletic upon further study. Molecular analysis can have a predictive function in these cases as resulting trees highlight taxa or groups that would benefit from additional morphological study.

Biogeography Phylogenetic structure within "true" Tryonia (Figure 6) suggests a complex biogeographic history. Note that of the three western drainages in which these snails are concentrated, only the Rio Grande-Pecos River basin contains a monophyletic fauna. Each of the two large sub-clades includes derived sets of species living in the Amargosa River drainage, which suggests that this area is composite, as also indicated by the basal position of yet another congener from this drainage (T. rowlandsi). Whereas two species living in widely separated components of the lower Colorado River basin (White and Gila River drainages) form a clade, placement of T. protea provides ambiguity with respect to this drainage. However, this snail is uniquely parthenogenetic in the genus and its broad distribution may in part reflect increased dispersal ability associated with this reproductive mode in hydrobiid snails (e.g., Ponder 1988a). Our molecular data indicate minimal sequence divergence among broadly disjunct populations of this species, which is congruent with recent dispersal-based origins. Although replete with several enigmatic aspects, Tryonia biogeography nevertheless exhibits partial congruence with timing of presumed vicariant events. We assume a broadly ancestral distribution of Tryonia which perhaps may be tied to the post-Laramide, Eocene- Oligocene erosional surface of low relief (and presumably highly integrated drainage) that covered much of the West (Epis and Chapin 1978; Gresens 1981), as was postulated for western American fishes (Minckley et al. 1986). Based on the Floridian distribution of T. aequicostata we assume that ancestral Tryonia also ranged along the northern Gulf of Mexico coastal plain eastward to the Atlantic margin, with subsequent vicariance effected by marine transgressions (Winker 1982) or development of major deltaic regimes (Salvador 1991) along this coast during

8 periods of the Cenozoic. Many other groups are similarly broadly disjunct across this region (Rosen 1978) and our phylogenetic hypothesis suggests that this pattern is repeated in other cochliopines (Figure 5, clade I). Our cladogram is not informative of the relative timing of this divergence, owing to the uncertain position of the clade composed of T. aequicostata and T. quitobaquitae. Tryonia phylogeny includes divergence into two large sub-clades. One is composed of species from Colorado River and the upper (eastern) Amargosa River drainages that are associated with margins of the Colorado Plateau. The other is composed of snails from a western area, including the lower (western) Amargosa River drainage, Great Basin, California coast, lower Colorado River drainage; and from Pecos River drainage well to the east. We conjecture a relationship between this vicariance and mid-Tertiary uplift of the Colorado Plateau and formation of the Mogollon Rim, which effectively bisected Arizona and profoundly disrupted existing regional drainage (Nations et al. 1985). Recent studies suggest a minimally Oligocene age of uplift for at least the eastern Colorado Plateau (Peirce et al., 1979; Elston and Young 1991). Subsequent vicariance of Pecos River species (T. circumstriata and T. cheatumi) within one of the two large clades may reflect Miocene-Pliocene (Seager et al. 1984; Dickerson and Muehlberger 1994) graben development along the Rio Grande rift in northern Texas, or, alternatively, might be tied to late Miocene-Pliocene development of the Pecos River Valley resulting from solution collapse of this region (Osterkamp et al. 1987; Gustayson and Winker 1988). As noted above, Tryonia phylogeny suggests that the Amargosa River basin is composite. On a local scale this area is divisible into two non-overlapping areas of endemism (upper and lower segments of this drainage) having clearly different sister relationships, thus implying that the river basin is not only a biotic, but also a geologic composite (sensu Platnick and Nelson 1984). Species from the upper drainage segment are most closely related to snails living in areas to the east and southeast that were destined to become Colorado River drainage, which is congruent with a geology-based hypothesis that these areas were early integrated in the form of an Amargosa-Colorado "paleoriver" prior to diversion of the Amargosa River into Death Valley in (Howard 1996), a relatively youthful feature which formed only 2-6 Ma (Serpa and Pavlis 1996) by "pull-apart" opening along major strike-slip faults (Burchfiel and Stewart 1966). (The

