Phylogeny of North American amblemines (Bivalvia, Unionoida): prodigious polyphyly proves pervasive across genera

Authors


aAuthor for correspondence.
E-mail: amblema@bama.ua.edu

Abstract

Abstract. The subfamily Ambleminae is the most diverse subfamily of fresh-water mussels (order Unionoida), a globally diverse and ecologically prominent group of bivalves. About 250 amblemine species occur in North America; however, this diversity is highly imperiled, with the majority of species at risk. Assessing and protecting this diversity has been hampered by the uncertain systematics of this group. This study sought to provide an improved phylogenetic framework for the Ambleminae. Currently, 37 North American genera are recognized in Ambleminae. Previous phylogenetic studies of amblemines highlighted the need for more extensive sampling due to the uncertainties arising from polyphyly of many currently recognized taxa. The present study incorporated all amblemine genera occurring in North America north of the Rio Grande, with multiple species of most genera, including the type species for all but seven genera. A total of 192 new DNA sequences were obtained for three mitochondrial gene regions: COI, 16S, and ND1. In combination with published data, this produced a data matrix incorporating 357 gene sequences for 143 operational taxonomic units, representing 107 currently recognized species. Inclusion of published data provides additional taxa and a summary of present molecular evidence on amblemine phylogeny, if at the cost of increasing the amount of missing data. Parsimony and Bayesian analyses suggest that most amblemine genera, as currently defined, are polyphyletic. At higher taxonomic levels, the tribes Quadrulini, Lampsilini, and Pleurobemini were supported; the extent of Amblemini and the relationships of some genera previously assigned to that tribe remain unclear. The eastern North American amblemines appear monophyletic. Gonidea and some Eurasian taxa place as probable sister taxa for the eastern North American Ambleminae. The results also highlight problematic taxa of particular interest for further work.

The subfamily Ambleminae is a diverse group of fresh-water bivalve mollusks found throughout North America east of the continental divide. They are also highly imperiled due to habitat specificity, limited ranges, and complex life history (Strayer et al. 2004). Many species require free-flowing medium to large rivers, a habitat type that is extensively impacted by human activity. In addition, the need for suitable host fish for the parasitic larval stage (the glochidium) contributes to mussels' vulnerability to habitat disturbance (Lydeard et al. 2004). As a result, geqslant R: gt-or-equal, slanted12.6% of the North American species and subspecies of Ambleminae are believed to be extinct, 22.9% are federally endangered or threatened, and many others are locally or globally rare (Turgeon et al. 1998).

Conservation efforts are hampered, however, by our limited understanding of their systematics. High levels of ecophenotypic plasticity may result in intraspecific variation in shell form exceeding intergeneric differences (Davis 1983). Thus, Roe (2000) used molecular sequences to demonstrate that several putative specimens of highly imperiled Ptychobranchus species actually represented other genera. Likewise, morphological identification of glochidia is often difficult, whereas molecular data readily identify them (White et al. 1996). This morphological variability has made both taxonomic definition and identification problematic, but both are essential to establishing conservation needs.

Well-supported phylogenetic hypotheses can provide valuable information for conservation. Many aspects of the biology of rare mussel species, such as host fish choice, habitat preferences, and breeding cycles, are often inferred based on better-studied common species (e.g., many species accounts in Parmalee & Bogan [1998] have only tentative suggestions for these features, based on other species in the genus). The closest relatives of the species of interest are likely to be the best basis for such inference. If the species is misclassified, the inferences are less likely to be accurate. Also, incorrect synonymization may lead to the neglect of phylogenetically distinctive taxa. Incorrect assumptions may lead to inappropriate research or management approaches, with potentially disastrous results for the rarest species (Minton & Lydeard 2003). Likewise, an accurate general phylogenetic framework is necessary for detailed phylogenetic studies to correctly identify relevant taxa that should be included. Ecological studies may also be misled by incorrectly grouping unrelated taxa. Such issues, along with the development of new techniques, have prompted a renewed interest in the systematics of unionids (Roe & Hoeh 2003; Strayer et al. 2004). Nevertheless, molecular data for many genera and species are still lacking. Previous analyses have generally focused either on several species from a few genera or on a broad sampling of genera represented by one or two species a piece. They also have relied on only one or two genes. The present analyses double to triple the taxonomic coverage of previous studies, incorporating all presently recognized genera of North American Ambleminae.

Both molecular and morphological data have influenced the most recent classifications of the Ambleminae. Molecular studies indicate that all studied North American unionoids fall into three categories, corresponding to the family Margaritiferidae and the unionid subfamilies Ambleminae and Unioninae (Graf 2002); however, classifications in the past few decades have varied widely in detail (see the discussion below). The present paper treats the subfamily Anodontinae, referred to by earlier authors, as a tribe within the subfamily Unioninae. Molecular data (Hoeh et al. 2001, 2002a; Graf 2002; Huang et al. 2002; Roe & Hoeh 2003) and the presence of hooked, subtriangular glochidia (Nagel et al. 1998; Hoeh et al. 2001; Roe & Hoeh 2003) suggest that Anodonta belongs in the same subfamily as Unio. Ambleminae includes ∼80% of North American species and ∼75% of the genera. Some East Asian taxa appear closely related to Ambleminae (Huang et al. 2002); however, other Asian taxa once assigned to Ambleminae appear to be only distantly related (Graf 2002). A few European species have also been assigned to Ambleminae (Nagel et al. 1998). However, the apparent similarities may reflect convergent shell form or plesiomorphic features rather than true affinity. The present study included data for the Asian Hyriopsis and Inversidens, and the European Psilunio, to represent Old World “amblemines.”Gonidea, from northwestern North America, is exceptionally problematic in its affinities. If it is an amblemine, it is the only one in Pacific drainages of North America. Existing molecular data suggest that it may be a sister taxon to the remaining North American Ambleminae (Graf 2002). Amblema, the type genus of Ambleminae, and all species that are undisputed close relatives of it occur in the Atlantic and Gulf of Mexico drainages of North America. Within Ambleminae, the North American species have been divided among the tribes Amblemini, Pleurobemini, and Lampsilini, plus Gonideini, with some authors also recognizing Quadrulini or other taxa (Graf 2002).

The great biological diversity of North American amblemines was first recognized in the early 1800s, by workers such as Say, Rafinesque, Lea, and Conrad. However, this work focused primarily on describing species. A few authors recognized distinct genera within what is now the Ambleminae, but others placed them, along with most of the rest of the global unionid fauna, into the single genus Unio.Rafinesque (1820) named the Ambleminae (as “Amblemidia”), the only suprageneric group proposed specifically for amblemines before 1900. The 19th century classifications were based almost entirely on shell characters. Simpson (1891, p. 86) observed that existing classifications were largely artificial but left the work of revising them “to some capable student of the future.” In fact, Simpson himself took up this challenge.

Beginning in the late 1800s, many workers recognized the distinctiveness of the American amblemines, adding both new genera and higher taxa. Greater emphasis on anatomical characters contributed to this development. Simpson (1900) provided the first thorough anatomy-based classification of amblemines and other Unionoida, but did not use formal subfamilial nomenclature, introducing vernacular names instead. He also grouped several taxa with disparate but relatively simple patterns of gill brooding (present Anodontini, Pleurobemini, and Margaritiferidae). Many authors have overlooked previous names and made redundant ones (e.g., 7 of the 11 families and subfamilies Modell [1964] used for North American Unionidae are junior synonyms). Different authors treat the same taxon as a tribe, subfamily, or family. Thus, the suprageneric nomenclature is confused. For consistency, the present paper treats Unioninae and Ambleminae as subfamilies of Unionidae, with both divided into tribes. Among the most widely used higher taxa in Ambleminae, albeit often under junior synonyms, are von Ihering's (1901) Quadrulini and Lampsilini, Hannibal's (1912) Pleurobemini, and Ortmann's (1916) Gonideini (all proposed as subfamilies). Additional tribes or subfamilies were proposed by several workers, especially Modell (1964 and references therein), Starobogatov (1970), and Heard & Guckert (1971), but these higher taxa have been synonymized or ignored by other workers. The proposed relationships of amblemine tribes to true Unionini (characterized by the European Unio, although the name has been widely misapplied to North American Pleurobemini), Anodontini, and Alasmidontini (now considered a synonym of Anodontini) also varied greatly, with many authors suggesting that Ambleminae is paraphyletic or polyphyletic. Also, authors have varied in including or excluding Eurasian and African genera in the amblemine tribes with North American types. Table 1 compares the assignments of North American amblemine genera to higher taxa in several recent classifications.

Table 1.   Suprageneric classifications of North American Ambleminae.
GenusModell (1964)Haas (1969)Heard & Guckert (1971)
ActinonaiasElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
AmblemaElliptionidae, AmbleminaeUnionidae, QuadrulinaeAmblemidae, Ambleminae
CyclonaiasUnionidae, QuadrulinaeUnionidae, QuadrulinaeUnionidae, Pleurobeminae
CyprogeniaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, mesogenae
CyrtonaiasElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Popenaiadinae
DromusElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, eschatigenae
EllipsariaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
ElliptioElliptionidae, ElliptioninaeUnionidae, UnioninaeUnionidae, Pleurobeminae
ElliptoideusUnionidae, QuadrulinaeUnionidae, UnioninaeAmblemidae, Ambleminae
EpioblasmaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
FusconaiaElliptionidae, PleurobeminaeUnionidae, QuadrulinaeAmblemidae, Ambleminae
GlebulaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
GonideaMargaritiferidae, PseudodontinaeUnionidae, UnioninaeAmblemidae, Gonideinae
HemistenaElliptionidae, AlasmidontinaeUnionidae, UnioninaeUnionidae, Pleurobeminae
LampsilisElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
LemioxElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
LeptodeaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
LexingtoniaElliptionidae, PleurobeminaeUnionidae, UnioninaeUnionidae, Pleurobeminae
LigumiaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
MedionidusElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
MegalonaiasUnionidae, QuadrulinaeUnionidae, QuadrulinaeAmblemidae, Megalonaiadinae
ObliquariaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, mesogenae
ObovariaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
PlectomerusUnionidae, QuadrulinaeUnionidae, QuadrulinaeAmblemidae, Ambleminae
PlethobasusElliptionidae, PleurobeminaeUnionidae, UnioninaeUnionidae, Pleurobeminae
PleurobemaElliptionidae, PleurobeminaeUnionidae, UnioninaeUnionidae, Pleurobeminae
PopenaiasNot mentionedUnionidae, UnioninaeUnionidae, Popenaiadinae
PotamilusElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
PtychobranchusElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, ptychogenae
QuadrulaUnionidae, QuadrulinaeUnionidae, QuadrulinaeAmblemidae, Ambleminae
QuincuncinaUnionidae, QuadrulinaeUnionidae, QuadrulinaeAmblemidae, Ambleminae
ToxolasmaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
TritogoniaUnionidae, QuadrulinaeUnionidae, QuadrulinaeAmblemidae, Ambleminae
TruncillaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
UniomerusElliptionidae, ElliptioninaeUnionidae, UnioninaeUnionidae, Pleurobeminae
VenustaconchaElliptionidae, LampsilinaeUnionidae, LampsilinaeNot mentioned
VillosaElliptionidae, LampsilinaeUnionidae, LampsilinaeUnionidae, Lampsilinae, heterogenae
GenusStarobogatov (1970)Davis & Fuller (1981) (Unionidae)Previous molecular studies (Unionidae)Present study (Unionidae)
ActinonaiasLampsilidae, Lampsilinae, LampsiliniAmbleminae, LampsiliniAmbleminae, LampsiliniAmbleminae, Lampsilinia
AmblemaAmblemidae, AmbleminaeAmbleminae, AmbleminiAmbleminae, AmbleminiAmbleminae, Amblemini
CyclonaiasLampsilidae, PleurobeminaeAmbleminae, AmbleminiAmbleminae, Lampsilini?Ambleminae, Quadrulini
CyprogeniaLampsilidae, CyprogeniinaeNot sampledAmbleminae, LampsiliniAmbleminae, Lampsilini
CyrtonaiasLampsilidae, Lampsilinae, LampsiliniNot sampledAmbleminae, Lampsilini?Ambleminae, Lampsilini?
DromusLampsilidae, Medionidinae, DrominiNot sampledAmbleminae, LampsiliniAmbleminae, Lampsilini
EllipsariaLampsilidae, Lampsilinae, GlebuliniNot sampledAmbleminae, LampsiliniAmbleminae, Lampsilini
ElliptioLampsilidae, ElliptioninaeAmbleminae, PleurobeminiAmbleminae, PleurobeminiAmbleminae, Pleurobeminia
ElliptoideusAmblemidae, AmbleminaeNot sampledAmbleminae, PleurobeminiAmbleminae, Pleurobemini?
EpioblasmaLampsilidae, Lampsilinae, PilaeiniNot sampledAmbleminae, LampsiliniAmbleminae, Lampsilini
FusconaiaAmblemidae, Quadrulinae, QuadruliniAmbleminae, PleurobeminiaAmbleminae, PleurobeminiaAmbleminae, Pleurobeminia
GlebulaLampsilidae, Lampsilinae, GlebuliniAmbleminae, LampsiliniAmbleminae, Lampsilini?Ambleminae, Lampsilini
GonideaMargaritiferidae, Pseudodontinae, PseudodontiniAmbleminae, GonideiniAmbleminae, GonideiniAmbleminae, Gonideini
HemistenaLampsilidae, Medionidinae, MedionidiniNot sampledAmbleminae, PleurobeminiAmbleminae, Pleurobemini
LampsilisLampsilidae, Lampsilinae, LampsiliniAmbleminae, LampsiliniAmbleminae, LampsiliniaAmbleminae, Lampsilinia
LemioxLampsilidae, Lampsilinae, LampsiliniNot sampledNot sampledAmbleminae, Lampsilini
LeptodeaLampsilidae, Lampsilinae, LampsiliniAmbleminae, LampsiliniAmbleminae, LampsiliniAmbleminae, Lampsilini
LexingtoniaLampsilidae, PleurobeminaeNot sampledNot sampledAmbleminae, Pleurobemini
LigumiaLampsilidae, Lampsilinae, LampsiliniAmbleminae, LampsiliniAmbleminae, LampsiliniaAmbleminae, Lampsilinia
MedionidusLampsilidae, Medionidinae, MedionidiniNot sampledAmbleminae, LampsiliniAmbleminae, Lampsilini
MegalonaiasAmblemidae, AmbleminaeAmbleminae, AmbleminiAmbleminae, QuadruliniAmbleminae, Quadrulini
ObliquariaLampsilidae, CyprogeniinaeNot sampledAmbleminae, Lampsilini?Ambleminae, Lampsilini
ObovariaLampsilidae, Lampsilinae, GlebuliniNot sampledAmbleminae, LampsiliniaAmbleminae, Lampsilinia
PlectomerusAmblemidae, AmbleminaeAmbleminae, AmbleminiAmbleminae, Amblemini?Ambleminae, Lampsilini?
PlethobasusLampsilidae, PleurobeminaeNot sampledNot sampledAmbleminae, Pleurobemini
PleurobemaLampsilidae, PleurobeminaeAmbleminae, PleurobeminiAmbleminae, PleurobeminiAmbleminae, Pleurobeminia
PopenaiasLampsilidae, ElliptioninaeNot sampledNot sampledAmbleminae, Amblemini
PotamilusLampsilidae, Lampsilinae, LampsiliniAmbleminae, LampsiliniAmbleminae, LampsiliniAmbleminae, Lampsilini
PtychobranchusLampsilidae, PtychobranchinaeAmbleminae, LampsiliniAmbleminae, LampsiliniAmbleminae, Lampsilini
QuadrulaAmblemidae, Quadrulinae, QuadruliniAmbleminae, AmbleminiAmbleminae, QuadruliniaAmbleminae, Quadrulinia
QuincuncinaAmblemidae, Quadrulinae, QuadruliniAmbleminae, AmbleminiAmbleminae, PleurobeminiaAmbleminae, Pleurobeminia
ToxolasmaLampsilidae, Lampsilinae, LampsiliniAmbleminae, LampsiliniAmbleminae, Lampsilini?Ambleminae, Lampsilini?
TritogoniaAmblemidae, Quadrulinae, QuadruliniAmbleminae, AmbleminiAmbleminae, QuadruliniAmbleminae, Quadrulini
TruncillaLampsilidae, Lampsilinae, GlebuliniNot sampledAmbleminae, LampsiliniAmbleminae, Lampsilini
UniomerusLampsilidae, ElliptioninaeAmbleminae, PleurobeminiAmbleminae, PleurobeminiAmbleminae, Quadrulini
VenustaconchaLampsilidae, Lampsilinae, LampsiliniNot sampledNot sampledAmbleminae, Lampsilinia
VillosaLampsilidae, Lampsilinae, LampsiliniAmbleminae, LampsiliniAmbleminae, LampsiliniAmbleminae, Lampsilinia

