Molecular Systematics of North American Phoxinin Genera (Actinopterygii: Cyprinidae) Inferred from Mitochondrial 12S and 16S Ribosomal RNA Sequences

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Blackwell Science, LtdOxford, UKZOJZoological Journal of the Linnean Society0024-4082The Lin- nean Society of London, 2003? 2003 1391 6380 Original Article A. M. SIMONS ET AL. NORTH AMERICAN PHOXININ SYSTEMATICS

Zoological Journal of the Linnean Society, 2003, 139, 63–80. With 7 figures

Molecular systematics of North American phoxinin genera (Actinopterygii: Cyprinidae) inferred from mitochondrial 12S and 16S ribosomal RNA sequences

ANDREW M. SIMONS1*, PETER B. BERENDZEN1 and RICHARD L. MAYDEN2

1University of Minnesota, Department of Fisheries, Wildlife, and Conservation Biology & Bell Museum of Natural History 1980 Folwell Avenue, St Paul, MN 55108, USA 2Saint Louis University, Department of Biology, 3507 Laclede Avenue, St Louis, MO 63103, USA

Received June 2002; accepted for publication December 2002

The cyprinid fish fauna of North America is relatively large, with approximately 300 species, and all but one of these are considered phoxinins. The phylogenetic relationships of the North American phoxinins continue to pose difficulties for systematists. Results of morphological analyses are not consistent owing to differences interpreting and coding characters. Herein, we present phylogenetic analyses of mitochondrial 12S and 16S ribosomal RNA sequence data for representatives of nearly all genera of North American phoxinins. The data were analysed using parsimony, weighted parsimony, maximum likelihood and bayesian analyses. Results from weighted parsimony, likelihood and the bayesian analysis are largely consistent as they all account for differing substitution rates between transitions and transversions. Several major clades within the fauna can be recognized and are strongly supported by all analyses. These include the western clade, creek chub–plagopterin clade and the open posterior myodome clade. The shiner clade is nested in the open posterior myodome clade and is the most species-rich clade of North American phoxinins. Relationships within this clade were not well resolved by our analyses. This may reflect the inability of the mitochondrial RNA genes to resolve recent speciation events or taxon sampling within the shiner clade. © 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 139, 63–80.

ADDITIONAL KEYWORDS: bayesian estimation – evolution – maximum likelihood – parsimony.

INTRODUCTION with all North American genera in the subfamily Leu- ciscinae. Leuciscinae was diagnosed by two charac- The family Cyprinidae is native to all continents ters: pharyngeal arch with one or two rows of teeth, except for Australia, Antarctica and South America and the loss of the suborbital–infraorbital connection. (Mayden, 1991). Over 2000 species have been Within Leuciscinae, two lineages of cyprinids were described (Nelson, 1994), 300 of which are from North recognized, both of which are present in North Amer- America (Mayden et al., 1992). Their high diversity ica, leuciscins and phoxinins. The leuciscins are pri- and widespread distribution have made cyprinids one marily Eurasian, with a single North American of the most difficult groups of fishes to understand tax- representative, the golden shiner (Notemigonus cryso- onomically and systematically (Mayden, 1989, 1991). leucas [Mitchill]). Phoxinins make up the remaining Various researchers have attempted to decipher the North American cyprinid diversity. There are several phylogenetic history and classify these fishes. One of Eurasian phoxinin genera known but their relation- the latest efforts employing phylogenetic systematics ship to the North American taxa is currently was by Cavender & Coburn (1992). Cavender & unresolved. Coburn recognized several subfamilies worldwide, The North American phoxinins are one of the most taxonomically difficult groups of fishes on the conti- nent. The large number of taxa, their diverse and com- *Corresponding author. E-mail: [email protected] plex morphological evolution, and their widespread

64 A. M. SIMONS ET AL. distribution has hampered an understanding of their Relationships and classification of North Ameri- phylogenetic relationships, essential for comprehend- can phoxinins have been chaotic since Rafinesque ing the evolution of biodiversity of these fishes. The (1818) described Notropis atherinoides (Fig. 2a). A tremendous breadth of trophic morphologies, breeding proliferation of generic and specific names has behaviours and habitat preferences (to name a few resulted in complex synonymies; some taxa have characteristics) is unmatched by any other group of been described a number of times, even within the North American fishes. Like cyprinid groups from same paper, e.g. Girard (1856). The confusion con- other continents, North American phoxinins contain tinued through the early part of the 20th century, algivores, herbivores (Fig. 1c,d), planktivores and pis- reflecting the absence of a coherent philosophy gov- civores (Fig. 1a,f), often with specialized oral morphol- erning classification and taxonomy. The cyprinid ogies for obtaining food. The most bizarre of these is fauna was considered to contain two distinct Exoglossum maxillinguae (Lesueur), a species that groups, a western fauna and an eastern fauna. The has been reported to use its modified lower jaw to western fauna contained taxa native to Pacific pluck eyes out of other fishes (Scott & Crossman, 1973; drainages whereas the eastern fauna contained Page & Burr, 1991). Breeding behaviours are very taxa native to the Mississippi, other Gulf of Mexico diverse; these include various courtship displays with drainages and Atlantic drainages. Although several spawning over unprepared substrate, in rock crevices, taxa obviously did not conform to this pattern, this cavities beneath rocks and on large, gravel nests. view has persisted. Members of the genus Nocomis (Fig. 2h) construct In the 1950s, Reeve Bailey dramatically revised the nests over 1 m in diameter and up to one-third of a taxonomic structure of several groups of North Amer- metre in height (Johnston & Page, 1992). Other spe- ican fishes. In an attempt to simplify the classification cies are known as nest associates and spawn over cyp- of minnows, Bailey (1951, 1956) synonymized several rinid or non-cyprinid fish species nests and leave genera based on a few morphological characters. parental care to the nest-constructing species. Pter- These characters include the presence and position of onotropis hubbsi (Bailey & Robison) and Notropis barbels, scalation and gut morphology. His simplifica- cummingsae Myers spawn over nests of sunfishes and tion of cyprinid classification was paralleled by basses (Centrarchidae) (Fletcher & Burr, 1992, 1993), similar changes in the classification of the North species that are normally considered predators, and American darters (Bailey, 1951; Bailey, Winn & initiate their spawning only after the presumed Smith, 1954; Bailey & Gosline, 1955). These taxo- release of pheromones contained in the milt of the cen- nomic changes were not proposed in papers on classi- trarchid species male (Hunter & Hasler, 1965). North fication but as footnotes in keys (Bailey, 1951, 1956) or American phoxinins are found in a wide range of hab- as commentaries in papers on distribution (Bailey itats, including high-velocity headwater streams, et al., 1954). Although there were some immediate large, silt-laden rivers, swamps and lakes and exhibit reactions to these taxonomic changes (Lachner & Jen- a wide range of morphologies allowing them to persist kins, 1967; McPhail & Lindsey, 1970; Jenkins & Lach- in these habitats (Figs 1, 2). ner, 1971), they strongly influenced work on North Many early classifications relied on morphological American fish systematics. Hubbs & Miller (1977: characters such as scalation, position of fin insertion, 275) effectively summarized the confusion: ‘It is abun- gut morphology and pharyngeal tooth counts to delin- dantly obvious that much of the generic placement in eate taxonomic boundaries. These classifications were American cyprinids is in a chaotic state, and that the not phylogenetic and were generally based on subjec- prime significance attributed to intestinal coiling vs. tive evaluations of the degree of taxonomic differenti- the single compressed-S configuration, and to the ation provided by various morphological features. presence vs. absence of a maxillary barbel, in the tax- Some characters were considered indicative of generic onomy of the group has been very considerably dis- differences, whereas others were indicative of specific credited.’ Hubbs focused on morphological differences or subspecific differentiation. This approach generated whereas Bailey focused on similarities. The differ- taxonomic instability and confusion. The emergence of ences between Hubbs’ and Bailey’s views perhaps phylogenetic systematics has altered the arguments reflects the tensions of systematic thought of the time, from the value of particular characters or character that classifications should reflect both history and systems to phylogeny reconstruction and appropriate adaptation. interpretation of characters. Although disagreements The introduction of a fundamentally phylogenetic still exist, recent phylogenetic studies are converging approach to classification marked the first real change on a consensus of North American phoxinin relation- in systematics of North American fishes (reviewed by ships. Herein, we review historical approaches and Mayden & Burr, 1992). Mayden (1989) published a problems, and present an analysis of molecular data phylogenetic classification of primarily eastern North for nearly all North American phoxinin genera. American cyprinids, resurrecting nine genera. This