9 northern portion of the valley opened somewhat earlier; Axen et al. 1993.) The sub-clade composed of congeners from the Death Valley trough, together with species from the Lahontan basin to the north and California coast to the west, which instead is most closely related to snails from the remote Rio Grande drainage to the southeast. We conjecture a southern derivation of this sub-clade, congruent with the tectonic history of this and surrounding regions. This parallels a proposal for origin of Death Valley pupfish, which have been shown to be most closely related to taxa from northern Mexico based on mtDNA variation (Echelle and Dowling 1992; Parker and Kornfield 1995):

If the genus was not already present on land surfaces now comprising the Death Valley region, which were then located to the east and south, a parsimonious explanation for origin of Cyprinodon is provided by formation of near coastal and inland shear zones from Miocene to Recent. A long zone of right- lateral wrench-faulting developed in Mexico, extending northward into the Great Basin and northwest as the San Andreas Transform. Schollen ranging to blocks as large as the Transverse Range were displaced north-northwestward along this splintered alignment. Movements of more than 300 km are indicated for schollen along and across the Salton Sea region. Springs rising directly or indirectly along such fractures now support most fishes of the Death Valley region. Transport of fishes on schollen or in spring zones associated with migrating schollen seems reasonable, and potentials for passive or active dispersal through this mechanism have existed for longer than 20 My (Minckley et al. 1986:605).

The two segments of Amargosa River drainage lie on different structural blocks (Inyo- Mono, Goldfield; Stewart 1988:fig. 25-3) in the Walker Lane tectonic belt which, "for the most part, acted independently of adjacent blocks" (ibid:686). These blocks are separated by the Furnace Creek Fault Zone, which extends >300 km and has accommodated ca. 80 km of pre- Quaternary right-lateral slip (Stewart 1988; Brogan et al. 1991). On a more regional level, late Cenozoic dextral shear and associated strike-slip faulting characterize not only the Walker Lane belt (Carr 1984; Stewart 1988, 1992), but also the Eastern California Shear Zone (Dolcka and Travis 1990) in the Mohave Desert immediately to the south. If these zones have acted as throughgoing strike-slip systems, tectonic transport of snails into the Death Valley area from more southern locations in California could have been effected between 6-20 Ma. Possible transport of snails into the region from even more southerly locations requires rafting on terrain west of the San Andreas Fault Zone, which has much more strike-slip displacement than that to the east (Dokka and Travis 1990), and appears unlikely as only a single, highly derived species of Tryonia (T. imitator) lives in this western area. Subsequent vicariance of species in coastal drainage to the west and Lahontan drainage to the north may be attributed to events contributing to closure of the Death Valley hydrographic system, including Pliocene severence of westward drainage across the present Sierra Nevada divide as a result of uplift of this range (Huber 1981), and structural differentiation from the Lahontan basin as a result of Quaternary vulcanism (Russell 1889). Note that a freshwater origin for Tryonia is implied by habitats of the sister group (inland springs) and basal in-group taxa whereas the highly derived position of T. imitator suggests that this snail secondarily invaded estuarine habitats, conflicting with an earlier, non-phylogenetic proposal that main groups of cochliopine snails were ancestrally estuarine (Hershler and Thompson 1992). Our interpretation of Tryonia biogeography obviously is largely speculative and severely constrained in several ways. Vicariance in the West usually can be attributed to alternative geological events of different ages: selection from among these in the absence of a well- documented fossil record is decidedly arbitrary. We also have been hampered by absence of information on the phylogenetic relationships of fauna of northern Mexico, which may prove critical in corroborating our hypothesis concerning origin of the Death Valley Tryonia. The concentration of living fauna in broadly disjunct sub-regions of the West also poses problems, but this is probably not attributable to sampling inadequacy, but instead to the fact that thermal springs are neither uniformly nor randomly distributed in the region (Waring 1965). Development of these habitats is controlled by magmatic sources of heat, and faulting that results in vertical permeability. Further evaluation of Tryonia biogeography in relation to distribution in space and time of these geological features, as opposed to drainage patterns as traditionally viewed, may prove instructive. Although there is a suggestion of commonality of snail biogeographic pattern with that of pupfish, there obviously is a need for additional studies of other groups. It is hoped that this paper may encourage more to respond to the "call" for such issued by Minckley et al. (1986) in their seminal biogeographic synthesis.