The many morphological studies have provided much additional data, but relatively few characters have shaped most classifications. Particularly important anatomical features include the structure of the gills and their modification for brooding. Other studies focused on hinge and shell features. Heard & Guckert (1971) summarized the traditional distinguishing suprageneric characters in Ambleminae from many previous sources. Lampsilini was distinguished by the distinctive modifications of the female posterior outer gills for brooding and by the sexually dimorphic shells. Quadrulini (used interchangeably with Amblemini) was based on the frequently sculptured shells and the use of all 4 gills for brooding (“tetrageny”). Pleurobemini was characterized by the generally smooth shells and the use of only the outer gills for brooding (“ectobranchy”), without the gill specializations of Lampsilini. Gonideini was based on the lack of hinge teeth and distinctive gill anatomy. Even within this rather short list, most authors emphasized only one or two characters. For example, Modell (1964) relied heavily on beak sculpture, and Haas (1969) emphasized shell form. In contrast, Heard & Guckert (1971, p. 337) “selectively elected to ignore one entire array of characters,” namely shell features, and instead emphasized reproductive features such as larval brooding periods and brooding structures of the gills.

This focus on character states for selected features produced an emphasis on grades rather than clades. Putatively primitive characters, as well as putative synapomorphies, were frequently used in defining genera and higher taxa. Thus, Hannibal (1912) proposed that Pleurobemini evolved from Quadrulini (including the present Amblemini), and that Unioninae (including the present Gonideini) and Lampsilini evolved from Pleurobemini. Heard & Guckert (1971) devised a very similar system, except that Gonideini was seen as the sister taxon to Amblemini. Such paraphyletic taxa are likely to be a poor guide to evolutionary relationships. Likewise, basing higher taxa on one or a few characters runs the risk of possible homoplasy in that character (Roe & Hoeh 2003). Currently used morphological characters may not provide enough data to resolve relationships within Ambleminae (Graf & Ó Foighil 2000a). Recent studies performing cladistic analyses of morphological features have found little or no resolution of relationships of Ambleminae within Unionidae, apart from the distinctive gill structures of Lampsilini (Hoeh et al. 2001; Roe & Hoeh 2003). This paucity of morphological characters has resulted in relatively few morphology-based hypotheses about the phylogenetic interrelationships of individual genera. Apart from general agreement about the monophyly and constituent genera of Lampsilini, there have been many differences between classifications. Several species have also varied in their generic assignment from author to author. Also, the use of paraphyletic taxa in non-cladistic classifications makes it unsafe to assume that previous authors thought that the genera and higher taxa that they used were monophyletic. This pattern of classification prevailed until the advent of molecular data and cladistic methodologies prompted thorough re-examination of the taxonomy.

The fossil record provides limited help in resolving the relationships of Ambleminae, due to the sporadic nature of their fossil record and the problems of convergence in shell form. Probable amblemines occur in the Cretaceous faunas of central North America (Hartman 1998), suggesting that the North American amblemines have a long evolutionary history separate from other unionoids. However, these might represent unrelated, morphologically convergent taxa (Watters 2001).

The development of molecular techniques provided a novel source of information on the systematics of the Ambleminae. Some traditional classifications were supported, whereas others were called into question and new ideas were suggested. In the first molecular study to include many amblemines, Davis & Fuller (1981) used immunoelectrophoresis to assess the relationships of North American unionoidean genera (Table 1). This study demonstrated the distinctiveness of Ambleminae from “Anodontinae” (Unioninae). The early immunological work was followed by numerous studies using DNA sequencing, as well as a few other genetic techniques, e.g., RFLP analysis (White et al. 1996). Rosenberg et al. (1994) found little variation in the D6 region of the 28S gene within the Unionidae, but subsequent studies have identified more variable genes (16S: Lydeard et al. 1996; COI: Roe & Lydeard 1998; ITS: King et al. 1999; D2 region of 28S: Graf & Ó Foighil 2000b; Graf 2002; ND1: Buhay et al. 2002; Serb et al. 2003; male mitotype COI: Hoeh et al. 1996, 2002b; COII: Curole & Kocher 2002; cytB: Mock et al. 2004). Studies using DNA sequencing have generally supported the higher taxa recognized by Davis & Fuller (1981), with the exception of their Amblemini, which appears to be a polyphyletic group sharing plesiomorphic features (Lydeard et al. 1996). In general, traditional species-level classification has been upheld, but genera and higher taxonomic categories often appear polyphyletic (Lydeard et al. 2000; Roe et al. 2001; Serb et al. 2003; Huff et al. 2004). However, sampling issues remain a problem, with many genera and type species unavailable to previous studies. The frequent polyphyly of genera makes data for type species especially important; otherwise, it is unclear which group of species actually belongs in the genus. Although these studies provide explicit hypotheses about the phylogenetic relationships of the included taxa, the patchy taxonomic coverage makes it impossible to extrapolate phylogenetic relationships for the Ambleminae as a whole.

The modern concept of Ambleminae as a monophyletic group has only arisen with the advent of molecular and cladistic studies. Most workers before Davis & Fuller (1981) placed Pleurobemini as closely related to, if not synonymous with, the Old World Unionini. However, Davis & Fuller (1981) found that Unioninae (as Anodontinae) were genetically very distinct from the Ambleminae (including Pleurobemini), a conclusion substantiated by all subsequent molecular studies. In these studies (e.g., Lydeard et al. 1996; Bogan & Hoeh 2000; Graf 2002; Graf & Ó Foighil 2000a; Hoeh et al. 2002a, b; Krebs et al. 2003; Roe & Hoeh 2003), the relationships between the tribes in Ambleminae have varied. Not all studies included Gonideini or Quadrulini. Those including Gonideini placed it basal to the other tribes or outside of Ambleminae. The interrelationships of Amblemini, Quadrulini, Pleurobemini, and Lampsilini differed from analysis to analysis, and Lampsilini was not consistently monophyletic. Often, these groupings did not have strong bootstrap support, and some, if not all, of the tribes were represented by only a few taxa in each analysis.

Thus, Simpson's (1891) challenge remains a problem: what are natural groups in the Unionidae? We sought to answer this for the Ambleminae by addressing three main questions: (1) Are the North American Ambleminae a monophyletic group? (2) What are the relationships among the North American genera of Ambleminae? (3) Are these genera, as currently recognized, natural entities?

Methods

Taxa were selected to represent all 37 currently recognized North American amblemine genera. Some nomenclatural disagreements exist within the literature (e.g., Smith [2000a] synonymized Cumberlandia with Margaritinopsis; this is not supported by molecular data, however [Huff et al. 2004]). The nomenclature of Turgeon et al. (1998) is followed here for convenience of reference. Revision of the nomenclature is outside the scope of this paper, and ongoing molecular studies of several taxa make an overall revision premature. Putative amblemines from outside North America, including Asian Hyriopsis and Inversidens, and European Psilunio, as well as the western North American Gonidea, were also included in the analyses to test the monophyly of the eastern North American tribes. Outgroups included margaritiferids (Cumberlandia and Margaritifera) and unionines (Anodontini) (Anodonta, Lasmigona, Pyganodon, and Strophitus). When possible, the type species for each ingroup genus was sequenced. Suitable data were not available for the type species of Actinonaias (A. sapotalensis [Lea 1841], from Central America), Cyrtonaias (C. berlandieri [Lea 1857], from Mexico, possibly synonymous with C. tampicoensis analyzed herein [Howells et al. 1996]), Epioblasma (E. rangiana [Lea 1838], endangered), Lexingtonia (L. subplana [Conrad 1837], very rare and taxonomically problematic), Obovaria (although the nearly extinct O. retusa [Lamarck 1819] has traditionally been considered the type species, the type designation is problematic [Graf, pers. comm.]). Toxolasma (T. lividus [Rafinesque 1831], failed to amplify), and Uniomerus (U. tetralasmus [Say 1831]), failed to amplify. For the outgroups, taxa were chosen based on the availability of sequences or material. Appendix 1 lists the taxa and GenBank accession numbers, and Appendix 2 lists the locality and collection information for the new sequences. A total of 137 COI, 119 16S, and 101 ND1 sequences, representing 107 currently recognized species, were analyzed. Just over half of the sequences are new, and many of the previously published sequences had not been integrated into a single analysis.

DNA was extracted from fresh, frozen, or ethanol-preserved specimens using standard CTAB and chloroform-isoamyl alcohol protocols (Winnepenninckx et al. 1993). Foot, mantle, or adductor tissue was used to avoid the risk of sampling male mitotypes from gonadal tissue (Hoeh et al. 2002b). Portions of the COI, 16S, and ND1 genes were amplified, as they were known to show species-level variation in Ambleminae (Lydeard et al. 1996; Roe & Lydeard 1998; Buhay et al. 2002; Serb et al. 2003). The present ND1 fragment is much longer than that analyzed in previous studies and is correspondingly more informative. Primers used were:

  • COI: 5′-GTTCCACAAATCATAAGGATATTGG-3′

  • 5′-TACACCTCAGGGTGACCAAA AAACCA-3′

  • 16S:5′-CCGTTCTGAACTCAGCTCATGT-3′

  • 5′-CGACTGTTTAACAAAAACAT-3′

  • ND1:5′-TGGCAGAAAAGTGCATCAGA TTTAAGC-3′

  • 5′-GCTATTAGTAGGTCGTATCG-3′

(COI modified from Folmer et al. 1994; 16S from Lydeard et al. 1996; ND1 from Buhay et al. 2002 and Serb & Lydeard 2003). PCR cycle parameters for COI and ND1 were: 92°C for 2 min; 92°C for 40 s, 40°C for 40 s, 72°C for 90 s, × 5; 92°C for 40 s, 50°C for 40 s, 72°C for 90 s, × 25; 72°C for 10 min; hold 4°C. For 16S, they were: 92°C for 5 min; 92°C for 40 s, 50°C for 60 s, 68°C for 90 s, × 35; 72°C for 10 min; hold 4°C. PCR products were purified using Qiagen QIAquick PCR purification kits (Valencia, CA). Cycle sequencing used ABI Big Dye Terminator kits (Foster City, CA) with thermal cycle parameters of 1°C s−1 ramp speed, starting with 1 min at 96°C followed by 26 cycles of 96°C for 10 s, 49°C for 5 s, and 60°C for 4 min, then 10 min at 60°C and hold at 4°C. The cycle sequencing products were purified with sephadex columns or Qiagen DyeEx kits, and then run on an ABI 3100 automated sequencer.