NORTH AMERICAN PHOXININ SYSTEMATICS 65

Figure 1. Representative members of the western (a–d) and creek chub clades (e,f). Numbers in parentheses indicate approximate maximum size. (a) Colorado River pikeminnow, Ptychocheilus lucius (180 cm); (b) humpback chub, Gila cypha (46 cm); (c) Sacramento blackfish, Orthodon microlepidotus (55 cm); (d) southern redbelly dace, Phoxinus erythrogaster (91 mm); (e) flame chub, Hemitremia flammea (72 mm); (f) creek chub, Semotilus atromaculatus (30 cm).

apparent reversal of Bailey’s previous changes was the nal characters. His primary interest was in species result of the profound change in philosophy of classi- relationships within the genus Cyprinella; however, ﬁcation from a neosynthetic approach to a phyloge- absence of explicit phylogenetic hypotheses at higher netic paradigm (Hull, 1988). Mayden’s work was based levels of relationship prompted an extensive search for on morphology, including both osteological and exter- appropriate outgroup taxa. Ironically, the search for

66 A. M. SIMONS ET AL.

Figure 2. Representative members of the open posterior myodome clade. Numbers in parentheses indicate approximate maximum size. (a) Emerald shiner, Notropis atherinoides (13 cm); (b) rainbow shiner, Notropis chrosomus (81 mm); (c) sil- verjaw minnow, Ericymba buccata (98 mm); (d) pugnose minnow, Opospoeodus emiliae (64 mm); (e) Tennessee shiner, Not- ropis leuciodus (82 mm); (f) spotﬁn chub, Erimonax monachus (11 cm); (g) rifﬂe minnow, Phenacobius catostomus (12 cm); (h) bluehead chub, Nocomis leptocephalus (26 cm); (i) Lahontan redside, Richardsonius egregius (17 cm); (j) splittail, Pogon- ichthys macrolepidotus (44 cm).

NORTH AMERICAN PHOXININ SYSTEMATICS 67 outgroups generated more attention than the phylog- ossify medially, occluding the opening to the myo- eny of Cyprinella itself. dome (Mayden, 1989: fig. 8). Mayden recognized any Mayden (1989) recognized a large clade supported ventral opening into the posterior myodome as by a single synapomorphy, an opening in the poste- derived and potentially synapomorphic, and consid- rior myodome (Fig. 3). The posterior myodome is a ered ontogenetic closure to be derived within the cavity in the posterior of the cranium that is the site OPM clade. The OPM clade contained primarily east- of origin of the lateral rectus muscles. The parasphe- ern taxa, but did include Agosia, Oregonichthys, Rich- noid and basioccipital form the floor of the myodome. ardsonius (Fig. 2i) and Yuriria, genera typically In most members of the open posterior myodome considered part of the western fauna (Uyeno, 1961; (OPM) clade, the floor of the myodome is open to the Coburn & Cavender, 1992). Concurrent with recogni- ventral aspect of the cranium, albeit sealed by con- tion of the OPM clade, Mayden elevated several taxa nective tissue. The OPM was first described by from synonymy with Notropis (Lythrurus, Luxilus, Coburn (1982), who reported its presence in Notropis Cyprinella, Pteronotropis) and Hybopsis (Erimystax, s.l., Ericymba (Fig. 2c), Erimystax, Extrarius (now Macrhybopsis, Extrarius, Platygobio). included in Macrhybopsis), Pimephales, as well as the Coburn & Cavender (1992) published the first phy- Hybopsis labrosa (Cope) group. Small openings in logenetic analysis of all North American phoxinin the myodome were observed in Hybognathus and taxa at the generic level (Fig. 4). Their analysis was Phenacobius (Fig. 2g). Mayden (1989) redescribed also based on osteological and external characters. this opening including a description of ontogenetic Coburn & Cavender recognized three major clades of closure of the OPM in several large taxa such as North American phoxinins, a shiner clade sister to a Campostoma. In juveniles of these taxa a large open- chub clade plus a western clade (Fig. 4). Coburn & ing in the posterior myodome is visible. During devel- Cavender argued for the dismemberment of Mayden’s opment, the parasphenoid apparently continues to OPM clade and placed members of the OPM clade in all three major clades they recognized. All taxa contained in their shiner clade were members of the

Cyprinella OPM clade sensu Mayden (Fig. 4). Membership of their exoglossin clade is the same as Mayden’s chub Luxilus clade, except for Agosia. Relationships among taxa Lythrurus within this clade differ greatly between the two Notropis hypotheses. The only commonality is the sister taxon relationship between Campostoma and Dionda. Pteronotropis Mayden (1989) considered two genera contained in Hybopsis Clade Coburn & Cavender’s western clade, Yuriria and Ago- Yuriria sia, part of the OPM clade. Pimephales Mayden’s and Coburn & Cavender’s analyses were based on morphological characters, but there were Richardsonius many disagreements regarding interpretations and Clinostomus codings of morphological characters including the Oregonichthys OPM. Coburn & Cavender (1992) restricted their Platygobio interpretation of the OPM to a large ventral opening that is bounded equally by the parasphenoid and Macrhybopsis basioccipital (Coburn & Cavender, 1992: ﬁg. 18). A Extrarius small opening was considered plesiomorphic and of lit- Erimystax tle phylogenetic signiﬁcance. Mayden, however, recog- Chub Clade Phenacobius nized any opening between the basioccipital and parasphenoid as potentially synapomorphic regard- Campostoma less of size or ontogenetic fate of the opening. Because Dionda of disagreements of interpretation of this and other Nocomis morphological characters, no strong consensus of relationship emerged from these papers. Exoglossum Hybognathus

Agosia MOLECULAR APPROACH Relationships among North American phoxinins have Figure 3. Phylogeny of the open posterior myodome clade been addressed using molecular characters by a num- based on morphology (Mayden, 1989). ber of investigators. Most of these studies focused on

68 A. M. SIMONS ET AL.

Agosia Rhinichthys Moapa plagopterins Gila G. (Gila)

Algansea Clade G. (Temeculina) Eremichthys Relictus G. (Siphateles) Western clade Iotichthys G. atraria G. crassicauda Ptychocheilus Mylopharodon Pogonichthys Yuriria Acrocheilus Mylocheilus Orthodon Lavinia Hesperoleucas Exoglossum Phenacobius

Nocomis C hub Clade Erimystax Macrhybopsis Campostoma Exoglossin Clade Dionda Hybognathus Platygobio Couesius Hemitremia Semotilus Margariscus Phoxinus Rhynchocypris Tribolodon

Opsopoeodus Notropin Clade Pimephales Cyprinella Shiner Clade Lythrurus Luxilus Notropis (s.l.) Oregonichthys Richardsonius Clinostomus

Figure 4. Phylogeny of North American phoxinins based on morphology (Coburn & Cavender, 1992).