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17 FIGURE CAPTIONS

Figure 1. Map showing location of sample localities for Tryonia species. 1=Moro Cojo Lagoon (T. imitator); 2=Penasquitos Lagoon (T. imitator); 3=Whitmore Hot Springs, Long Valley (T. protea); 4=Grapevine Springs, Death Valley (T. margae, T. rowlandsi); 5=Cottonball Marsh, Death Valley (T. sauna); 6=Travertine Springs, Death Valley (T. robusta); 7=Saratoga Spring, Death Valley (T. variegata); 8=Ash Meadows (T. angulata, T. elata, T. ericae, T. variegata); 9="Oasis Spring," Salton Trough (T. protea); 10=Moapa National Wildlife Refuge (T. clathrata); 11=Potts Ranch, Monitor Valley (T. n. sp. 1); 12=southeast Steptoe Valley (T. n. sp. 2); 13=Blue Lake, Bonneville basin (T. protea); 14=Fish Springs National Wildlife Refuge, Bonneville basin (T. protea). 15=Slcull Valley, Bonneville basin (T. protea); 16=Tooele Valley, Bonneville basin (T. protea); 17=Quitobaquito Spring, Rio Sonoyta drainage (T. quitobaquitae); 18=Tom Niece Spring, Gila River drainage (T. gilae); 19=0jo Caliente, Rio Grande drainage (T. alamosae); 20=Lost River, Pecos River drainage (T. kosteri); 21=Phantom Lake, Pecos River drainage (T. cheatumi); 22=Diamond Y Spring, Pecos River drainage (T. adamantina); 23=Salt Spring, Tombigbee River drainage (T. n. sp. 3); 24=Alexander Springs, St. Johns River drainage (T. aequicostata); 25=Lake Eustis, St. Johns River drainage (7'. aequicostata); 26=Lake Panasoffkee, Withlacoochie River drainage (T. brevissima).

Figure 5. One of eight trees of shortest length based on maximum parsimony analysis of mtCOI sequence data. Numbers are bootstrap percentages for well-supported clades.

Figure 6. Portion of preferred tree depicting phylogenetic structure within clade IV. The tree is simplified for the purpose of discussion in that all species are represented by single samples, with the exception of T. variegata (T. aequicostata, sample=LE; T. cheatumi, PL1; T. circumstriata, DY; 7'. imitator, PE; T. protea, WH). Numbers represent branch lengths.

18 •r

Table 1 List of analyzed taxa, with sampling localities. If multiple samples were analyzed for a given species, locality abbreviations (in parentheses) are supplied for use in Figure 5 and 6.