The results for each strand were compared and aligned with published sequences using BioEdit (Hall 1999). No indels were found within the protein-coding genes. Two short variable regions in the 16S gene and all indels were excluded, as positional homology was unclear, thus excluding a total of 79 base pairs. The exact length of published sequences and of readable sequences obtained in the present study varies. We used 602 base pairs (bp) for COI, 315 bp for 16S, and 753 bp for ND1. The high evolutionary rate of mitochondrial genes may produce problems due to saturation for older divergences (Graf 2002), thus potentially limiting the resolution of higher-level relationships. However, they provide appropriate evolutionary rates to resolve relationships within Ambleminae. Sequences were analyzed using PAUP* 4.10 (Swofford 1998) and TNT (Goloboff et al. 2000). All taxa with data for at least two of the three genes were analyzed. This ensured that any two taxa would share some sequence information.

GenBank data for these genes come from several studies (COI: Hoeh et al. 1998; Roe & Lydeard 1998; King et al. 1999; Bogan & Hoeh 2000; Lydeard et al. 2000; Graf & Ó Foighil 2000a; Roe et al. 2001; Buhay et al. 2002; Giribet & Wheeler 2002; Hoeh et al. 2002b; Machordom et al. 2003; Serb & Lydeard 2003; Okazaki & Ueshima, unpubl. data; 16S: Lydeard et al. 1996; Mulvey et al. 1997; Lydeard et al. 2000; Turner et al. 2000; Roe et al. 2001; Huang et al. 2002; Krebs et al. 2003; Machordom et al. 2003; Serb & Lydeard 2003; Okazaki & Ueshima, unpubl. data; ND1: Buhay et al. 2002; Serb et al. 2003; Serb & Lydeard 2003;; Okazaki & Ueshima, unpubl. data). The sequences were concatenated to provide a greater number of informative characters for the analyses. Although this approach of concatenating gene sequences produced many taxa with extensive missing data, this should not pose a problem for analyses as long as an adequate number of characters are represented in all included taxa (Wiens 2003). Concatenation of multiple sequences raises a risk of misleading results due to non-monophyly of the source taxa (Malia et al. 2003). However, in the present study, only sequences from the same species were concatenated; in many cases, a single individual supplied all the sequences. Multiple published sequences for the same species that were nearly or entirely identical and that formed a monospecific polytomy in the published analyses were eliminated as redundant. This only affected the few species that have three or more published sequences for the same gene. Polytomies that included multiple species were retained.

To test for the compatibility of the different gene sequences, a partition homogeneity test (sensu Swofford 1998; PILD of Dowton & Austin 2002) was run in PAUP* using all taxa with data for all three genes with 100 replicates of 10 random addition replicates each. The maximum number of trees per replicate was set to 10,000. This test is sensitive to other factors, such as partition size and evolutionary model, besides data compatibility (Dowton & Austin 2002), but may provide a rough idea of agreement between data sets. Despite the problems of the ILD type of tests, no better alternative has gained wide acceptance. Sampling of 200,000 random trees yielded g1 values of −0.257194 for the whole data set, −0.219740 for COI alone, −0.298611 for 16S alone, and −0.278602 for ND1 alone.

The missing data made PAUP* inefficient; however, parsimony and bootstrap analyses in TNT finished quickly. Parsimony analyses used 500 replicates of random sequence addition with TBR branch swapping, holding 10 shortest trees at each replicate. Bootstrap analysis used 1000 replicates of standard bootstrapping; each replicate used heuristic searches of 10 random addition sequence replicates.

Bayesian analysis provided a second phylogenetic technique. MrBayes 3.0b4 (Huelsenbeck & Ronquist 2001) was used to run MCMCMC searches. This was run with Nst=6; rates=invgamma; partitions corresponding to the genes; revmat, shape, pinvar, and statefreq unlinked; 2,000,000 generations and 8 chains. These parameters were chosen to maximize model flexibility. Other parameters were set to default values, saving every 100th tree. Bootstrap values typically underestimate support, whereas Bayesian analyses tend to overestimate it (Erixson et al. 2003; Simmons et al. 2004), so comparison of the results of the two provides a useful check.

Results

The present data set greatly expands the molecular data available for Ambleminae. Table 2 summarizes previous molecular analyses. In comparison, our analyses include a total of 37 genera, 30 type species, and 96 species of North American Ambleminae, thus doubling to tripling the taxonomic coverage. (These tallies include Gonidea and accept the taxonomy within each paper in cases of disagreement, e.g., whether two forms are valid species.)

Table 2.   Number of amblemine taxa in selected molecular studies.
ReferenceNumber of generaNumber of speciesNumber of type species
  1. Gonidea is counted, but not Eurasian species, and the taxonomy of the respective paper is followed for counting the number of taxa (e.g., if one paper synonymizes two species treated as distinct in another paper).

Davis & Fuller (1981)214015
Lydeard et al. (1996)16208
Bogan & Hoeh (2000), Hoeh et al. (2001, 2002a), and Roe & Hoeh (2003)12129
Graf & Ó Foighil (2000a)12176
Lydeard et al. (2000)6133
Krebs et al. (2003)182410
Serb et al. (2003)173613
Present study379630

Among the three genes, the 16S sequence, being the shortest and most conservative, also had the fewest informative characters (240 parsimony-informative sites for COI, 132 for 16S, and 377 for ND1). The PILD test gave a value of 0.88, thus not indicating rejection of compatibility, and separate parsimony analyses for each gene yielded similar results to the patterns found with the combined data sets (not shown). This agrees with expected evolutionary similarity due to the functioning of the mitochondrial genome as a single evolutionary locus. The analyses yielded trees supporting several major clades, as well as providing resolution within these clades. Figure 1 shows the strict consensus parsimony trees with bootstrap percentages; Fig. 2 shows the Bayesian likelihood tree.

Figure 1a.

 Strict consensus of 2304 most parsimonious trees of length 7447 (CI=0.2062, RI= 0.7938). Numbers above branches represent bootstrap percentages. Numbers after species names reflect multiple individuals of the same species. The largest clade corresponding with a particular tribe, subfamily, etc. is labeled. There is some variation between the trees as to the exact taxa included, e.g., Plectomerus is within Lampsilini, and Fusconaia ebena and Obovaria rotulata are within Amblemini in the Bayesian tree, but the parsimony analyses placed all three together in their own clade outside of Amblemini and of Lampsilini. See text for discussion of these taxa.

Figure 1b.

 Continuation of Fig. 1a.

Figure 2a.

 50% majority-rule consensus of 17,312 Bayesian likelihood trees (burn-in=268,800, mean log likelihood=−32,760). Numbers above branches indicate posterior probabilities.

Figure 2b.

 Continuation of Fig. 2a.

The eastern North American Ambleminae (hereafter “Ambleminae s.s.”) form a monophyletic group. However, Gonidea appears more closely related to Eurasian species than to other North American taxa. These clades had high Bayesian probability, but were unresolved in the bootstrap analysis. In turn, the clade including Gonidea was the sister taxon to the eastern North American Ambleminae. Within the Ambleminae s.s. clade, most taxa fall into Quadrulini, Pleurobemini, or Lampsilini, but a few species were not clearly assigned to one of these three main tribes. These species appear closely related to the Lampsilini+Amblemini group, but their exact relationships differed between the analyses. All phylogenetic analyses supported Lampsilini, Quadrulini, and Pleurobemini as major clades within Ambleminae, along with several subclades within these large groups. Within the eastern North American amblemines, Quadrulini appeared as the basal taxon. Pleurobemini appears to be the sister taxon of a Lampsilini+Amblemini clade. Quadrulini and Lampsilini received 89% posterior probability from the Bayesian analysis; Pleurobemini received 100%. Although an Amblemini clade received 99% support in the Bayesian analysis, the parsimony analyses supported a different topology.

Several smaller clades occur in both the parsimony and the Bayesian analyses. Many of these clades do not correspond with current genus-level nomenclature. Twelve genera (out of 25 that have multiple species) are clearly polyphyletic, and the status of several more remains ambiguous. Fusconaia ebena, F. succissa, Obovaria rotulata, Quincuncina infucata, and Q. kleiniana are currently assigned to genera in the wrong tribe. Previous molecular analyses (Lydeard et al. 2000; Serb et al. 2003) indicated that they were assigned to the wrong genera, but the present data set confirms the tribe-level differences. Our analyses also suggest possible sister-taxon relationships for several genera.

Discussion

Possible extralimital amblemines

The analyses support a relationship of western North American Gonidea, European Psilunio, and Asian Inversidens with eastern North American Ambleminae, although this failed to receive bootstrap support. Both Bayesian and parsimony analyses placed these three taxa in a clade sister to the eastern North American amblemine clade. Within this basal amblemine group, the Eurasian Psilunio and the Asian Inversidens were supported as close relatives. Psilunio also resembles Gonidea morphologically (Nagel et al. 1998). This closely resembles the biogeographic pattern for pleurocerid gastropods (Lydeard et al. 2002). In both, the North American Pacific drainage fauna is depauperate compared with the eastern drainages and shows closest relationships with Eurasian taxa. In turn, the Eurasian–western North American clade is sister to a diverse eastern North American clade (in the Pleuroceridae, some Eurasian taxa are basal members of the eastern North American clade). The similarity between eastern Asian and western North American faunas may reflect interchange via the Bering land bridge. The pre-Pleistocene fossil record of Gonidea and the pleurocerid Juga in North America (Hannibal 1912) indicate that this interchange occurred before 4.8 million years ago (Marinkovich & Gladenkov 1999). This dating also fits with their climatic preferences. Gonidea and Juga range north only to southern British Columbia (Clarke 1981); records of Juga north of Washington State are dubious (Goodrich 1937). This suggests that the younger, ice-age land bridge would have been inhospitable.

The affinities of Hyriopsis were poorly resolved: close to Anodontini with low Bayesian probability and unresolved in the parsimony tree. Further sampling of Eurasian genera will be needed to resolve whether it is a basal amblemine, unionine, or neither. Graf (2002) also placed Gonidea as the sister taxon to eastern North American Ambleminae. Hyriopsis, Inversidens, and Psilunio were not included in his study; the Asian genera that he included placed outside of both Unioninae and Ambleminae. As he used a more slowly evolving gene, his results are probably more reliable at this taxonomic level; conversely, he observed that his 28S data did little to resolve relationships within the eastern North American taxa. Other Eurasian taxa that appear possibly assignable to Ambleminae, based on molecular evidence, include Lamprotula, Ptychorhynchus, and Solenaia (Huang et al. 2002). Inversidens and Psilunio had not previously been included in phylogenetic analyses incorporating multiple amblemines.

Many previous classifications have assigned Eurasian and African taxa to eastern North American tribes (or ambiguous tribes, in the case of “Unionini” that included Pleurobemini). Among the sampled taxa (standardizing the taxon names to the usage in this paper), Psilunio littoralis was assigned to Unionini (Simpson 1900), Quadrulini (Haas 1969), and Gonideini (Nagel et al. 1998); Hyriopsis cumingii to Lampsilini (Simpson 1900), Unionini (Haas 1969), Anodontini (Starobogatov 1970), and Pleurobemini (Heard & Guckert 1971); and Inversidens japanensis to Unionini (Haas 1969) and Quadrulini (Starobogatov 1970). Modell (1964) excluded non-North American genera from the North American amblemine tribes; however, Gonidea was placed in Margaritiferidae with some Asian unionids. Also, he did not unite the amblemine tribes as a monophyletic group, with Quadrulini assigned to Unioninae, and Alasmidontini (which Heard & Guckert 1971, and subsequent workers, synonymize with Anodontini) assigned to Ambleminae.

Graf (2002) suggested three reasons for the overuse of North American tribes. First, the current taxonomic framework for the unionids is based largely on the work of Simpson and Ortmann, who, working in North America, naturally relied heavily on the regional fauna in developing their classifications. In large part, worldwide unionids have been fit into classifications based primarily on North American taxa. Secondly, relatively few morphological characters are known for many taxa, and the characters used to group taxa are in many cases homoplaseous. Although there are many disagreements as to which characters are reliable (including molecular characters), the fact that different characters yield different classifications demonstrates that at least some of them are homoplaseous. Thirdly, traditional classifications have often used symplesiomorphies as well as synapomorphies to group taxa. The present results agree with Graf (2002) and Huang et al. (2002) in excluding non-North American taxa from Quadrulini, Amblemini, Pleurobemini, and Lampsilini, with each study sampling a slightly different set of taxa. However, many additional Old World genera have no published molecular data and little or no anatomical study.

Ambleminae

The general division of taxa among Amblemini, Lampsilini, Pleurobemini, and Quadrulini corresponds well to patterns found in Lydeard et al. (1996), Huang et al. (2002), Graf & Ó Foighil (2000a), Krebs et al. (2003), and Serb et al. (2003). Tribe assignments resulting from the present study are shown in Table 1. Relationships within the amblemine tribes were consistent between analyses, with Quadrulini basal, and Amblemini and Lampsilini as sister taxa. This contrasts with morphology-based phylogenies (Hannibal 1912; Heard & Guckert 1971) that suggest a close relationship between Pleurobemini and Lampsilini based on the shared character of ectobranchy. However, the present data indicate that ectobranchy probably arose at least four times independently from tetragenous ancestors, once in Unioninae, once in Pleurobemini (with a reversal to tetrageny in Fusconaia), once in Lampsilini, and once in Cyclonaias. Although the separate origins of ectobranchy in Unioninae and Ambleminae had been recognized in many previous studies (e.g., Davis & Fuller 1981; Graf 2002; Roe & Hoeh 2003), the extent of convergence within Ambleminae was not previously evident.