NORTH AMERICAN PHOXININ SYSTEMATICS 69 single genera, or species groups within genera, and includes all taxa from Simons & Mayden (1997, 1998, thus do not provide information on phylogenetic rela- 1999) and additional taxa, with particular emphasis tionships among North American phoxinins as a on the shiner clade. Abramis, Rutilus and Notemigo- whole. In a series of papers, Simons & Mayden (1997, nus were included as outgroup taxa in all analyses. 1998, 1999) examined relationships of North Ameri- See Appendix for taxon and locality information. can phoxinin genera using molecular characters, spe- DNA extraction, amplification and sequencing were cifically the DNA sequences of the mitochondrial described in Simons & Mayden (1998). ribosomal 12S and 16S genes. These analyses found Multiple alignment was performed using CLUST- evidence for three major clades of North American ALW (Thompson, Higgins & Gibson, 1994) and subse- phoxinins: a western clade, a creek chub–plagopterin quently adjusted by eye. Secondary structure models clade and an OPM clade. Although they retained the for rRNA of Carp, Cyprinus carpio (De Rijk et al., names of the western and OPM clades, the make-up of 1994; Van de Peer et al., 1994), were used as aids in these clades differs somewhat from those proposed by adjusting alignments. Where possible, gaps were Mayden (1989) and Coburn & Cavender (1992). The shifted from stem to non-stem regions of the align- relationships proposed in those analyses supported ment. Gaps were considered missing data. Regions of aspects of both Coburn & Cavender’s (1992) and May- ambiguous alignment were omitted from analyses. den’s (1989) hypotheses but most clades were novel. Several included taxa were missing large amounts of Each of the analyses presented by Simons & May- sequence data owing to technical difficulties with den focused on one of the phoxinin clades and their sequencing. These include Campostoma ornatum 1999 paper, examining relationships of the OPM Girard, Codoma ornata Girard (UAIC 7904.02), Eri- clade, omitted many genera, particularly those repre- cymba buccata Cope (Fig. 2c), Erimonax monachus senting the shiner clade. The clade by clade approach Cope (UAIC 79242.03, Fig. 2f), Exoglossum maxilin- to previous analyses was taken because of computa- guae, Luxilus cerasinus Cope, Nocomis biguttatus tional constraints and availability of material. There- (Kirtland), Notropis chrosomus (Jordan) (Fig. 2b), fore, results presented in previous analyses may be Notropis harperi Fowler, Notropis leuciodus (Cope) compromised. The use of exemplar taxa is an unavoid- (Fig. 2e), Notropis maculatus (Hay), Notropis stilbius able aspect of phylogenetic analyses of large clades; Jordan, Opsopoeodus emiliae Hay (UAIC 10979.04, however, sampling strategy can cause problems with Fig. 2d), Oregonichthys crameri (Snyder), Phenacobius phylogeny estimation. First, sampling can result in catostomus Jordan (Fig. 2g), Pimephales notatus long branches causing inconsistency in parsimony (Rafinesque), Pimephales vigilax (Baird & Girard), analyses (Swofford et al., 2001). The break up of long Pteronotropis euryzonus (Suttkus), Pteronotropis branches by adding taxa can reduce the long branch hubbsi and Richardsonius balteatus (Richardson) (see effect (Graybeal, 1998). Increased sampling may also Appendix). Parsimony analyses were duplicated with decrease analysis time (Hillis, 1998; Soltis et al., 1998) all taxa included (complete data set) and with taxa and reduce error (Zwickl & Hillis, 2002). Second, missing >5% sequence data excluded (reduced data alignment of nucleotide sequence may be difficult to set). Parsimony analyses were performed using achieve, especially with ribosomal genes that contain PAUP* 4.0b8 (Swofford, 2000) with heuristic searches, a large number of insertions or deletions. Inaccurate random sequence addition with 100 replications and alignments generate inaccurate homology statements TBR branch swapping. All characters were weighted and will probably confuse analyses. Adding taxa often equally. Decay indices were calculated using MAC- helps to resolve difficult alignment issues. CLADE 4.0 (Maddison & Maddison, 2000) and PAUP* To address these potential problems we constructed 4.0b8. Bootstrap analyses were performed with 1000 a large data set that contains all taxa included in pre- pseudoreplications using heuristic searches, simple vious analyses as well as additional taxa representing addition sequence and TBR branch swapping. Pos- the shiner clade. The analyses and results presented sible incongruence between 12S and 16S genes was here represent another step toward an understanding assessed using the incongruence length difference of the historical relationships of North American (ILD) test (Farris et al., 1994), as implemented by the phoxinins. partition homogeneity test in PAUP*, with 100 replications. Previous analyses (Simons & Mayden, 1998) indicated that transitions showed signs of saturation. MATERIAL AND METHODS To account for possible saturation, weighted parsi- The phoxinins contain approximately 300 species in mony analyses were performed. Weighting may 50 genera (Mayden et al., 1992). The 12S and 16S improve the consistency of parsimony analysis where mitochondrial rRNA genes plus the intervening valine a transition/transversion bias exists (Huelsenbeck & tRNA gene were sequenced for 99 individuals repre- Hillis, 1993). The choice of a particular weighting senting 83 species among 47 genera. This data set scheme is difficult to justify a priori, and therefore a