Tryonia adamantina Diamond Y Spring, Pecos Co., TX Tryonia aequicostata Lake Eustis, Lake Co., FL (LE); Alexander Springs, Lake Co., FL (AS) Tryonia alamosae spring 100 m west of Ojo Caliente, Socorro Co., NM Tryonia angulata Big Spring, Ash Meadows, Nye Co., NV Tryonia brevissima Lake Panasoffkee, Sumter Co., FL Tryonia cheatumi Phantom Lake spring, Jeff Davis Co., TX (PL1); Phantom Lake spring outflow (first lateral canal)(PL2) Tryonia c ircumstr gala Diamond Y Spring, Pecos Co., TX (DY); Diamond 1( Draw, Pecos Co., TX (DD) Tryonia clathrata spring, Moapa National Wildlife Refuge, Moapa Valley, Clark Co., NV Tryonia data spring tributary to Kings Pool, Point of Rocks, Ash Meadows, Nye Co., NV Tryonia ericae spring, north of Collins Ranch, Ash Meadows, Nye Co., NV Tryonia gilae Tom Niece Spring, Graham Co., AZ Tryonia imitator Penasquitos Lagoon, San Diego Co., CA (PE); Moro Cojo Lagoon, Moss Landing, Monterey Co., CA (MO) Tryonia kosteri Lost River, Chaves Co., NM Tryonia margae Grapevine Springs (upper warm spring), Death Valley, Inyo Co., CA Tryonia protea Whitmore Hot Springs, Long Valley, Mono Co., CA (WH); "Oasis Spring," Salt Creek, Salton Trough, Riverside Co., CA (OA); spring, south end of Fish Springs National Wildlife Refuge, Juab Co., UT (FS); Horseshoe Springs, Skull Valley, Tooele Co., UT (HS); Warm Springs, Tooele Valley, Tooele Co., UT; spring, Blue Lake, Great Salt Lake Desert, Tooele Co., UT (BL) Tryonia quitobaquitc4e Quitobaquito Spring, Pima Co., AZ Tryonia robusta Travertine Springs, Death Valley, Inyo Co., CA Tryonia rowlandsi Grapevine Springs (lower warm spring), Death Valley, Inyo Co., CA Tryonia sauna spring, Cottonball Marsh, Death Valley, Inyo Co., CA Tryonia variegata Saratoga Spring, Death Valley, San Bernardino Co., CA (SA); , Ash Meadows, Nye Co., NV (DV) Tryonia n. sp. spring, Potts Ranch, Monitor Valley, Nye Co., NV Tryonia n. sp. 2 spring, southeast Steptoe Valley, north of Ely, White Pine Co., NV Tryonia n. sp. 3 Salt Spring, Fred T. Simpson Wildlife Refuge, 22 km south-southeast of Jackson, Clarke County, AL Aphaostracon sp. Alexander Springs, Lake Co., FL (AS); Lake Panasoffkee, Sumter Co., FL (LP) Heleobia dalmatica spring, Pirovac, Croatia Heleobops docimus pond at Chisholm Point, Grand Cayman Island Littoridinops monroensis St. Johns River, Fort Gates Ferry, 4.8 km southwest of Welaka, St. Johns Co., FL Littoridinops tenuipes brackish marsh, 4 km southwest of Yankeetown, Levy Co., FL Mezcipyrgus carranzae Mojarral West Laguna, Cuatro Cienegas basin, Coahuila, Mexico Onobops jacksoni brackish marsh, 4 km southwest of Yankeetown, Levy Co., FL Pyrgophorus platyrachis Lithia Springs, Hilsborough Co., FL Pyrgophorus sp. Simmons Bayou, Ocean Springs, Jackson Co., MS Ascorhis victoriae Manly Lagoon, New South Wales, Australia (ML); Careel Bay, Pittwater, New South Wales, Australia (CB) Phrantela marginata tributary of Thirteen Mile Creek, Tasmania, Australia • r "Tryonia" brevissima 93 93 "Tryonia'' n. sp. 3 9 1 "Tryonia'' adamantina 1— "Tryonia" alamosae 100 r Aphaostracon sp. (AS) I- Aphaostracon sp. (LP) Littoridinops tenuipes - 99 [I -- Littoridinops monroensis rc Pyrgophorus sp. Pyrgophorus platyrachis "Tryonia" kosteri 99 Heleobops docimus Heleobia dalmatica Et_ Onobops jacksoni ' Tryonia" robusta ■ 11•• 80 Tryonia imitator (PE) Tryonia imitator (MO) 76 Tryonia margae 94 Tryonia n. sp. 1 Tryonia sauna Tryonia circumstriata (DY) 100 Tryonia circumstriata (DD) Tryonia circumstriata (DY) 100 Tryonia cheatumi (PL2) L Tryonia cheatumi (PL I) Tryonia protea (FS) Tryonia protea (WS) 95 Tryonia protea (OA) 100 Tryonia protea (BL) Tryonia protea (WH) - Tryonia protea (HS) Tryonia clathrata Tryonia gilae Tryonia elata 96 Tryonia ericae Tryonia variegata (DV) 74 Tryonia variegata (SA) 100 Tryonia angulata Tryonia rowlandsi 100 91 Tryonia aequicostata (LE) 75 I Tryonia aequicostata (AS) Tryonia quitobaquitae Mexipyrgus carranzae "Tryonia" n. sp. 2 100 j Ascorhis victoriae (ML) Ascorhis victoriae (CB) Phrantela marginata T. imitator (Pacific)

64 T. margae (Amargosa R.)

T. n. sp. 1 (Lahontan)

T. sauna (Amargosa R.)

T. circumstriata (Pecos R.) 16 T. cheatumi (Pecos R.)

T. protea (Colorado R., Grt. Basin)

10 T. clathrata (White R.) 15 T. gilae (Gila R.)

T. elata (Amargosa R.)

T. ericae (Amargosa R.) 11

T. variegata (DV) (Amargosa R.)

T. variegata (SA) (Amargosa R.)

T. angulata (Amargosa R.)

T. rowlandsi (Amargosa R.)

T. aequicostata (Florida) 17 T. quitobaquitae (R. Sonoyta)

M. carranzae