Amblemini

Only Amblema can confidently be assigned to Amblemini, although Popenaias placed in the same clade as Amblema in both analyses. The affinities of Amblema with other genera remain poorly supported, and the composition of Amblemini remains uncertain. Both analyses suggested that Amblemini was the sister taxon to Lampsilini, but this was not strongly supported by the bootstrap. Based on the present analyses, other taxa that may be related to Amblemini include “Fusconaiaebena, “Obovariarotulata, and Plectomerus. The relationships among these taxa, as well as their relationships with Lampsilini and Pleurobemini, varied between analyses. Previous molecular analyses likewise had difficulty placing these taxa, although no sequence data have previously been published for Popenaias and not all of the others were included in any one previous analysis. The present analyses placed all of these taxa, along with Lampsilini, in a large clade, sister to Pleurobemini. However, bootstrap analysis failed to support this large clade.

Although Elliptoideus placed basal to Pleurobemini, with 100% posterior probability, this did not receive significant bootstrap support. The association of Elliptoideus with Pleurobemini agrees with the results of Lydeard et al. (1996) and Krebs et al. (2003) rather than with Serb et al. (2003), who placed Elliptoideus and Plectomerus close together as possible amblemines. Morphologically, Elliptoideus and Plectomerus are similar (Modell 1964; Heard & Guckert 1971); however, they are also morphologically similar to the quadruline Megalonaias, which suggests that their similarities may be plesiomorphic or convergent. Plectomerus placed as a basal lampsiline in the Bayesian analyses, but the parsimony analysis placed it outside of Lampsilini. Plectomerus lacks the specialized gill structures characteristic of Lampsilini and is tetragenous rather than ectobranchous. The COI sequence obtained in this study is definitely lampsiline in its affinities; its 16S and ND1 sequences are not clearly lampsiline. Popenaias tends to group with Amblema (not always exclusive of other taxa), but this received weak bootstrap support.

Both analyses strongly supported a clade of “Obovariarotulata and “Fusconaiaebena, although the exact placement of this clade varied from analysis to analysis. The close relationship of “O.”rotulata and “F.”ebena agrees with previous molecular (Lydeard et al. 2000) and morphological (Stansbery 1971; Williams & Butler 1994; Athearn 1998) studies. Davis & Fuller (1981) noted a difference between “F.”ebena and true Fusconaia, but did not have “O.”rotulata. However, they expressed reservations about this difference, noting the possibility of experimental error. The present results suggest that their results were correct in this regard. “Obovariarotulata was assigned to Obovaria by Simpson (1900), based on examination of the type and only known specimen at the time, a shell with no soft parts (Lydeard et al. 2000). No anatomical data have been published for “O.”rotulata, but work in progress (Garner, pers. comm.) indicates that it is anatomically similar to “F.”ebena. Both lack the strong posterior ridge and angulation characteristic of true Fusconaia. Although no molecular data are available for Obovaria retusa, the nearly extinct, putative type species of Obovaria, anatomical studies indicate that it is lampsiline, similar to O. olivaria and O. subrotunda (Ortmann 1911). Placement of the ebena-rotulata clade varied among analyses, although none put it near other species of Fusconaia or of Obovaria.

The presence of Popenaias among the poorly resolved taxa suggests that sampling of Mexican and Central American unionids may be necessary to resolve the relationships of the Amblemini. Likewise, the primarily tropical Cyrtonaias appears near the base of Lampsilini. Unfortunately, no unionid specimens from Latin America were available for molecular work.

Among these genera, Elliptoideus and Plectomerus are monotypic. “Fusconaiaebena and “Obovariarotulata are clearly not closely related to F. flava (the type species of Fusconaia) nor to the other available Obovaria species. None of the other species of Popenaias, from Mexico, have been available for molecular studies. Amblema appears monophyletic, in agreement with the analysis of Mulvey et al. (1997).

Morphological studies had assigned taxa to the Amblemini based on the pattern of gill use in brooding (tetragenous in Amblemini versus ectobranchous in Pleurobemini and Lampsilini). However, this is evidently a plesiomorphic feature for the eastern North American Ambleminae. Many recent analyses also found this (Lydeard et al. 1996; Graf & Ó Foighil 2000a; Hoeh et al. 2001, 2002a; Roe & Hoeh 2003). In fact, the morphology-based analyses that defined Amblemini based on tetrageny frequently suggested that the tribe was paraphyletic (e.g., Hannibal 1912; Heard & Guckert 1971; see also the cladistic morphological analyses in Graf 2000; Hoeh et al. 2001; Roe & Hoeh 2003). Graf (2002) suggested that tetrageny may be a synapomorphy for the entire Unionoidea, and thus plesiomorphic for Ambleminae; Roe & Hoeh (2003) suggested that tetrageny was plesiomorphic either for Unionoidea or the entire Unionoida in different analyses. Reliance on this character placed Quadrulini and Fusconaia in Amblemini, contrary to the present results, which recognize Quadrulini as a distinct tribe and assign true Fusconaia to Pleurobemini. Thus, the traditional morphological character distinguishing Amblemini is not reliable for distinguishing tribes. Given the uncertainty about the included taxa, speculation on alternative morphological characters is premature.

Contrary to Davis & Fuller (1981), Amblema, Megalonaias, and Plectomerus do not appear congeneric. As in the case of Margaritiferidae versus Unionidae (Smith & Wall 1984), low immunological differences apparently concealed the fact that they are on separate branches and in separate tribes.

Lampsilini

The present analyses consistently support a monophyletic Lampsilini, although with low bootstrap support. Lampsilini clearly includes the following genera: Actinonaias, Cyprogenia, Dromus, Ellipsaria, Epioblasma, Lampsilis, Lemiox, Leptodea, Ligumia, Medionidus, Obovaria (except “Obovariarotulata), Potamilus, Ptychobranchus, Truncilla, Venustaconcha, and Villosa. Cyrtonaias, Glebula, Obliquaria, and Toxolasma belong to Lampsilini based on anatomical features. The present analyses grouped these four as basal in Lampsilini. However, their association with Lampsilini did not receive significant bootstrap support, and the Bayesian analysis also placed Plectomerus with them as a basal lampsiline. Similarly, Lydeard et al. (1996), Bogan & Hoeh (2000), Hoeh et al. (2002a,b), and Krebs et al. (2003) found at most relatively weak bootstrap support for associating these four genera with Lampsilini; some of their analyses failed to group these genera with Lampsilini at all. (Although high neighbor-joining bootstrap values were found in some of these analyses, the phylogenetic significance of neighbor-joining bootstrap values is doubtful [Swofford et al. 1996]). No previous study on DNA sequence data included all four of the genera. Lydeard et al. (1996) and Krebs et al. (2003) also supported a close relationship of Plectomerus to these taxa. Hoeh et al. (2002b) found stronger support for lampsiline affinities for Cyrtonaias when they concatenated the two analyzed gene sequences in a single analysis, suggesting that the poor resolution is mainly a function of the limited amount of data. All four of these genera have the highly modified gills and other specialized reproductive characters that characterize Lampsilini.

The variable position of Plectomerus may have prevented bootstrap analyses from finding strong support for any one clade involving these taxa. Although Plectomerus lacks the anatomical characteristics of Lampsilini, it might be the sister taxon to Lampsilini or an atavistic basal lampsiline. Analysis of individual genes indicates that the COI sequence obtained for Plectomerus (obtained from two different specimens) shows definite lampsiline affinities, whereas the 16S and ND1 sequences do not. Sequences for additional genes will clarify its affinities.

Within Lampsilini, both analyses supported several subclades. The phylogeny of Epioblasma and of the clade of superconglutinate-producing “Lampsilis” species (L. altilis, L. australis, L. perovalis, L. subangulata) matches the results of previous studies (Roe et al. 2001; Buhay et al. 2002). The many additional taxa in the present analysis provide much broader support for the monophyly of these two clades. However, the present analyses suggest new ideas about the relationships of Epioblasma, consistently supporting a close relationship with Obovaria and Venustaconcha pleasii. The inclusion of several additional taxa in the present study strengthens the conclusion of Roe et al. (2001) that the superconglutinate group is distinct from true Lampsilis. Although the parsimony tree placed the superconglutinate clade as sister to the clade including the type of Lampsilis, this was not supported by bootstrap or Bayesian analyses. Actinonaias pectorosa appears closely related to Lampsilis ovata (the type species), L. cardium, and L. ornata. Lemiox, Medionidus, Dromus, Cyprogenia, Ptychobranchus, and Venustaconcha ellipsiformis appeared closely related, although this group did not receive much bootstrap support. Lampsilis teres, Actinonaias ligamentina, and Lampsilis siliquoides likewise formed a group with low bootstrap support; Villosa vanuxemensis placed in this clade in the parsimony analysis. Several pairs of genera appeared closely related, including Cyprogenia and Dromus, Ellipsaria and Truncilla, Leptodea and Potamilus, Ptychobranchus and Venustaconcha ellipsiformis (the type), and Cyrtonaias and Glebula. In general, these groups of taxa have not previously been included in a single analysis, apart from Leptodea and Potamilus (Roe & Lydeard 1998).

High polyphyly or paraphyly of genera within the Lampsilini raises questions about the characters currently used to define genera. A few genera (notably Potamilus and Leptodea) have very distinctive glochidia. Most genera are defined based on anatomical features, such as the form of the gill marsupium, form of the female posterior mantle edge (often modified to attract potential host fish for the glochidia), and attachment of the gill to the body (Heard & Guckert 1971; Burch 1975; Smith 2000b). However, these characters have rarely been examined in a phylogenetic context to determine whether similarities are synapomorphic, homoplaseous, or symplesiomorphic. A few genera have highly distinctive gill form (Cyprogenia, Cyrtonaias, Dromus, Obliquaria, and Ptychobranchus, among the sampled taxa), but the rest have been grouped as “heterogenae” (Simpson 1900; Heard & Guckert 1971; Fuller 1975; Lampsilinae of Starobogatov 1970). Even among the unusual gill types, Cyprogenia and Obliquaria do not appear closely related, despite similar gill form. Within Heterogenae, morphological characters are often problematic (Davis & Fuller 1981). Thus, some of the reproductive features used in traditional classifications probably represent symplesiomorphies for suprageneric groups within Lampsilini or convergent adaptation of similar reproductive strategies. Especially in groups based on a single feature, traditional taxa require corroboration from other lines of evidence.

Among lampsiline genera, Dromus, Ellipsaria, Lemiox, Glebula, and Obliquaria are monotypic, and Cyrtonaias and Truncilla are represented here by a single species. A published COI sequence (Bogan & Hoeh 2000) for the type species of Toxolasma, T. lividus, closely resembles the sequences reported on here, but no data for other genes of that species were available. Cyprogenia, Leptodea, and Toxolasma appear monophyletic based on species represented in our analyses. Medionidus appeared polyphyletic, but both species placed within the same small clade, and neither bootstrap support nor Bayesian probability for the apparent polyphyly was high. Obovaria rotulata is clearly not related to the other sampled species of Obovaria. Its placement in Obovaria was based solely on shell characters, and the anatomy is not distinctively lampsiline. Relationships of Obovaria olivaria to the other species of the Obovaria–Epioblasma–Venustaconchapleasii clade were not consistently resolved between analyses, but its morphological distinctiveness (Ortmann 1919), in conjunction with the molecular results, suggest that further study is advisable.

Modest support for Obovaria being paraphyletic to Epioblasma and “Venustaconchapleasii makes data for the type species of the genera particularly of interest, if it can be obtained. The RFLP data of White et al. (1996) suggest that E. rangiana, the type of Epioblasma, is genetically similar to species of Epioblasma represented in the present study. However, those data do not provide much resolution of its relationships. Although the currently included species of Potamilus form a monophyletic group, Roe & Lydeard (1998) found that Potamilus capax places outside of Potamilus. Only COI data were available for P. capax, so it was not included in the present analyses. Actinonaias, Lampsilis, Ligumia, Venustaconcha, and Villosa as presently used include taxa that are only distantly related. This finding of polyphyly agrees with Graf & Ó Foighil (2000a) for Lampsilis and Ligumia, Lydeard et al. (2000) for Obovaria, Roe (2000) for Lampsilis and Villosa, and Roe et al. (2001) and Krebs et al. (2003) for Lampsilis; an extensive survey of Villosa (Buhay, unpubl. data) confirms the polyphyly of this genus.

Pleurobemini

Pleurobemini includes Elliptio, Fusconaia flava (the type of the genus), and most other species currently assigned to Fusconaia, Hemistena, Lexingtonia, Plethobasus, Pleurobema, and Quincuncina burkei (the type of the genus) but not other sampled Quincuncina species. Elliptoideus placed as the sister taxon to Pleurobemini in the present analyses; however, this differs from anatomical classifications (Heard & Guckert 1971) and from the results of Serb et al. (2003). Quincuncina burkei appears assignable to Fusconaia, in agreement with previous molecular studies (Lydeard et al. 2000; Serb et al. 2003). Although the published 16S sequence for Uniomerus carolinianus (Lydeard et al. 1996) places with Pleurobemini, the present sequences for Uniomerus declivis place with Quadrulini. Uniomerus is taxonomically problematic, with some individuals nearly indistinguishable from Elliptio spp. (Davis 1983). Thus, the discrepant results for Uniomerus may reflect misidentification or polyphyly. The immunological analyses of Davis & Fuller (1981) and Davis (1983) suggested affinities of Uniomerus to Pleurobemini and Quadrulini.