© 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 139, 63–80 70 A. M. SIMONS ET AL. range of weights were applied. These weights spanned the complete data set, although relationships among the range of observed TS/TV ratios (1/2, 1/3, 1/5) and the western, creek chub–plagopterin and OPM clades allowed a qualitative assessment of the stability of the were not resolved and relationships among taxa data to weighting. within the shiner clade were less resolved. Conflicting MODELTEST (Posada & Crandall, 1998) was used relationships among taxa between the two analyses to determine parameters for a hierarchy of likelihood were restricted to groups with low bootstrap and decay models. The model that provided the best explanation support. Thus additional taxa, even with incomplete of the data was determined via the likelihood ratio test sequence data, improved resolution of the phylogeny. (Posada & Crandall, 2001). Weighting transversions over transitions created Maximum likelihood analysis was performed under minor changes in resolution within major clades but the model determined using MODELTEST. All taxa did not change relationships or taxa of major clades, were included in this analysis. Model parameters were except that the Mylocheilus clade (Simons & Mayden, set a priori based on values estimated by MODEL- 1999) was recovered as a monophyletic group. TEST. Available computing power was limited and did Weighted parsimony caused the greatest change not allow extensive searching under likelihood crite- within the shiner clade. Recovered relationships ria. Therefore, only a single starting tree, obtained via among these taxa are sensitive to taxon sampling as stepwise addition, was evaluated. No bootstrapping or well as weighting. other estimates of support were obtained. Results from MODELTEST (Posada & Crandall, An indirect estimate of support for the likelihood tree 1998) indicated the model HKY+I+G provided the best was obtained by comparison with the results of a baye- fit to the data. Model parameters were TI/TV = 4.9052, sian analysis. Analysis of the complete data set was car- A = 0.3584, C = 0.2335, G = 0.1686, T = 0.2396, ried out using MRBAYES 2.01 (Huelsenbeck & I = 0.4975, a= 0.5658. Maximum likelihood analysis Ronquist, 2001). The likelihood model was determined under this model recovered a tree that was very sim- by MODELTEST. Markov chains were run for 2000 000 ilar to the trees produced by weighted parsimony anal- generations starting with a random tree and default yses (–ln L = 30 587.898: Fig. 6). priors; trees were sampled every 1000 generations. Bayesian analyses using MRBAYES produced 2000 Branch lengths of sampled trees were saved. The burn- trees. Generation 500 000 was used as the burn-in in was determined by plotting –ln likelihood scores vs. point, leaving 1500 trees for estimating trees and tree generation. After the Markov chain reached stationar- parameters. The average –ln likelihood at stationarity ity, trees were sampled from the posterior probability was 32 448.454. The percentage of times a node occurs distribution. The posterior probability of trees and within these 1500 trees is interpreted as the posterior tree parameters were estimated from this distribution. probability of the node. Branch lengths of sampled trees were calculated and used to construct the tree shown in Figure 7. Model parameters estimated with RESULTS MRBAYES are presented in Table 1. Parsimony analysis of the complete taxon data set resulted in 21 equally parsimonious trees with tree DISCUSSION lengths of 5730 steps, a consistency index excluding uninformative characters of 0.24, and a retention The addition of taxa to the 12S and 16S data set has index of 0.62 (Fig. 5a,b). All major clades reported by increased resolution and support for various clades of Simons & Mayden (1999) were recovered. Bootstrap North American phoxinins. These analyses have also values and decay indices indicated strong support for raised further questions regarding this complex basal nodes, corroborating hypotheses of monophyly group. The North American Phoxinin clade was recov- for major clades. Similarly, terminal nodes supporting ered as monophyletic in all analyses. This clade con- monophyly of small species groups or genera were sists of all native North American cyprinid taxa with strongly supported. Support for internal nodes was the exception of the golden shiner, Notemigonus cry- weaker, particularly within the shiner clade where soleucas. Unweighted parsimony analysis recovered there is little support for various relationships among this clade with high (100%) bootstrap support and genera. Comparison between the 12S and 16S parti- Bremer support value of 14. Bayesian analysis recov- tions as measured by the ILD test did not result in a ered this clade with a posterior probability of 1.00. significant probability value (P = 0.20). Within the North American phoxinins, three major Analysis of the reduced taxon data set resulted in 24 clades are recognized, the western clade, the creek equally parsimonious trees with tree lengths of 4828 chub–plagopterin clade and the OPM clade (Simons & steps, consistency index excluding uninformative Mayden, 1998). We discuss interrelationships within characters of 0.26 and a retention index of 0.61. these clades and compare them with previous phylo- Results were largely consistent with the analysis of genetic analyses of these taxa.

Table 1. Likelihood values and model parameters from nuptial coloration and shared tolerance of brackish bayesian estimation of phylogeny. Parameters estimated water: rare among cyprinids. Coburn & Cavender from 1500 trees sampled after Markov chain reached (1992) suggested that Tribolodon and Rhynchocypris stationarity were nested within North American phoxinins as basal members of the chub clade. Parameter Mean Variance Gila and Ptychocheilus were not recovered as monophyletic groups in any analyses. The phylogenetic ln L -32448.45425 154.428068 position of P. oregonensis shifted somewhat depending k 10.708292 0.150756 upon the analysis but it was never sister to, or nested A 0.366375 0.000051 within, Gila. Ptychocheilus lucius, however, was C 0.2304 0.000029 always sister to Gila species exclusive of G. coerulea G 0.163576 0.000019 (Girard). The monophyly of this clade was strongly T 0.239649 0.000029 supported by all analyses. It is possible that introgres- a 0.545216 0.001231 I 0.455259 0.000267 sion between Gila sp. and P. lucius has occurred in the past, resulting in Gila haplotypes in P. lucius. We had access to only one sample of P. lucius for this study. Sequences of additional mitochondrial and nuclear genes from additional specimens should be examined WESTERN CLADE to test this result. The western clade is sister to remaining North Amer- ican phoxinins (Simons & Mayden, 1998) and takes its CREEK CHUB – PLAGOPTERIN CLADE name from the large number of taxa restricted to Simons & Mayden (1997) first proposed a close rela- drainages west of the continental divide. This clade tionship between the creek chubs and their relatives, contains the relatively widespread and species-rich and the plagopterins. The creek chubs and their rela- genus Gila, best known for the bizarre and endan- tives are classified in the genera Couesius, Hemitre- gered species endemic to the Colorado River (Fig. 1b). mia (Fig. 1e), Margariscus and Semotilus (Fig. 1f). Also contained in this clade are the piscivorous pike- These fishes have a number of morphological similar- minnows, Ptychocheilus lucius (Girard), which have ities and have long been thought to be related been reported to reach lengths of 1.8 m (Sigler & (McPhail & Lindsey, 1970; Jenkins & Lachner, 1971; Sigler, 1996; Fig. 1a) and herbivorous minnows such Etnier & Starnes, 1993). Coburn & Cavender (1992) as Orthodon microlepidotus (Fig. 1c). The monophyly identified Semotilus and Hemitremia as sister taxa of the western clade is strongly supported by high but did not find evidence for a monophyletic group con- bootstrap and Bremer support values. The western taining all four genera. Rather, these were paraphyl- clade supported by our analyses does not include etic with respect to the exoglossin clade (Fig. 4). The Mylocheilus, Pogonichthys, Rhinichthys, Agosia and plagopterins are very unusual cyprinids classified the plagopterins, all included in a western clade by in the genera Lepidomeda, Meda, Plagopterus and Coburn & Cavender (1992). Other genera included in Snyderichthys. All have very small scales and Lepi- their western clade (Hesperoleucas, Yuriria, Iotich- domeda, Meda and Plagopterus are the only North thys, Algansea, Moapa) were not available for our American cyprinids with spine-like modifications in analyses but may be part of this clade; Mayden (1989) the dorsal and pelvic rays (Miller & Hubbs, 1960). placed Yuriria within the OPM clade. In addition, In the unweighted analysis, the plagopterin taxa based on our analyses, the genus Phoxinus (Fig. 1d) is were resolved as closely related to Couesius and Mar- included in the western clade. Coburn & Cavender gariscus. This was unexpected given the similarity of (1992) considered Phoxinus part of the chub clade. Couesius and Margariscus to Hemitremia and Semo- Interestingly, this genus is always resolved as sister to tilus. Indeed, Margariscus was once placed in synon- the rest of the clade and is the only member of the ymy with Semotilus (Hubbs & Lagler, 1958). Similar clade not found in western drainages. Phoxinus is dis- results were obtained by Dowling et al. (2002). In the tributed widely in eastern North America and Laur- weighted, likelihood and bayesian analyses, these asia. This relationship and distribution suggests that taxa formed a monophyletic group sister to the pla- other North American phoxinin clades should also gopterins. This result is more consistent with mor- have relatives in Europe or Asia. We expect that these phology and biogeography of these taxa. will be sister to the creek chub–plagopterin clade plus the OPM clade or even sister to each of these clades. Simons & Mayden (1999) suggested that the Asian OPEN POSTERIOR MYODOME CLADE genus Tribolodon was related to Mylocheilus and This clade is named based on the opening in the Pogonichthys (Fig. 2j) within the OPM clade based on ventral floor of the posterior myodome between the

10 Shiner Clade

54 Platygobio 14 9 Macrhybopsis aestivalis 60 84 9 Macrhybopsis storeriana Clade 8 80 Macrhybopsis storeriana 69 Platygobio gracilis 26 6 30 77 Platygobio gracilis

99 Phenacobius 100 Platygobio gracilis

34 Erimystax dissimilis Clade 20 100 Erimystax x-punctatus OPM Clade 4 99 26 Phenacobius catostomus 100 Phenacobius mirabilis

25 Exoglossum laurae Exoglossum 100 Exoglossum maxilinguae 7 52 Oregonichthys crameri Clade 14 79 4 100 Oregonichthys kalawatseti 95 6 Tiaroga cobitis

5 Rhinichthys atratulus Campostoma 58 Rhinichthys osculus

59 Campostoma anomalum Clade 100 Campostoma ornatum 10 9 Nocomis biguttatus 90 68 10 96 Nocomis leptocephalus Mylocheilus

Clinostomus elongatus

12 Clinostomus funduloides Clade 96 23 Richardsonius balteatus 100 Richardsonius egregius 4 9 Mylocheilus caurinus 52 89 Pogonichthys macrolepidotus Couesius plumbeus

36 Plagopt

100 Couesius plumbeus Creek Chub - 4 21 Lepidomeda vittata 54 Meda fulgida 4 100 4 erin Snyderichthys copei 14 55 Margariscus margarita

95 Clade 53 Hemitremia flammea 31 100 Hemitremia flammea 100 18 Semotilus atromaculatus 99 Semotilus thoreauianus 4 Acrocheilus alutaceus Relictus solitarius 14 9 Gila atraria 100 99 Gila cypha 7 Gila conspersa 83 8 Gila ditaenia 10 89 Gila robusta 95 Gila nigrescens Gila pandora 4 4 50 Gila orcutti Western Ptychocheilus lucius Clade 12 Gila coerulea 5 100 Gila coerulea 16 Ptychocheilus oregonensis 5 100 Ptychocheilus oregonensis 5 Eremichthys acros Siphateles bicolor 9 31 Lavinia exilicauda 96 100 Lavinia exilicauda 22 51 Orthodon microlepidotus 100 100 Orthodon microlepidotus 37 Phoxinus erythrogaster 100 Phoxinus neogaeus

Figure 5a. Consensus of 21 trees (TL = 5730) from unweighted parsimony analysis of 12S and 16S sequences. Relation- ships among taxa in the shiner clade shown in Fig. 5b. Numbers above branches are Bremer support indices, numbers below branches are bootstrap values. Clade names follow Simons & Mayden (1999). Outgroups are not shown.

71 Agosia chrysogaster Mayden (1999). In the unweighted analysis, the 3 100 Agosia chrysogaster Mylocheilus clade was not resolved as a monophyletic 68 Erimonax monachus group (Fig. 5a). Instead, Mylocheilus plus Pogonichthys Erimonax monachus 100 (Fig. 2j), Clinostomus plus Richardsonius (Fig. 2i) and 52 Codoma ornata 9 100 Codoma ornata the remainder of the OPM clade formed an unresolved 9 68 37 Dionda erimyzonops trichotomy. Weighted, likelihood and bayesian analy- 100 Dionda ipni ses resolved this trichotomy and recovered the Notropis blennius Mylocheilus clade as a monophyletic group. Within the 3 Ericymba buccata Exoglossum clade, Exoglossum was resolved as the 9 3 Notropis nazas 5 Notropis leuciodus basal taxon in unweighted and weighted (TS/TV, 1/2, 3 31 Notropis volucellus 1/3) parsimony analyses. When the weight of transver- 100 Notropis volucellus sions was increased (TS/TV, 1/5) in the likelihood and 13 Hybognathus nuchalis 9 87 Hybognathus regius bayesian analyses, Rhinichthys was resolved as the 10 9 5 Luxilus chrysocephalus basal taxon in this clade. Relationships within the 16 Notropis atherinoides 54 9 Phenacobius and the Platygobio clades are consistent 98 Notropis stilbius 6 Lythrurus fumeus with results reported by Simons & Mayden (1999). 88 Lythrurus umbratilus Hybopsis amblops 9 68 Opsopoeodus emiliae SHINER CLADE 11 100 Opsopoeodus emiliae 4 63 Pimephales vigilax In the data set presented in this paper, representation Pimephales notatus within the shiner clade was greatly expanded from 29 Cyprinella venusta four (Simons & Mayden, 1999) to 35 OTUs. The taxa 9 100 Cyprinella formosa contained in this clade have not previously been con- 2 Notropis asperifrons 4 Notropis chrosomus sidered a monophyletic group. Within the shiner clade, Notropis maculatus relationships among taxa were largely unstable and Luxilus cerasinus changed a great deal depending upon the details of the 13 Notropis harperi analysis. This is probably a function of taxon sam- 82 17 Pteronotropis euryzonus 99 Pteronotropis hubbsi pling; there are at least 165 species that are part of the shiner clade and our analyses included only 29. How- Figure 5b. Consensus of 21 trees (TL = 5730) showing ever, some general observations can be made. The relationships within the shiner clade, from unweighted shiner clade contains the following genera: Agosia, parsimony analysis of 12S and 16S sequences. Number Codoma, Cyprinella, Dionda, Ericymba (Fig. 2c), above branches are Bremer support indices, numbers Erimonax (Fig. 2f), Hybognathus, Hybopsis, Luxilus, below nodes are bootstrap values. This figure is a continu- Lythrurus, Notropis (Fig. 2a,b,e), Opsopoeodus ation of Fig. 5a. (Fig. 2d), Pimephales and Pteronotropis. Many of these were formerly classified in the genus Notropis. May- den (1989) removed Pteronotropis, Cyprinella, Luxilus parasphenoid and the basioccipital. The functional and Lythrurus from synonymy with Notropis. Coburn significance of this opening is unknown, although its & Cavender (1992) removed Opsopoeodus from synon- presence may be correlated with increased eye size ymy with Notropis. Some of these genera may not be (Coburn, 1982). The posterior myodome is the site of monophyletic (Simons, Knott & Mayden, 2000; this attachment for the rectus muscles and the opening study); however, it is beyond the scope of this study to may be a secondary effect of increasing the volume of address critically the monophyly of all included gen- the myodome. The homology of this structure is com- era. The inclusion of the genera Codoma, Cyprinella, plex and unclear as not all taxa included in this clade Ericymba, Erimonax, Hybopsis, Luxilus, Lythrurus, have an opening between the parasphenoid and the Notropis, Opsopoeodus, Pimephales and Pteronotropis basioccipital, and in some taxa, the opening closes is not controversial, although Erimonax has been con- during ontogeny (Mayden, 1989). Simons & Mayden sidered a close relative of Erimystax (Mayden, 1989). (1999) discussed problems assessing homology of this However, the inclusion of Agosia, Dionda and Hybog- character. The results of analyses presented herein do nathus differs from previous hypotheses. Coburn & not resolve these issues. Cavender (1992) and Woodman (1992) considered the Simons & Mayden (1999) recognized six major clades monotypic genus, Agosia, closely related to Rhinich- within this group: the Mylocheilus, Campostoma, Exo- thys. Mayden (1989) placed Agosia as sister to the rest glossum, Phenacobius, Platygobio and shiner clades. of a chub clade, within his OPM clade. In our analyses, With a few exceptions, the interrelationships of these relationships of Agosia within the shiner clade are clades are the same as those reported by Simons & ambiguous. Agosia is resolved as sister to Erimonax