The present composition of Pleurobemini thus agrees more closely with Davis & Fuller (1981) than with Heard & Guckert (1971), by including Fusconaia and excluding Cyclonaias; however, the present placement of Uniomerus is unexpected. Few previous studies have included more than a few taxa for Pleurobemini. Most of the species included herein have no previously published sequence data. Relationships between the genera varied among the analyses and were largely unresolved, although the genus-level clades and many groups within the genera were well-supported. The analyses strongly supported a clade of “Fusconaiabarnesiana, “Lexingtoniadollabelloides, and “Pleurobemagibberum, not corresponding to any currently recognized genus.

Among the genera as currently recognized, Hemistena is monotypic, and only one species of Lexingtonia and Plethobasus was available. Uniomerus appears polyphyletic, but this is problematic in light of the taxonomic uncertainties discussed above. It may also have biochemical peculiarities, as extractions from multiple specimens persistently failed to amplify. The remaining genera in Pleurobemini are polyphyletic. However, the majority of Fusconaia and Pleurobema species each form a clade that includes the type species of the respective genus. Both Fusconaia and Quincuncina, as presently used, include species that belong in Quadrulini. This agrees with other recent molecular studies (Lydeard et al. 2000; Serb et al. 2003).

Quadrulini

Although long recognized on the basis of shell morphology, the tribe Quadrulini has been synonymized with Amblemini in most recent morphological classifications (Heard & Guckert 1971; Burch 1975) and the immunological analysis of Davis & Fuller (1981). Molecular sequencing studies, however, have supported separating it from Amblemini (Lydeard et al. 1996; Bogan & Hoeh 2000; Hoeh et al. 2001; Krebs et al. 2003; Roe & Hoeh 2003; Serb et al. 2003; see also discussion in Graf 2002). The present analyses placed Quadrulini basal within Ambleminae s.s.

Quadrulini includes Cyclonaias, “Fusconaiasuccissa, Megalonaias, Quadrula, “Quincuncinainfucata, “Quincuncinakleiniana, Tritogonia, and Uniomerus. Megalonaias and Uniomerus were the basal members of Quadrulini in all analyses. Quadrula appears paraphyletic to the other taxa in Quadrulini, except Megalonaias and Uniomerus. Cyclonaias, “F.”succissa, and the “Quincuncina” species appear closely related, placing with Quadrula kieneriana in the pustulosa group of Serb et al. (2003). Within Quadrula, Q. metanevra is basal to the remaining species.

These results agree closely with those of Serb et al. (2003), except in the placement of Cyclonaias and the prior lack of data for Uniomerus. Placing Cyclonaias in Quadrulini agrees with some morphological studies (e.g., Frierson 1927; Modell 1964) and with the results of Davis & Fuller (1981), although Heard & Guckert (1971) assigned it to Pleurobemini based on its ectobranchy. Cyclonaias tuberculata includes two distinctive morphologies (Parmalee & Bogan 1998); further investigation of this species is needed. Lydeard et al. (2000) also grouped “Fusconaiasuccissa, “Quincuncinainfucata, and “Quincuncinakleiniana with Quadrulini, and Davis & Fuller (1981) and Davis (1983) placed “Q.”infucata with Quadrulini.

Genus-level polyphyly

Most genera that currently include multiple species appear to be polyphyletic. This rampant polyphyly indicates that genera, as currently recognized, are a poor guide to phylogenetic affinity. Of the 37 currently recognized genera of North American Ambleminae (counting Gonidea), 12 are monotypic. The present study and previous molecular work (Roe & Lydeard 1998; Lydeard et al. 2000; Graf & Ó Foighil 2000a; Roe et al. 2001; Krebs et al. 2003; Serb et al. 2003) support paraphyly or polyphyly for 12 of the remaining genera. These include the highly diverse genera Elliptio, Lampsilis, Pleurobema, Quadrula, and Villosa, as well as Actinonaias, Fusconaia, Ligumia, Obovaria, Potamilus, Quincuncina, and Venustaconcha. Medionidus appeared polyphyletic, but with weakly supported relationships. Amblema, Cyprogenia, Epioblasma, Leptodea, and Toxolasma appeared monophyletic in the present analyses, but not all the species were included. Detailed analysis of Cyprogenia is in preparation (Serb, unpubl. data). Seven of the remaining genera have not yet received detailed phylogenetic study to test their monophyly. All were represented by a single sequence in the present analyses: Cyrtonaias, Lexingtonia, Plethobasus, Popenaias, Ptychobranchus, Truncilla, and Uniomerus. The problem of polyphyletic genera is not confined to Ambleminae as, within the outgroup, Lasmigona appears polyphyletic. This result agrees with King et al. (1999), who examined other Lasmigona species.

Thus, although the tribes Pleurobemini, Quadrulini, and Lampsilini appear to be supported by molecular data, the genera of Ambleminae appear overwhelmingly polyphyletic. A few species, such as “Fusconaiasuccissa and “F.”ebena, are presently assigned to the wrong tribe. Reclassifying the species will require detailed studies on species groups, including intraspecific sampling to examine phylogeographic patterns, especially in putatively widespread, variable species. These analyses will also require a wide range of outgroups to ensure that misassigned taxa are recognized.

Problematic taxa

Some taxa show relatively poor resolution of relationships, especially many lampsilines. Most of these are taxa with limited data; in particular, many lack ND1 data. However, other taxa seem problematic despite relatively complete data. Several possible reasons exist. First, various mistakes are possible, including misidentification and lab errors. For example, Krebs et al. (2003) noted that the 16S sequence for Quadrula quadrula from Lydeard et al. (1996) appeared very different from their newly generated sequence. Analysis of ND1 data for the specimen sequenced by Lydeard et al. suggested that it was in fact Q. nobilis (Serb et al. 2003). Although the two are genetically distinctive, they are morphologically so similar that few studies have recognized them as distinct (Howells et al. 1996). The present analyses using data for COI, 16S, and ND1 support this re-identification. The specimen is morphologically intermediate between standard Q. nobilis and Q. quadrula, and is from just outside the previously reported range of Q. nobilis (Howells, pers. comm.), so the molecular data provided critical evidence on the species-level affinity of the specimen. By greatly increasing the number of taxa with published sequences, the present study facilitates molecular identification in future studies.

However, it is also possible that an accurate DNA sequence may give anomalous phylogenetic results. Hybridization may produce disparate evolutionary patterns for individual genes versus for the organism as a whole (Makarenkov et al. 2004). Gene duplication and subsequent independent evolution may result in multiple similar sequences being present in a single individual. PCR may preferentially amplify one of these, producing misleading results due to non-orthology of the sampled genes (e.g., Williams & Knowlton 2001). A specialized case of non-orthologous genes results from the doubly uniparental inheritance pattern for bivalve mitochondria (Hoeh et al. 1996). The doubly uniparental inheritance pattern appears to be followed very strictly in unionoids (Curole & Kocher 2002; Hoeh et al. 2002b), although cases of mixing or switching are known in other bivalves (Hoeh et al. 1996). Because the male and female mitotypes have remained independent in unionids, the sequences are very different (Curole & Kocher 2002; Hoeh et al. 2002b). Comparison of the present sequences with published male and female unionoid sequences confirmed that all of the present sequences group with the female sequences, as expected for DNA extracted from somatic tissue. Nevertheless, other paralogous sequences might still cause problems, such as a pseudogene derived from the female mitochondrial sequence.

Gene rearrangements may also explain the failure of some species to amplify for ND1. The forward primer used in the present study is complementary to the tRNA immediately upstream of the ND1 gene itself. Although the two published unionid female mitochondrial genomes have only one major rearrangement, bivalves vary extensively in mitochondrial gene order (Serb & Lydeard 2003). The failure of some DNA extractions (such as Uniomerus declivis) to amplify for ND1, despite successful amplification using internal primers for the other genes, might reflect a different arrangement of genes from that assumed in the choice of primers. Similarly, tRNA gene remolding (Rawlings et al. 2003) could produce a mismatch with the primer.

A few species appeared paraphyletic or polyphyletic, but with all sequences placing within a single genus or small clade (e.g., Lampsilis altilis and Pleurobema rubrum). Resolution of such problems will require further sampling of closely related species and different populations, within what is presently considered a single species. However, four species (all from monotypic genera) appeared in very different relationships based on different DNA sequences. The present study obtained identical COI sequences for two Plectomerus dombeyanus specimens. The newly generated 16S and ND1 sequences for P. dombeyanus are similar to published sequences. However, the COI sequences show strong affinity for basal lampsilines, whereas the 16S and ND1 sequences appear outside of Lampsilini when analyzed individually. Similarly, published sequences for Obliquaria reflexa yield different affinities, with the COI data suggesting quadruline, and 16S and ND1 suggesting basal lampsiline, affinities. The new COI and 16S sequences for O. reflexa differ from published sequences; the new ND1 sequence closely resembles the ND1 sequence of Serb et al. (2003). All of the new O. reflexa sequences support assignment to Lampsilini. Two O. reflexa specimens, separately sequenced, yielded the same COI sequence (Roe & Lydeard 1998; Roe et al. 2001). The use of a slightly modified primer in the present analysis may have promoted amplification of a different copy of the gene. No evidence currently available indicates which, if either, is the functional copy. Multiple independent sequences for Plectomerus and Obliquaria suggest that these disparate COI genes represent some sort of gene duplication, rather than precisely replicated lab errors. Data for additional genes or other parts of COI (to test whether the presently known partial sequences form part of a functional copy) may help identify which sequences are homologous to the amplified sequences of other amblemines. Nuclear versions of mitochondrial genes are not uncommon and may show only slight differentiation from the mitochondrial paralogue (Zhang & Hewitt 1996).

The present Elliptoideus sloatianus sequences agree with the 16S sequence of Lydeard et al. (1996), but differ from the ND1 sequence of Serb et al. (2003) in grouping with Pleurobemini rather than with Plectomerus. The new data for Cyclonaias tuberculata place it in the pustulosa group of Quadrula; the sequence of Serb et al. (2003) appears lampsiline, similar to Potamilus. The new sequences for Elliptoideus and the published sequences for Elliptoideus and Cyclonaias from Serb et al. (2003) are derived from tissue clips obtained from other labs, adding more opportunities for error. All these sequences reflect single specimens, so error or non-homologous sequences are both plausible possibilities. Again, additional data for these taxa will better constrain their affinities.

Summary

The polyphyly of most currently recognized genera has important implications for all aspects of unionid research. Studies that assumed that current genera are biologically coherent units appear to have mixed apple snails and orangefoot pimplebacks. Such assumptions underlie most biological studies on unionids. Omission of key ingroups or misidentification of ingroups as outgroups may mislead phylogenetic analyses. Current genus-level taxonomy in Ambleminae conceals the relationships among species, so that taxa of interest for a particular study may lurk under multiple genera whereas putative close relatives may have little in common. Thus, a phylogenetic analysis of a genus as currently used may be nearly meaningless unless a large selection of outgroups is included. Likewise, biogeographic analyses that treat the current genera as natural groups rest on foundations that are at best misleading and at worst entirely erroneous. Exclusion of Eurasian taxa from the eastern North American tribes provides greater geographic consistency in the distribution of the tribes and new impetus to systematic work on Eurasian genera.

The improved phylogenetic understanding of the Ambleminae generated by this study will also improve our understanding of other aspects of their biology. Again, a major result is caution about interpreting the current generic assignments as necessarily indicative of close similarity. This is a particular problem because information about many taxa has been inferred from what is known about putative close relatives, due to the rarity of many species and the patchiness of our knowledge about unionid biology. However, if the presumed relationships are wrong, these inferences are also likely to be incorrect. Effective conservation requires protection of all aspects of the habitat that are needed at any stage of the mussel life cycle. As fish host preference may vary between populations of a single species (Haag et al., pers. comm.), inferred fish hosts based on incorrect assumptions about generic affinity will probably be incorrect. Likewise, extrapolation of ecological preferences, physiological tolerances, or breeding patterns will be more reliable in the context of a well-supported phylogenetic framework.

We hope that these results will inspire and direct further research on this diverse, imperiled clade of invertebrates, especially on taxa that remain poorly resolved.

Note added in proofs

New data indicate that specimen UAUC 136 was mislabeled, and is Obovaria olivaria rather than Venustaconcha pleasii. In addition, a paper just published proposes a new genus for some of the included taxa; Hamiota (Roe & Hartfield 2005) includes the superconglutinate-forming “Lampsilis” species.

Roe KJ & Hartfield PD 2005. Hamiota, a new genus of fresh-water mussel (Bivalvia: Unionidae) from the Gulf of Mexico drainages of the southeastern United States. Nautilus 119(1): 1–10.

Acknowledgments

Acknowledgments. In addition to the authors, C. R. Dean, Q. Parham, K. K. Small, J. Moseley, and A. Wethington helped with lab work; K. K. Small and Q. Parham were supported as Hughes Undergraduate Research Interns by a Howard Hughes Medical Institute Undergraduate Biological Sciences Education Program grant to The University of Alabama. Many people provided specimens; they are acknowledged in Appendix 2. These include tissue samples from Leetown Science Center and Auburn University collections. The ABI 3100 automated sequencer was funded by an NSF equipment grant to C. Lydeard, R. Mayden, M. Powell, and P. Harris (DBI-0070351). A grant from the U.S. Fish and Wildlife Service to C. Lydeard supported this work. D. Graf and two anonymous reviewers provided helpful feedback. This manuscript was completed while C. Lydeard served as a Program Officer at the National Science Foundation under the Intergovernmental Personnel Agreement Act and was supported in part by the IR/D program. The version of TNT used is registered to Frank Pezold of the University of Louisiana-Monroe Museum of Natural History.