Agosia chrysogaster Agosia chrysogaster Opsopoeodus emiliae Opsopoeodus emiliae Pimephales notatus Pimephales vigilax Codoma ornata Codoma ornata Dionda erimyzonops Dionda ipni Erimyonax monachus Erimonax monachus Hybognathus nuchalis Hybognathus regius Luxilus chrysocephalus Notropis atherinoides Notropis stilbius Notropis leuciodus Notropis volucellus Notropis volucellus Lythrurus fumeus Lythrurus umbratilus Notropis nazas Ericymba buccata Hybopsis amblops Notropis asperifrons Notropis chrosomus Notropis blennius Notropis maculatus Luxilus cerasinus Notropis harperi Pteronotropis euryzonus Pteronotropis hubbsi Cyprinella venusta Cyprinella formosa Macrhybopsis aestivalis Macrhybopsis storeriana Macrhybopsis storeriana Platygobio gracilis Platygobio gracilis Platygobio gracilis Erimystax dissimilis Erimystax x-punctatus Phenacobius catostomus Phenacobius mirabilis Exoglossum laurae Exoglossum maxilinguae Oregonichthys crameri Oregonichthys kalawatseti Tiaroga cobitis Rhinichthys atratulus Rhinichthys osculus Campostoma anomalum Campostoma ornatum Nocomis bigutattus Nocomis leptocephalus Clinostomus elongatus Clinostomus funduloides Richardsonius balteatus Richardsonius egregius Mylocheilus caurinus Pogonichthys macrolepidodus Couesius plumbeus Couesius plumbeus Margariscus margarita Hemitremia flammea Hemitremia flammea Semotilus atromaculatus Semotilus thoreauianus Lepidomeda vittata Meda fulgida Snyderichthys copei Acrocheilus alutaceus Gila atraria Gila cypha Gila conspersa Gila nigrescens Gila pandora Gila orcutti Gila ditaenia Gila robusta Ptychocheilus lucius Gila coerulea Gila coerulea Relictus solitarius Eremichthys acros Siphateles bicolor Ptychocheilus oregonensis Ptychocheilus oregonensis Lavinia exilicauda Lavinia exilicauda Orthodon microlepidotus Orthodon microlepidotus Phoxinus erythrogaster Phoxinus neogaeus

0.01 substitutions/site

Figure 6. Tree produced by maximum likelihood analyses under HKY+I+Γ (–ln L = 30 587.898). Model parameters were set a priori. Outgroups are not shown.

1.00 Agosia chrysogaster Agosia chrysogaster 1.00 Opsopoeodus emiliae Opsopoeodus emiliae Pimephales vigilax Pimephales notatus 1.00 Erimonax monachus Erimonax monachus Hybopsis amblops .86 Notropis asperifrons .92 Notropis chrosomus Notropis blennius Notropis maculatus 1.00 Codoma ornata 1.00 Codoma ornata 1.00 Dionda erimyzonops Dionda ipni Ericymba buccata .84 1.00 Lythrurus fumeus Lythrurus umbratilus .96 Hybognathus nuchalis .83 Hybognathus regius Notropis nazas Luxilus chrysocephalus 1.00 Notropis atherinoides .80 Notropis stilbius Notropis leuciodus 1.00 Notropis volucellus Notropis volucellus 1.00 Luxilus cerasinus 1.00 Notropis harperi 1.00 Pteronotropis euryzonus Pteronotropis hubbsi .98 1.00 Cyprinella venusta Cyprinella formosa 1.00 Macrhybopsis aestivalis Macrhybopsis storeriana 1.00 1.00 Macrhybopsis storeriana 1.00 Platygobio gracilis 1.00 Platygobio gracilis Platygobio gracilis Erimystax dissimilis 1.00 1.00 Erimystax x-punctatus .99 1.00 Phenacobius catostomus Phenacobius mirabilis 1.00 Exoglossum laurae .92 Exoglossum maxilinguae 1.00 Oregonichthys crameri 1.00 Oregonichthys kalawatseti .96 Tiaroga cobitis 1.00 Rhinichthys atratulus Rhinichthys osculus 1.00 Campostoma anomalum 1.00 Campostoma ornatum .96 1.00 Nocomis biguttatus Nocomis leptocephalus .91 Clinostomus elongatus 1.00 Clinostomus funduloides 1.00 Richardsonius balteatus Richardsonius egregius 1.00 Mylocheilus caurinus Pogonichthys macrolepidotus 1.00 Couesius plumbeus Couesius plumbeus 1.00 Hemitremia flammea 1.00 Hemitremia flammea 1.00 Semotilus atromaculatus 1.00 Semotilus thoreauianus Margariscus margarita Lepidomeda vittata 1.00 Snyderichthys copei Meda fulgida Acrocheilus alutaceus .90 1.00 Gila atraria Gila cypha Gila conspersa 1.00 1.00 .98 Gila nigrescens Gila pandora Gila orcutti Gila ditaenia Gila robusta Ptychocheilus lucius Gila coerulea Gila coerulea Relictus solitarius .80 Siphateles bicolor Eremichthys acros 1.00 1.00 Ptychocheilus oregonensis Ptychocheilus oregonensis 1.00 1.00 Lavinia exilicauda Lavinia exilicauda 1.00 1.00 Orthodon microlepidotus Orthodon microlepidotus 1.00 Phoxinus erythrogaster Phoxinus neogaeus

0.01 substitutions/site

Figure 7. Tree produced by Bayesian analysis of 12S and 16S sequences under HKY+I+G (–ln L = 32 448.454). Posterior probabilities of 0.80 or greater are shown on branches. Outgroups are not shown.

(unweighted and weighted parsimony, TS/TV, 1/2); sis- particular, the groups represented by P. hubbsi and ter to Cyprinella (weighted parsimony, TS/TV, 1/3); or P. euryzonus were never resolved as sister taxa. Anal- sister to Opsopoeodus plus Pimephales (weighted par- yses presented here always recovered P. hubbsi and simony, TS/TV, 1/5, maximum likelihood, bayesian P. euryzonus as a monophyletic group, sister to Notro- analysis). pis harperi. This result may be a function of increased Both Mayden (1989) and Coburn & Cavender (1992) taxon sampling, although preliminary results from considered Dionda sister to Campostoma. In all our cytochrome b analysis of the shiner clade, including a analyses, Dionda was nested within the shiner clade, large number of taxa, do not recover a monophyletic sister to Codoma. Morphological similarity of Dionda Pteronotropis (R. L. Mayden, pers. comm.). to Campostoma is probably a function of convergence due to herbivory. Hybognathus was considered part of CONCLUSION a chub clade (Mayden, 1989) as were Dionda and Campostoma (Coburn & Cavender, 1992). The phylo- Addition of taxa to the 12S and 16S data set has genetic position of Hybognathus varies among our increased resolution and support for various clades of analyses; however, it is always nested within the North American phoxinins. However, a clear under- shiner clade. It was never recovered as sister to standing of the relationships within and among gen- Dionda or Dionda plus Codoma. We found no evidence era is still elusive. It is still unclear whether genera in for a chub clade as recognized by previous authors. the shiner clade represent monophyletic groups; in Different authors have variously classified Erimonax some cases it is clear that they do not. Although the monachus (Fig. 2f). Bailey (1956) placed this taxon in taxon sampling in this data set is much higher than Hybopsis based on the presence of a maxillary barbel. previous molecular hypotheses of relationship for Mayden (1989) removed E. monachus from Hybopsis to North American phoxinins, it is still too low, with only Erimystax based on ultrastructure of the barbel. This 80 of over 300 North American cyprinid species species was subsequently placed in Cyprinella based on included in these analyses. The 12S and 16S ribosomal osteology and scale morphology (Coburn & Cavender, RNA genes provide a great deal of resolution to the dif- 1992), biochemical data (Dimmick, 1993) and spawn- ficult phylogenetic relationships among North Ameri- ing behaviour (Jenkins & Burkhead, 1993). In our can phoxinins. However, these genes may not exhibit work, the phylogenetic position of E. monachus was the variation necessary to resolve relationships among highly sensitive to the particular analysis; however, we species within monophyletic groups identified in our never recovered it as sister to Cyprinella. We concur analyses. Several other mitochondrial genes have with Mayden et al.’s (1992) resurrection of the genus been used at this level including ND2 and ND4L Erimonax (Jordan, 1924) to contain this species. within the genus Cyprinella (Broughton & Gold, 2000) The genus Luxilus was represented by two species and cytochrome b within Notropis (Bielawski & Gold, in our analysis, L. cerasinus and L. chrysocephalus. 2001; Raley & Wood, 2001), Lythrurus (Schmidt, These were never recovered as a monophyletic group. Bielawski & Gold, 1998) and Luxilus (Dowling & Nay- This result is consistent with cytochrome b data (R. L. lor, 1997). A robust phylogenetic hypothesis of rela- Mayden, pers. comm.) that also indicate that Luxilus, tionships for the North American phoxinin fauna will as currently recognized, is not a monophyletic group. require denser taxon sampling and inclusion of genes Similarly, included members of the genus Notropis with a higher rate of nucleotide substitution. were not recovered as a monophyletic group. This taxon is very large and has generally served as a ACKNOWLEDGEMENTS dumping ground for any small, silvery North Ameri- can minnow of uncertain phylogenetic affinity. The We thank the following for help with this paper: M. taxonomic sampling in this study is not sufficient to Andersen, W. Dimmick, T. Dowling, P. Harris, G. Haas, resolve relationships of taxa currently included within J. D. McPhail, D. Markle and M. Sabaj provided spec- Notropis. However, it is evident from our analyses that imens; B. Kuhajda, R. Matson, E. B. Jones, R. Wood and any future study of Notropis relationships should S. R. Layman provided assistance in the field. J. Tomel- include representatives of all major lineages within leri graciously allowed us to use his illustrations of the shiner clade. North American phoxinins. We are especially grateful The genus Pteronotropis was represented in our for help from K. E. Knott and B. Kuhajda. S. P. Dins- analyses by two species, P. hubbsi and P. euryzonus, more provided concrete advice. We thank T. Gamble, T. representing the two species groups within the genus Kittel and B. Nagle for their comments on the manu- (Simons et al., 2000). The monophyly of this group is script. This research was supported by the Department controversial. Simons et al. (2000) found no evidence of Fisheries, Wildlife, and Conservation Biology at the for monophyly of the genus based on parsimony and University of Minnesota and by National Science Foun- likelihood analyses of cytochrome b sequence. In dation grant DEB-9307132 (to R.L.M.).

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APPENDIX Specimens examined. Institutional abbreviations are as listed in Leviton et al. (1985) and as corrected in Leviton & Gibbs (1988).

GenBank accession Species Locality Voucher no. no.

Abramis bjoerkna (Linnaeus) Brunnsviken Bay, Stockholm, Sweden UAIC 8533.03 AF038468 Agosia chrysogaster Girard San Francisco River, Catron Co., NM no voucher AF081838 Agosia chrysogaster No locality data UAIC 13018.01 AY216528 Acrocheilus alutaceus Agassiz & Pickering Kettle River, BC, Canada UAIC11366.01 AF023183, AF038469 © 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 139, 63–80 NORTH AMERICAN PHOXININ SYSTEMATICS 79

APPENDIX Continued

GenBank accession Species Locality Voucher no. no.

Campostoma anomalum (Rafinesque) Rolling Fork River, Nelson Co., KY UAIC 10147.02 AF023184, AF081839 Campostoma ornatum Rio San Juan, Durango, Mexico UAIC 7904.03 AF081843 Clinostomus elongatus (Kirtland) Craney Creek, Rowan Co., KY UAIC 7971.02 AF081840 Clinostomus funduloides Girard Mill Creek, McDowell Co., NC UAIC 7920.02 AF081842 Codoma ornata Rio San Juan, Durango, Mexico UAIC 7904.02 AY216529 Codoma ornata Rio Santa Isabel, Chihuahua, Mexico UAIC 7910.06 AY216530 Couesius plumbeus (Agassiz) Lizard Hot Springs, BC, Canada UAIC 11367.01 AF023185 Couesius plumbeus Mill Creek, BC, Canada UAIC 11366.01 AF023186, AF038470 Cyprinella formosa (Girard) Rio Santa Clara, Chihuahua, Mexico UAIC 7887.03 AF081841 Cyprinella venusta Girard Cahaba River, Bibb Co., AL no voucher AY216531 Dionda erimyzonops Hubbs & Miller Rio Matlapa, San Louis Potosi, Mexico UAIC 9153.01 AY216532 Dionda ipni Alvarez & Navarro Rio Matlapa, San Louis Potosi, Mexico UAIC 7901.05 AY216533 Eremichthys acros Hubbs & Miller Soldier Meadows, Humboldt Co., NV no voucher AF038471 Ericymba buccata Hashuqua Creek, Noxubee Co., MS UAIC 12994.01 AY216534 Erimonax monachus Buffalo River, Lewis Co., TN UAIC 10655.02 AY216535 Erimonax monachus Holston River, Scott Co., VA UAIC 7922.03 AY216536 Erimystax dissimilis (Kirtland) Copper Creek, Scott Co., VA UAIC 7953.03 AF081844 Erimystax x-punctatus (Hubbs & Crowe) Shoal Creek, Cherokee Co., KS UAIC 11551.01 AF081847 Exoglossum laurae (Hubbs) Sinking Creek, Craig Co., VA UAIC 11011.02 AF081845 Exoglossum maxillinguae Mettawee River, Washington Co., NY UAIC 10239.02 AF081846 Gila atraria (Girard) Sevier River, Piute Co., UT no voucher AF038472 Gila coerulea (2) Upper Klamath Lake, Klamath Co., OR OS 015083 AF038473, AF038474 Gila conspersa Garman Rio San Isabel, Chihuahua, Mexico UAIC 7910.01 AF038475 Gila cypha Miller Colorado River, Coconino Co., AZ no voucher AF023188, AF038477 Gila ditaenia Miller Sycamore Canyon, Santa Cruz Co., AZ no voucher AF038476 Gila nigrescens (Girard) Mimbres River, NM no voucher AF038478 Gila orcutti (Eigenmann & Eigenmann) CA, USA UAIC 11562.01 AF038479 Gila pandora (Cope) Rio Chama, NM no voucher AF038480 Gila robusta Baird & Girard Aravaipa Creek, Graham Co., AZ no voucher AF038481 Hemitremia flammea (Jordan & Gilbert) Unnamed spring, Madison Co., AL UAIC 10687.01 AF023189 Hemitremia flammea King Spring, Lauderdale Co., AL UAIC 10697.01 AF023190 Hybognathus nuchalis Agassiz Black River, Wayne Co., MO UAIC 10294.04 AF081848 Hybognathus regius Girard Turkey Creek, Chester Co., SC UAIC 7930.01 AY216538 Hybopsis amblops (Rafinesque) Clinch River, Scott Co., VA UAIC 7942.02 AY216537 Lavinia exilicauda Baird & Girard (2) Putah Creek, Yolo Co., CA OS 015075 AF038482, AF038483 Lepidomeda vittata Cope Rudd Creek, Apache Co., AZ no voucher AF023191 Luxilus cerasinus Roanoke River, Mongomery Co., VA UAIC 9859.03 AY216539 Luxilus chrysocephalus Rafinesque Conasauga River, Polk Co., TN UAIC 9820,21 AY216540 Lythrurus fumeus (Evermann) Tabby Creek, Lafayette Co., MS UAIC 10672.03 AY216541 Lythrurus umbratilus (Girard) Shawnee Creek, Cape Girardeau Co., MO UAIC 8416.02 AY216542 Macrhybopsis aestivalis (Girard) Rio Grande, Presido Co., TX UAIC 11563.01 AF081850 Macrhybopsis storeriana (Kirtland) Uphappee Creek, Macon Co., AL UAIC 10474.05 AF081852 Macrhybopsis storeriana Mississippi River, Jackson Co., IL UAIC 11552.01 AF081852 Margariscus margarita (Cope) Plover River, Marathon Co., WI UAIC 10241.07 AF023192, AF038485 Meda fulgida Girard Aravaipa Creek, Graham Co., AZ no voucher AF023193 Mylocheilus caurinus (Richardson) North Thompson River, BC, Canada UAIC 11553.01 AF081851 Nocomis biguttatus Black River, Reynolds Co., MO UAIC 11554.01 AF081854 Nocomis leptocephalus (Girard) Buffalo River, Wilkinson Co., MS UAIC 11555.01 AF081855