Appendix

Appendices

Appendix 1. Taxa analyzed and GenBank accession numbers

SpeciesAccession no.Reference
COI
Actinonaias ligamentina (Lamarck 1819)AF231730Bogan & Hoeh (2000)
Actinonaias pectorosa (Conrad 1834)AY654990UAUC880
Amblema elliottii (Lea 1856)AY654991UAUC2511
Amblema plicata 1T (Say 1817)U56841Hoeh et al. (1998)
Amblema plicata 2TAF156512Graf & Ó Foighil (2000a)
Anodonta cygneaT (Linnaeus 1758)U56842Hoeh et al. (1998)
Anodonta oregonensis (Lea 1838)AY493480Mock et al. (2004)
Cumberlandia monodonta 1T (Say 1829)AF156497Graf & Ó Foighil (2000a)
Cumberlandia monodonta 2AF156498Graf & Ó Foighil (2000a)
Cyprogenia stegariaT (Rafinesque 1820)AY654992UAUC1499
Cyrtonaias tampicoensis (Lea 1838)AF231749Bogan & Hoeh (2000)
Dromus dromasT (Lea 1834)AY654993UAUC3156
Ellipsaria lineolataT (Rafinesque 1820)AY654994UAUC450
Elliptio arca (Conrad 1834)AY654995UAUC498
Elliptio crassidensT (lamarck 1819)AY613820UAUC1493
Elliptio dilatata 1 (Rafinesque 1820)AF231751Bogan & Hoeh (2000)
Elliptio dilatata 2AF156506Graf & Ó Foighil (2000a)
Elliptoideus sloatianusT (Lea 1840)AY613822Specimen Es
Epioblasma brevidens (Lea 1831)AF156527Graf & Ó Foighil (2000a)
Epioblasma capsaeformis (Lea 1834)AY654996UAUC1527 (inc. AY094372 Buhay et al. 2002)
Epioblasma florentina walkeri 1 (Wilson & Clark 1914)AY094373Buhay et al. (2002)
Epioblasma florentina walkeri 2AY094374Buhay et al. (2002)
Epioblasma triquetra (Rafinesque 1820)AF156528Graf & Ó Foighil (2000a)
Fusconaia barnesiana (Lea 1838)AY613822UAUC1553
Fusconaia cerina 1 (Conrad 1838)AY613823UAUC3233
Fusconaia cerina 2AF049522Roe & Lydeard (1998)
Fusconaia cor (Conrad 1834)AY654997UAUC2606
Fusconaia cuneolus (Lea 1840)AY654998UAUC1552
Fusconaia ebena (Lea 1831)AY654999UAUC71 (inc. AF232815 Lydeard et al. 2000)
Fusconaia escambia 1 (Clench & Turner 1956)AF232816Lydeard et al. (2000)
Fusconaia escambia 2AF232817Lydeard et al. (2000)
Fusconaia flava 1T (Rafinesque 1820)AF231733Bogan and Hoeh (2000)
Fusconaia flava 2TAF232822Lydeard et al. (2000)
Fusconaia subrotunda (Lea 1831)AY613824UAUC1554
Fusconaia succissa 1 (Lea 1852)AF232819Lydeard et al. (2000)
Fusconaia succissa 2AF232820Lydeard et al. (2000)
Glebula rotundataT (Lamarck 1819)AF231729Bogan and Hoeh (2000)
Gonidea angulataT (Lea 1838)AF231755Bogan and Hoeh (2000)
Hemistena lataT (Rafinesque 1820)AY613825UAUC2797
Hyriopsis cumingii (Lea 1852)AY655000UAUC3160
Inversidens japanensis (Lea 1859)AB055625Okazaki & Ueshima, unpubl. data
Lampsilis altilis 1 (Conrad 1834)AF385105Roe et al. (2001)
Lampsilis altilis 2AF385108Roe et al. (2001)
Lampsilis altilis 3AF385092Roe et al. (2001)
Lampsilis australis 1 Simpson (1900)AF385101Roe et al. (2001)
Lampsilis australis 2AF385098Roe et al. (2001)
Lampsilis australis 3AF385099Roe et al. (2001)
Lampsilis cardium (Rafinesque 1820)AF120653Giribet & Wheeler (2002)
Lampsilis ornata 2AF049520Roe & Lydeard (1998)
Lampsilis ornata 1 (Conrad 1835)AY365193Serb & Lydeard (2003)
Lampsilis ovataT (Say 1817)AY613826UAUC108 (inc. AF385111 Roe et al. 2001)
Lampsilis perovalis 1 (Conrad 1834)AF385094Roe et al. (2001)
Lampsilis perovalis 2AF385096Roe et al. (2001)
Lampsilis siliquoidea 1 (Barnes 1823)AF156521Graf & Ó Foighil (2000a)
Lampsilis siliquoidea 2AF156522Graf & Ó Foighil (2000a)
Lampsilis subangulata 1 (Lea 1840)AF385104Roe et al. (2001)
Lampsilis subangulata 2AF385102Roe et al. (2001)
Lampsilis teres 1 (Rafinesque 1820)AF385113Roe et al. (2001)
Lampsilis teres 2AF406803Hoeh et al. (2002b)
Lasmigona costataT (Rafinesque 1820)AF093848King et al. (1999)
Lasmigona holstonia etowahensis (Lea 1858)AY655001UAUC3159
Lemiox rimosusT (Rafinesque 1831)AY655002UAUC1528
Leptodea fragilis 1 (Rafinesque 1820)AF049518Roe & Lydeard (1998)
Leptodea fragilis 2AF049519Roe & Lydeard (1998)
Leptodea leptodonT (Rafinesque 1820)AY655003UAUC135
Lexingtonia dolabelloides 1 (Lea 1840)AY655004UAUC1488
Lexingtonia dolabelloides 2AY613827UAUC2819
Ligumia nasuta (Say 1817)AF156515Graf & Ó Foighil (2000a)
Ligumia rectaT (Lamarck 1819)AF385110Roe et al. (2001)
Margaritifera margaritiferaT (Linnaeus 1758)U56847Hoeh et al. (1998)
Medionidus accutissimus (Lea 1831)AY655005UAUC82
Medionidus conradicusT (Lea 1834)AY655006UAUC10
Megalonaias nervosaT (Rafinesque 1820)AY655007UAUC266
Obliquaria reflexaT (Rafinesque 1820)AY655008UAUC2508
Obovaria jacksoniana (Frierson 1912)AY655009UAUC680
Obovaria olivaria (Rafinesque 1820)AF232812Lydeard et al. (2000)
Obovaria rotulata 1 (Wright 1899)AF232814Lydeard et al. (2000)
Obovaria rotulata 2AF232813Lydeard et al. (2000)
Obovaria subrotunda (Rafinesque 1820)AY655010UAUC2838
Obovaria unicolor (Lea 1845)AF232811Lydeard et al. (2000)
Plectomerus dombeyanusT (Valenciennes 1827)AY655011UAUC2536
Plethobasus cyphusT (Rafinesque 1820)AY613828UAUC1639
Pleurobema chattanoogaense 1 (Lea 1858)AY655012UAUC1621
Pleurobema chattanoogaense 2AY613829UAUC3194
Pleurobema clavaT (Lamarck 1819)AY655013UAUC1477
Pleurobema collina (Conrad 1837)AY613830UAUC1074
Pleurobema cordatum (Rafinesque 1820)AY613831UAUC2572
Pleurobema decisum 1 (Lea 1831)AF232801Lydeard et al. (2000)
Pleurobema decisum 2AY655014UAUC253
Pleurobema decisum 3AY613832UAUC3196
Pleurobema furvum (Conrad 1834)AY613833UAUC678
Pleurobema georgianum 2 (Lea 1841)AY613834UAUC3193
Pleurobema georgianum 3AY655015UAUC3084
Pleurobema gibberum (Lea 1838)AY613835UAUC3319
Pleurobema hanleyianum 1 (Lea 1852)AY655016UAUC273
Pleurobema hanleyianum 2AY613836UAUC1622
Pleurobema oviforme 1 (Conrad 1834)AY655017UAUC1402
Pleurobema oviforme 2AY613837UAUC1642
Pleurobema perovatum (Conrad 1834)AY613838UAUC1640
Pleurobema pyriforme (Lea 1857)AY613839A29
Pleurobema rubellum (Conrad 1834)AY613840UAUC679
Pleurobema rubrum 1 (Rafinesque 1820)AY655018UAUC2719
Pleurobema rubrum 2AY613841UAUC3229
Pleurobema sintoxia (Rafinesque 1820)AY655019UAUC1714
Pleurobema strodeanum (Wright 1898)AY613843UAUC1110
Pleurobema taitianum (Lea 1834)AY613844UAUC885
Pleurobema troschelianum (Lea 1852)AY613845UAUC516
Popenaias popeiiT (Lea 1857)AY655020UAUC3161
Potamilus alatusT 1 (Say 1817)AF049510Roe & Lydeard (1998)
Potamilus alatusT 2AF049511Roe & Lydeard (1998)
Potamilus purpuratus (Lamarck 1819)AF049507Roe & Lydeard (1998)
Psilunio littoralisT 1 (Cuvier 1798)AF303348Machordom et al. (2003)
Psilunio littoralisT 2AF303349Machordom et al. (2003)
Ptychobranchus fasciolarisT (Rafinesque 1820)AF156514Graf & Ó Foighil (2000a)
Pyganodon grandis (Say 1829)AF156504Graf & Ó Foighil (2000a)
Quadrula nobilis (Conrad 1854)AF232823Lydeard et al. (2000)
Quadrula quadrulaT 1 (Rafinesque 1820)AF231757Bogan & Hoeh (2000)
Quadrula quadrulaT 2AF156511Graf & Ó Foighil (2000a)
Quincuncina burkeiT 1 (Walker 1922)AF232804Lydeard et al. (2000)
Quincuncina burkeiT 2AF232803Lydeard et al. (2000)
Quincuncina burkeiT 3AF232802Lydeard et al. (2000)
Quincuncina infucata 1 (Conrad 1834)AF232807Lydeard et al. (2000)
Quincuncina infucata 2AF232806Lydeard et al. (2000)
Quincuncina infucata 3AF232805Lydeard et al. (2000)
Quincuncina kleiniana 1 (Lea 1852)AF232808Lydeard et al. (2000)
Quincuncina kleiniana 2AF232809Lydeard et al. (2000)
Strophitus subvexus (Conrad 1834)AY655021UAUC2715
Toxolasma parvus (Barnes 1823)AY655022UAUC3331
Toxolasma texasiensis (Lea 1857)AY655023UAUC80
Tritogonia verrucosaT (Rafinesque 1820)AY655024UAUC3195
Truncilla truncataT (Rafinesque 1820)AF156513Graf & Ó Foighil (2000a)
Uniomerus declivus (Say 1831)AY613846UAUC3290
Venustaconcha ellipsiformisT (Conrad 1836)AY655025UAUC2596-2598
Venustaconcha pleasii (Marsh 1891)AY655026UAUC136
Villosa iris (Lea 1829)AF156524Graf & Ó Foighil (2000a)
Villosa vanuxemensis (Lea 1838)AF156526Graf & Ó Foighil (2000a)
Villosa villosaT (Wright 1898)AF385109Roe et al. (2001)
 16S 
Actinonaias ligamentinaAY655027UAUC241
Actinonaias pectorosaAY655028UAUC880
Amblema elliottiiAY655029Mulvey et al. (1997)
Amblema plicataT 1U72547Lydeard et al. (1996)
Amblema plicataT 2U72548Lydeard et al. (1996)
Anodonta cygneaTAF232799Lydeard et al. (2000)
Cumberlandia monodontaT 1AF232800Lydeard et al. (2000)
Cumberlandia monodontaT 2U72546Lydeard et al. (1996)
Cyclonaias tuberculataT (Rafinesque 1820)AY655030UAUC3158
Cyprogenia aberti (Conrad 1850)AY655031UAUC75
Cyrtonaias tampicoensisAY655032UAUC78
Dromus dromasTAY655033UAUC3156
Ellipsaria lineolataTU72567Lydeard et al. (1996)
Elliptio crassidensTAY655034UAUC3150
Elliptio dilatata 1U72557Lydeard et al. (1996)
Elliptoideus sloatianusTAY655035Specimen Es
Epioblasma brevidensAY655036UAUC509
Epioblasma capsaeformisAY655037UAUC1527
Fusconaia barnesianaAY655038UAUC1553
Fusconaia cerina 1AY655039UAUC073
Fusconaia corAY655040UAUC2606
Fusconaia ebenaAF232790Lydeard et al. (2000)
Fusconaia escambia 1AF232791Lydeard et al. (2000)
Fusconaia escambia 2AY655041UAUC1449 (inc. AF232792 Lydeard et al. 2000)
Fusconaia flavaT 1AY238481Krebs et al. (2003)
Fusconaia flavaT 2AY655042UAUC146
Fusconaia subrotundaAY655043UAUC1554
Fusconaia succissa 1AF232794Lydeard et al. (2000)
Fusconaia succissa 2AF232795Lydeard et al. (2000)
Glebula rotundataTAY655044UAUC502
Gonidea angulataTAY655045UAUC3147
Hemistena lataTAY655046UAUC2797
Hyriopsis cumingiiAY655047UAUC3160
Inversidens japanensisAB055625Okazaki & Ueshima, unpubl. data
Lampsilis altilis 1AF385129Roe et al. (2001)
Lampsilis altilis 2AF385132Roe et al. (2001)
Lampsilis altilis 3AF385116Roe et al. (2001)
Lampsilis australis 1AF385125Roe et al. (2001)
Lampsilis australis 2AF385122Roe et al. (2001)
Lampsilis australis 3AF385123Roe et al. (2001)
Lampsilis cardiumAF191574Turner et al. (2000)
Lampsilis ornata 2AF385136Roe et al. (2001)
Lampsilis ornata 1AY365193Serb & Lydeard (2003)
Lampsilis ovataTAY655048UAUC108
Lampsilis perovalis 1AF385118Roe et al. (2001)
Lampsilis perovalis 2AF385120Roe et al. (2001)
Lampsilis siliquoidea 1AF191571Turner et al. (2000)
Lampsilis siliquoidea 2U72571Lydeard et al. (1996)
Lampsilis subangulata 1AF385128Roe et al. (2001)
Lampsilis subangulata 2AF385126Roe et al. (2001)
Lampsilis teres 1AF232785Lydeard et al. (2000)
Lampsilis teres 2U72568Lydeard et al. (1996)
Lasmigona costataTAY238488Krebs et al. (2003)
Lemiox rimosusTAY655049Unnumbered specimen
Leptodea fragilis 1U72570Lydeard et al. (1996)
Leptodea fragilis 2AY238483Krebs et al. (2003)
Leptodea leptodonTAY655050UAUC135
Lexingtonia dolabelloides 1AY655051UAUC3148
Ligumia nasutaAY655052No specimen data
Ligumia rectaTAY655053UAUC89
Margaritifera margaritiferaTAF303297Machordom et al. (2003)
Medionidus accutissimusAY655054UAUC82
Medionidus conradicusTU72572Lydeard et al. (1996)
Megalonaias nervosaTU72555Lydeard et al. (1996)
Obliquaria reflexaTAY655055UAUC2508
Obovaria olivariaAF232787Lydeard et al. (2000)
Obovaria rotulata 1AF232788Lydeard et al. (2000)
Obovaria rotulata 2AF232789Lydeard et al. (2000)
Obovaria subrotundaAY655056UAUC2838
Obovaria unicolorAF232786Lydeard et al. (2000)
Plectomerus dombeyanusTAY655057UAUC2536
Plethobasus cyphusTAY655058UAUC3157
Pleurobema chattanoogaense 1AY655059UAUC1621
Pleurobema clavaTAY655060UAUC1477
Pleurobema collinaAY655061UAUC1074
Pleurobema decisum 1AF232776Lydeard et al. (2000)
Pleurobema georgianum 1AY655062UAUC1623
Pleurobema georgianum 2AY655063UAUC3193
Pleurobema gibberumAY655064UAUC3153
Pleurobema hanleyianum 1AY655065UAUC273
Pleurobema hanleyianum 2AY655066UAUC1622
Pleurobema oviforme 1AY655067UAUC3238
Pleurobema oviforme 2AY655068UAUC1642
Pleurobema rubellumAY655069UAUC679
Pleurobema strodeanumAY655070UAUC1818
Pleurobema taitianumAY655071UAUC885
Pleurobema troschelianumAY655072UAUC516
Popenaias popeiiTAY655073UAUC3161
Potamilus alatusT 1AY655074UAUC41
Potamilus alatusT 2AY238484Krebs et al. (2003)
Potamilus purpuratusU72573Lydeard et al. (1996)
Psilunio littoralisT 1AF3033078Machordom et al. (2003)
Psilunio littoralisT 2AF3033078Machordom et al. (2003)
Ptychobranchus fasciolarisTAY655075LSC23701-001
Pyganodon grandisAY238490Krebs et al. (2003)
Quadrula apiculata (Say 1829)U72554Lydeard et al. (1996)
Quadrula kieneriana (Lea 1852)AY655076UAUC334
Quadrula metanevra (Rafinesque 1820)U72551Lydeard et al. (1996)
Quadrula nobilisAF232798Lydeard et al. (2000)
Quadrula quadrulaT 1AY238485Krebs et al. (2003)
Quadrula quadrulaT 2U72552Lydeard et al. (1996)
Quincuncina burkeiT 1AF2327779Lydeard et al. (2000)
Quincuncina burkeiT 2AF2327779Lydeard et al. (2000)
Quincuncina burkeiT 3AF2327779Lydeard et al. (2000)
Quincuncina infucata 1AF232782Lydeard et al. (2000)
Quincuncina infucata 2AF232781Lydeard et al. (2000)
Quincuncina infucata 3AF232780Lydeard et al. (2000)
Quincuncina kleiniana 1AF232783Lydeard et al. (2000)
Quincuncina kleiniana 2AF232784Lydeard et al. (2000)
Strophitus subvexusAY655077UAUC2715
Toxolasma parvusAY238482Krebs et al. (2003)
Toxolasma texasiensisAY655078UAUC80
Tritogonia verrucosaTAY655079UAUC3195
Truncilla truncataTAY655080Unnumbered specimen
Uniomerus declivusAY655081UAUC3290
Venustaconcha ellipsiformisTAY655082UAUC2596-2598
Villosa irisAY655083UAUC260
Villosa vanuxemensisAY655084UAUC3046
Villosa villosaTAF385133Roe et al. (2001)
 ND1 
  1. T indicates the type of a genus. If more than one sequence is listed, this indicates multiple identical sequences. For new sequences, the collection number is listed; more details about the new material are in Appendix 2. With new sequences, “inc.” indicates that the new sequence is a longer version of a published sequence.UAUC, University of Alabama Unionid Collection; LSC, Leetown Science Center.