APPENDIX Continued

GenBank accession Species Locality Voucher no. no.

Notemigonus crysoleucas Mill Creek, Portage Co., WI UAIC 10527.02 AF023194, AF038487 Notemigonus crysoleucas Hunter Creek, Conecah Co., AL UAIC 10849.15 AY216543 Notropis asperifrons Suttkus & Raney Copperas Creek, Bibb Co., AL UAIC 9841.01 AY216544 Notropis atherinoides Rafinesque Waupaca River, Waupaca Co., WI UAIC 10485.06 AF023195, AF038486 Notropis blennius (Girard) Mississippi River, Jackson Co., IL UAIC 13017.01 AY216545 Notropis chrosomus Gurley Creek, Blount Co., AL UAIC 8425.01 AY216546 Notropis harperi Crooked Creek, Covington Co., AL UAIC 11026.03 AY216547 Notropis leuciodus Spring Creek, Polk Co., TN UAIC 9819.07 AY216548 Notropis maculatus Cowarts Creek, Houston Co., AL UAIC 10857.03 AY216549 Notropis nazas Meek Rio Saina Alto, Zacatecas, Mexico UAIC 7895.03 AY216550 Notropis stilbius Little Warrior River, Blount Co., AL UAIC 10046.04 AY216551 Notropis volucellus (Cope) Red Creek, Stone Co., MS UAIC 12913.01 AY216552 Notropis volucellus Bogue Chitto River, Washington Pa., LA UAIC 10466.03 AY216553 Opsopoeodus emiliae Spring Creek, Colbert Co., AL UAIC 10979.04 AY216554 Opsopoeodus emiliae Big Black River, Webster Co., MS UAIC 12995.01 AY216555 Orthodon microlepidotus (Ayres) (2) Putah Creek, Yolo Co., CA OS 015075 AF038488, AF038489 Oregonichthys crameri Shady Dell Pond, Lane Co., OR UAIC 11556.01 AF081856 Oregonichthys kalawatseti (Markle, Calapooia Creek, Douglas Co., OR UAIC 11556.01 AF081857 Pearsons & Bills) Phenacobius catostomus Cahaba River, Bibb Co., AL UAIC 7884.07 AF023196, AF081858 Phenacobius mirabilis (Girard) Cottonwood River, Chase Co., KS UAIC 11564.01 AF038492 Phoxinus erythrogaster (Rafinesque) Spring River, Lawrence Co., MO UAIC 11560.01 AF038490 Phoxinus neogaeus Cope Orange River, ME no voucher AF038493 Pimephales notatus Big Black River, Webster Co., MS UAIC 12995.02 AY216556 Pimephales vigilax Sipsey River, Tuscaloosa Co., AL no voucher AY216557 Platygobio gracilis (Richardson) Mississippi River, Jackson Co., IL UAIC 11558.01 AF023197, AF038491 Platygobio gracilis Canadian River, Quay Co., NM UAIC 11559.01 AF081859 Platygobio gracilis Sandoval Co., NM UAIC 12304.01 AY216558 Pogonichthys macrolepidotus (Ayres) Suisun Marsh, Solano Co., CA no voucher AF081860 Pteronotropis euryzonus Snake Creek, Russell Co., AL UAIC10493.04 AY216559 Pteronotropis hubbsi Chemin-a-haut, Moorehouse Pa., LA UAIC 7988.01 AY216560 Ptychocheilus lucius Dexter Fish Hatchery no voucher AY216561 Ptychocheilus oregonensis (Richardson) Errock Lake, BC, Canada UAIC 11561.01 AF038494 Ptychocheilus oregonensis Kettle River, BC, Canada UAIC 11365.02 AY216562 Relictus solitarius Hubbs & Miller Phalan Spring, Elko Co., NV no voucher AF038496 Rhinichthys atratulus (Hermann) Russell Creek, Clairborne Co., TN UAIC 9850.01 AF023198, AF038495 Rhinichthys osculus (Girard) Rock Springs Creek, Elko Co., NV no voucher AF081863 Richardsonius balteatus Sweltzer Creek, BC, Canada UAIC 11524.02 AF081861 Richardsonius egregius (Girard) Walker River, Lyon Co., NV no voucher AF081862 Rutilus rutilus (Linnaeus) Brunnsviken Bay, Stockholm, Sweden UAIC 8533.04 AF038484 Semotilus atromaculatus (Mitchill) Masterson Creek, Lawrence Co., AL UAIC 10875.13 AF023199 Semotilus thoreauianus Jordan Snake Creek, Russell Co., AL UAIC 10858.05 AF023200 Siphateles bicolor (Girard) Pyramid Lake, Washoe Co., NV no voucher AF038497 Snyderichthys copei (Jordan & Gilbert) Sevier River, Garfield Co., UT no voucher AF023201, AF038498 Tiaroga cobitis Girard Aravaipa Creek, Graham Co., AZ no voucher AF081863