Actinonaias ligamentinaAY655085UAUC241
Amblema elliottiiAY655086UAUC2511
Amblema plicataT 1AY158796Serb et al. (2003)
Anodonta oregonensisAY655087UAUC3169
Cyclonaias tuberculataTAY655088UAUC3158
Cyprogenia abertiAY158749Serb et al. (2003)
Cyprogenia stegariaTAY655089UAUC1499
Cyrtonaias tampicoensisAY655090UAUC314
Dromus dromasTAY655091UAUC3156
Ellipsaria lineolataTAY655092UAUC450
Elliptio arcaAY655093UAUC501
Elliptio crassidensTAY613788UAUC3150
Elliptio dilatata 1AY613789UAUC2735
Elliptio dilatata 2AY655094UAUC2721
Elliptoideus sloatianusTAY613790Specimen Es
Epioblasma brevidensAY094378=AY094377Buhay et al. (2002)
Epioblasma capsaeformisAY094381=AY094379Buhay et al. (2002)
Epioblasma florentina walkeri 1AY094383Buhay et al. (2002)
Epioblasma florentina walkeri 2AY094384Buhay et al. (2002)
Epioblasma triquetraAY094375Buhay et al. (2002)
Fusconaia barnesianaAY613791UAUC1553
Fusconaia cerina 1AY655095UAUC073
Fusconaia cerina 2AY613792UAUC3234
Fusconaia corAY655096UAUC2606
Fusconaia cuneolusAY655097UAUC1552
Fusconaia ebenaAY655098UAUC71
Fusconaia flavaT 1AY613793UAUC2864
Fusconaia flavaT 2AY158781Serb et al. (2003)
Fusconaia subrotundaAY613794UAUC1554
Fusconaia succissa 1AY158792Serb et al. (2003)
Fusconaia succissa 2AY158809Serb et al. (2003)
Glebula rotundataTAY613795UAUC502
Gonidea angulataTAY655099UAUC3147
Hemistena lataTAY613796UAUC2797 (inc. AY158787 Serb et al. 2003)
Hyriopsis cumingiiAY655100UAUC3160
Inversidens japanensisAB055625Okazaki & Ueshima, unpubl. data
Lampsilis altilis 1AY655101UAUC125
Lampsilis ornata 2AY158748Serb et al. (2003)
Lampsilis ornata 1AY365193Serb & Lydeard (2003)
Lampsilis ovataTAY613797UAUC1681
Lampsilis siliquoidea 2AY158747Serb et al. (2003)
Lampsilis teres 1AY655102UAUC3330
Lasmigona holstonia etowahensisAY655103UAUC3159
Lemiox rimosusTAY655104UAUC1528
Leptodea leptodonTAY655105UAUC135
Lexingtonia dolabelloides 1AY613798UAUC3148
Lexingtonia dolabelloides 2AY655106UAUC2819
Medionidus accutissimusAY655107UAUC82
Medionidus conradicusTAY158746Serb et al. (2003)
Megalonaias nervosaTAY158794Serb et al. (2003)
Obliquaria reflexaTAY655108UAUC2508 (incl. AY158751 Serb et al. 2003)
Obovaria jacksonianaAY655109UAUC775
Obovaria rotulata 2AY158799Serb et al. (2003)
Plectomerus dombeyanusTAY655110UAUC2536
Plethobasus cyphusTAY613799UAUC3157
Pleurobema chattanoogaense 1AY655111UAUC1621
Pleurobema chattanoogaense 2AY613801UAUC3194
Pleurobema clavaTAY613802UAUC1477
Pleurobema collinaAY613803UAUC1074
Pleurobema cordatumAY613804UAUC2572
Pleurobema decisum 2AY655112UAUC2997
Pleurobema decisum 3AY613805UAUC3196
Pleurobema furvumAY613806UAUC678
Pleurobema georgianum 1AY655113UAUC1623
Pleurobema georgianum 2AY613807UAUC3193
Pleurobema georgianum 3AY655114UAUC3084
Pleurobema gibberumAY613808UAUC3153
Pleurobema hanleyianum 1AY655115UAUC273
Pleurobema hanleyianum 2AY613809UAUC1622
Pleurobema oviforme 1AY613810UAUC3238
Pleurobema oviforme 2AY655116UAUC1642
Pleurobema perovatumAY613811UAUC1640
Pleurobema pyriformeAY613812A29
Pleurobema rubellumAY613813UAUC679
Pleurobema rubrum 1AY655117UAUC2719
Pleurobema rubrum 2AY613814UAUC3229
Pleurobema sintoxiaAY613815UAUC1714
Pleurobema strodeanumAY613817UAUC1110
Pleurobema taitianumAY613818UAUC885
Pleurobema troschelianumAY613819UAUC516
Popenaias popeiiTAY655118UAUC3161
Potamilus alatusT 1AY655119UAUC3329
Ptychobranchus fasciolarisTAY655120LSC23701-001
Quadrula apiculataAY158805Serb et al. (2003)
Quadrula kienerianaAY158769Serb et al. (2003)
Quadrula metanevraAY158771Serb et al. (2003)
Quadrula nobilisAY158789Serb et al. (2003)
Quadrula quadrulaT 1AY158790Serb et al. (2003)
Quadrula quadrulaT 2AY158774Serb et al. (2003)
Quincuncina burkeiT 3AY158793Serb et al. (2003)
Quincuncina infucata 1AY655121UAUC3283
Quincuncina infucata 3AY158810Serb et al. (2003)
Quincuncina kleiniana 1AY158795Serb et al. (2003)
Strophitus subvexusAY655122UAUC2715
Toxolasma parvusAY655123UAUC3331
Toxolasma texasiensisAY655124UAUC80
Tritogonia verrucosaTAY158791Serb et al. (2003)
Truncilla truncataTAY655125Unnumbered specimen
Venustaconcha pleasiiAY655126UAUC136
Villosa irisAY655127UAUC2701
Villosa villosaTAY094387Buhay et al. (2002)

Appendix 2. Locality and collection information for new sequences

SpeciesGeneCollection numberCollectorLocality
  1. UAUC, University of Alabama Unionid Collection; LSC, Leetown Science Center.

Actinonaias ligamentina16S, ND1UAUC241K. J. RoeKankakee River, Aroma Park, T30N R13W Sec 23, Kankakee Co. IL
Actinonaias pectorosaCOI, 16SUAUC880H. McCullaghClinch River, Pendleton Island, Rt. 72 bridge, Ft. Blackmore, Scott Co. VA
Amblema elliottiiCOI, ND1UAUC2511M. Pierson, K. Chalk, R. JamesCoosa River 2.7 mi. downstream of Jordan Dam, Elmore Co. AL
Anodonta oregonensisND1UAUC3169T. J. Frest, E. J. JohannesLake Washington at Magnuson Park, Seattle, King Co. WA
Cyclonaias tuberculata16S, ND1UAUC3158S. Ahlstedt, R. BigginsPowell River, RM 111.8 Bales Ford, Hancock Co. TN
Cyprogenia aberti16SUAUC75J. L. Harris, R. Doster, J. Fleming, K. StobaughSaline River, downstream of Hwy 229 in Benton, Saline Co. AR
Cyprogenia stegariaCOI, ND1UAUC1499S. Ahlstedt, S. FraleyClinch River, Brooks Island, RM 184.5, Hancock Co. TN
Cyrtonaias tampicoensis16SUAUC78R. G. HowellsLeon River, Belton Reservoir, Bell Co. TX
Cyrtonaias tampicoensisND1UAUC314R. G. HowellsNueces River, Lake Corpus Christi, Live Oak Co. TX
Dromus dromasCOI, 16S, ND1UAUC3156UnknownPowell River, McDowell Shoal, Hancock Co. TN
Ellipsaria lineolataCOI, ND1UAUC450S. McGreggor, P. O'NeilCahaba River, below Cooper Island, Bibb Co. AL
Elliptio arcaCOIUAUC498M. HughesOostanaula River, 0.8 river mi upstream from Armuchee Creek, Floyd Co. GA
Elliptio arcaND1UAUC501P. Hartfield and othersSipsey Fork, Black Warrior River at mouth of Hurricane Creek, Winston Co. AL
Elliptio crassidensCOIUAUC1493C. Lydeard, C. R. Merrell, J. M. Serb, J. T. GarnerTennessee River, upstream of US Rt. 43 in Florence, Lauderdale Co. AL
Elliptio crassidens16S, ND1UAUC3150S. AhlstedtCoosa River, above Wetumpka below Pipeline Shoals, Elmore Co. AL
Elliptio dilatataND1UAUC2735S. AhlstedtObed River at Alley Ford, Morgan Co. TN
Elliptio dilatataND1UAUC2721S. Ahlstedt, C. HubbsDuck River, Venable Spring, Marshall Co. TN
Elliptoideus sloatianusCOI, 16S, ND1Specimen EsJ. Brim-Box and J. D. WilliamsAppalachicola River, Gadsden Co. FL
Epioblasma brevidens16SUAUC509J. KhymClinch River, Kyles Ford, RM 189.6, Hancock Co. TN
Epioblasma capsaeformisCOI, 16SUAUC1527L. KochDuck River, Lillard Mill Dam, RM 179, Marshall Co. TN
Fusconaia barnesianaCOI, 16S, ND1UAUC1553S. AhlstedtDuck River, Lillard Mill Dam, RM 179, Marshall Co. TN
Fusconaia cerinaCOIUAUC3233M. GangloffTallapoosa River, Choctafaula Creek at FR906, Tuskegee NF, Macon Co. AL
Fusconaia cerina16S, ND1UAUC73P. HartfieldTombigbee River, Coal Fire Creek at CR 26, Pickens Co. AL
Fusconaia cerinaND1UAUC3234S. ShivelyBogue Chitto River, Little Silver Creek at Pleasant Hill Road, Washington Pa. LA
Fusconaia corCOI, 16S, ND1UAUC2606J. Fridell, M. Cantrell, S. FraleyHolston River, North Fork above SR633 crossing, Smyth Co. VA
Fusconaia cuneolusCOI, ND1UAUC1552S. Ahlstedt, S. FraleyClinch River, Pendleton Island, Rt. 72 bridge, Ft. Blackmore, Scott Co. VA
Fusconaia ebenaCOI, ND1UAUC71D. HubbsTennessee River, Kentucky Reservoir, RM 88.1, Humpheys Co. TN
Fusconaia escambia16SUAUC1449J. Williams et al.Conecuh River, CR28 1 mi. E Goshen, Pike Co. AL
Fusconaia flava16SUAUC146P. MorrisonOhio River, Rosewood Bend, RM625, Harris Co. IN, Jefferson Co. KY
Fusconaia flavaND1UAUC2864W. R. Haag, D. Thurmond, J. G. McWhirterBig Sunflower River, end of FS Rd 717A, N of Green Ash/Greentree Reservoir, 6 mi E of Rolling Fork, Sharkey Co. MS
Fusconaia subrotundaCOI, 16S, ND1UAUC1554S. Ahlstedt, S. FraleyPowell River, McDowell Ford, RM 106.7, Hancock Co. TN
Glebula rotundata16S, ND1UAUC502UnknownApalachicola River, Rm 21.8; tip of Brickyard Island, 5 air mi SSW Sumatra, Franklin Co. FL
Gonidea angulata16S, ND1UAUC3147T. J. Frest, E. J. JohannesSnake River, RM 569.5 upstream of Dritch Bowler's house/studio, Gooding Co. ID
Hemistena lataCOI, 16S, ND1UAUC2797S. AhlstedtClinch River, Frost Ford, RM 181.2, Hancock Co. TN
Hyriopsis cumingiiCOI, 16S, ND1UAUC3160H. LiuPoyang Lake, Jiangxi Province, China
Lampsilis altilisND1UAUC125K. J. Roe et al.Etowah River, Shoal Ck., Pine Glen Recreation Area, Talladega NF, Cleburne Co. AL
Lampsilis ovataCOI, 16SUAUC108J. GarnerElk River, fish trap above Hwy 127 near state line, Limestone Co. AL
Lampsilis ovataND1UAUC1681H. McCullaghClinch River, near Pendleton Island and Rt. 72 bridge, Ft. Blackmore, Scott Co. VA
Lampsilis teresND1UAUC3330S. ClarkTennessee River, Decatur, Morgan Co. AL
Lasmigona holstonia etowahensisCOI, ND1UAUC3159P. JohnsonConasauga River, Poplar Spring Creek, Whitfield Co. GA
Lemiox rimosusCOI, ND1UAUC1528L. KochDuck River at Lillard Mill Dam, Marshall Co. TN
Lemiox rimosus16SUnnumberedUnknownDuck River, TN
Leptodea leptodonCOI, 16S, ND1UAUC135A. RobertsMeramec River, MO
Lexingtonia dolabelloidesCOI, ND1UAUC2819S. AhlstedtDuck River, Lillard Mill Dam, RM 179, Marshall Co. TN
Lexingtonia dolabelloidesCOI, 16S, ND1UAUC3148S. AhlstedtElk River, RM 105.5 Dickey Bridge, Lincoln Co. TN
Ligumia recta16SUAUC89UnknownOhio River, near Louisville, Jefferson Co. KY
Medionidus accutissimusCOI, 16S, ND1UAUC82UnknownTombigbee River, Lubbub Ck. at CR 24, Pickens Co. AL
Medionidus conradicusCOIUAUC10C. LydeardClinch River, Kyles Ford, RM 189.6, Hancock Co. TN
Megalonaias nervosaCOIUAUC266K. RoeCoosa River, near Leesburg, downstream from mouth of Terrapin Ck., Cherokee Co. AL
Obliquaria reflexaCOI, 16S, ND1UAUC2508M. Pierson, K. Chalk, R. JamesCoosa River 2.7 mi downstream of Jordan Dam, Elmore Co. AL
Obovaria jacksonianaCOIUAUC680D. N. SheldonPascagoula River, confluence of Brewton Lake, Jackson Co. MS
Obovaria jacksonianaND1UAUC775H. McCullagh, C. LydeardSipsey River at CR2, Pickens Co. AL
Obovaria subrotundaCOI, 16SUAUC2838S. AhlstedtDuck River, Lillard Mill Dam, RM 179, Marshall Co. TN
Plectomerus dombeyanusCOI, 16S, ND1UAUC2536M. Pierson, K. Chalk, R. JamesCoosa River 2.7 mi downstream of Jordan Dam, Elmore Co. AL
Plethobasus cyphusCOIUAUC1639S. Ahlstedt, S. FraleyClinch River, Brooks Island, RM 184.5, Hancock Co. TN
Plethobasus cyphus16S, ND1UAUC3157S. AhlstedtClinch River, Frost Ford, RM 181.2, Hancock Co. TN
Pleurobema chattanoogaenseCOI, 16S, ND1UAUC1621S. A. Ahlstedt, R. R. EvansConasauga River, below Mitchell Bridge, Murray Co. GA
Pleurobema chattanoogaenseCOI, ND1UAUC3194P. JohnsonDead River, 500 m below Terrapin Creek confluence, Cherokee Co. AL
Pleurobema clavaCOI, 16S, ND1UAUC1477UnknownAllegheny River, Kennerdell and Clear Ck. SP, Venango Co. PA
Pleurobema collinaCOI, 16S, ND1UAUC1074M. A. McGregor, P. BurgessJames River, Wards Creek, CR 665 1.5 mi NE Millington, Albemarle Co. VA
Pleurobema cordatumCOI, ND1UAUC2572J. Buhay, A. WethingtonGreen River, Munfordville, River Road, Hart Co. KY
Pleurobema decisumCOIUAUC253P. HartfieldTallapoosa River, Chewacla Ck. S of CR 22 bridge crossing, 5 mi E Tuskegee, Macon Co. AL
Pleurobema decisumND1UAUC2997H. McCullaghTallapoosa River, Chewacla Creek at Rt 71 bridge, 5 mi E Tuskegee, Macon Co. AL
Pleurobema decisumCOI, ND1UAUC3196P. JohnsonDead River, 500 m below Terrapin Creek confluence, Cherokee Co. AL
Pleurobema furvumCOI, ND1UAUC678P. Hartfield et al.Black Warrior River, Brushy Creek, FS Rd 255, upstream from Capsey Creek, Bankhead NF, Winston Co. AL
Pleurobema georgianum16S, ND1UAUC1623S. A. Ahlstedt, R. R. EvansConasauga River, Holly Creek, N Hwy 52 bridge, Murray Co. GA
Pleurobema georgianumCOI, 16S, ND1UAUC3193P. JohnsonConasauga River 200m above Jacks river confluence, Murray Co. GA
Pleurobema georgianumCOI, ND1UAUC3084M. GangloffCoosa River, Big Canoe Creek between CR 36 & Rt 231, St. Clair Co. AL
Pleurobema gibberumCOIUAUC3319S. Ahlstedt, B. Butler, Rob TowesCollins River, Barren fork, Hwy 287 Bridge, Trousdale, Warren Co. TN
Pleurobema gibberum16S, ND1UAUC3153S. Ahlstedt, B. Butler, Rob TowesCollins River, Barren fork, Hwy 287 Bridge, Trousdale, Warren Co. TN
Pleurobema hanleyianumCOI, 16S, ND1UAUC273K. J. Roe et al.Coosa River, near Leesburg, downstream from mouth of Terrapin creek, Cherokee Co. AL
Pleurobema hanleyianumCOI, 16S, ND1UAUC1622P. D. Johnson, R. R. EvansConasauga River, below Beaverdale crossing (GA 2), Upper Kings Bridge, Murray Co. GA
Pleurobema oviforme16S, ND1UAUC3238Steve Ahlstedt, Steve BakaletyBig South Fork Cumberland River, Lower Rough Shoals, Scott Co. TN
Pleurobema oviformeCOIUAUC1402Steve Ahlstedt, Steve BakaletyBig South Fork Cumberland River, Lower Rough Shoals, Scott Co. TN
Pleurobema oviformeCOI, 16S, ND1UAUC1642S. Fraley, R ButlerHolston River, Beech Creek near Keplar Elementary at Webster Road, Hawkins Co. TN
Pleurobema perovatumCOI, ND1UAUC1640J. Williams et al.Alabama River, Sturdivant Creek, Hwy 10 2.4 mi E Awin, Wilcox Co. AL
Pleurobema pyriformeCOI, ND1A29J. WilliamsChipola River, Big Creek, Houston Co. AL
Pleurobema rubellumCOI, 16S, ND1UAUC679P. Hartfield et al.Black Warrior River, Brushy Creek, FS Rd 255, upstream from Capsey Creek, Bankhead NF, Winston Co. AL
Pleurobema rubrumCOI, ND1UAUC2719S. Ahlstedt, C. HubbsDuck River, Venable Spring, Marshall Co. TN
Pleurobema rubrumCOI, ND1UAUC3229W. R. Haag, A. M. CommensSt. Francis River, Hwy 64 bridge at Parkin, Cross Co. AR
Pleurobema sintoxiaCOI, ND1UAUC1714S. AhlstedtCumberland River, Big South Fork at Station Camp Creek, Scott Co. TN
Pleurobema strodeanumCOI, ND1UAUC1110K. J. Roe et al.Choctawhatchee River, West Fork, Hwy 10, Blue Springs SP, Barbour Co. AL
Pleurobema strodeanum16SUAUC1818K. J. Roe, K. S. CummingsPea River, CR 77 2 mi NW Ariton, Barbour Co. AL
Pleurobema taitianumCOI, 16S, ND1UAUC885J. T. GarnerAlabama River, Selma just below AL Hwy 80 bypass, E bank, Dallas Co. AL
Pleurobema troschelianumCOI, 16S, ND1UAUC516A. Wyss, M. HughesConasauga River, RM49.05, 0.8 RM upstream of Sumac Ck., E of Sumac, Murray Co. GA
Popenaias popeiiCOI, 16S, ND1UAUC3161Tom MillerRio Grande River, Lincoln-Juarez Bridge, Laredo, Webb Co. TX
Potamilus alatus16SUAUC41UnknownElk River, Limestone Co. AL
Potamilus alatusND1UAUC3329S. ClarkTennessee River, Decatur, Morgan Co. AL
Ptychobranchus fasciolaris16S, ND1LSC23701-001W. TolinElk River, Clendenin, Kanawah Co. WV
Quadrula kieneriana16SUAUC334M. Hughes et al.Coosawattee River 2.5 mi upstream from Hwy 225 bridge, Gordon Co. GA
Quincuncina infucataND1UAUC3283C. O'BrienFlint River, Cooleewahee Ck., GA Rt. 91 bridge, Baker Co. GA
Strophitus subvexusCOI, 16S, ND1UAUC2715S. Fraley, J. BaxterTombigbee River, Sucarnoochie Creek, Old Scooba Crossing, Kemper Co. MS
Toxolasma parvusCOI, ND1UAUC3331D. WillisTennessee River, Decatur, Morgan Co. AL
Toxolasma texasiensisCOI, 16S, ND1UAUC80R. G. HowellsColorado River, Giddings State School Lake, Lee Co. TX
Tritogonia verrucosaCOI, 16SUAUC3195P. JohnsonConasauga River, below Mitchell Bridge, Whitfield Co. GA
Truncilla truncata16S, ND1UnnumberedB. SietmanMississippi River, Hannibal, Marion Co. MO
Uniomerus declivusCOI, 16SUAUC3290W. R. Haag, J. L. StantonBig Sunflower River, Farrell Rd. Bridge, ∼5 mi N of Clarksdale, Coahoma Co. MS
Venustaconcha ellipsiformisCOI, 16SUAUC2596-8B. SietmanBig Piney River, Texas Co. MO
Venustaconcha pleasiiCOI, ND1UAUC136UnknownMeramec River, Fish Trap Rapids, Franklin Co. MO
Villosa iris16SUAUC260Louis LevineCollins River, Highway 56 bridge near Beersheba Springs, Grundy Co. TN
Villosa irisND1UAUC2701S. AhlstedtDuck River, Lillard Mill Dam, RM 179, Marshall Co. TN
Villosa vanuxemensis16SUAUC3046S. J. FraleyLittle River, Telb at old Wallard Hwy access 1.5 RM upstream of Melrose Rd, Blount Co. TN

Ancillary