COPEIA February 1

2000, No. 1COPEIA February 1 Copeia, 2000(1), pp. 1±10 Phylogenetic Relationships in the North American Cyprinid Genus Cyprinella (Actinopterygii: Cyprinidae) Based on Sequences of the Mitochondrial ND2 and ND4L Genes RICHARD E. BROUGHTON AND JOHN R. GOLD Shiners of the cyprinid genus Cyprinella are abundant and broadly distributed in eastern and central North America. Thirty species are currently placed in the genus: these include six species restricted to Mexico and three barbeled forms formerly placed in different cyprinid genera (primarily Hybopsis). We conducted a molecular phylogenetic analysis of all species of Cyprinella found in the United States, using complete nucleotide sequences of the mitochondrial, protein-coding genes ND2 and ND4L. Maximum-parsimony analysis recovered a single most-parsimonious tree for Cyprinella. Among historically recognized, nonbarbeled Cyprinella, the mitochondrial (mt) DNA tree indicated that basal lineages in Cyprinella are comprised largely of species with linear breeding tubercles and that are endemic to Atlantic and/or Gulf slope drainages, whereas derived lineages are comprised of species broadly distributed in the Mississippi basin and the American Southwest. The Alabama Shiner, C. callistia, was basal in the mtDNA tree, although a monophyletic Cyprinella that included C. callistia was not supported in more than 50% of bootstrap replicates. There was strong bootstrap support (89%) for a clade that included all species of nonbarbeled Cyprinella (except C. callistia) and two barbeled species, C. labrosa and C. zanema. The third barbeled species, C. monacha, fell outside of Cyprinella sister to a species of Hybopsis. Within Cyprinella were a series of well-supported species groups, although in some cases bootstrap support for relationships among groups was below 50%. A derived clade consisting of C. spiloptera, C. whipplei, C. venusta, and the southwestern C. lutrensis group was strongly supported. The species C. lutrensis and C. lepida were not monophyletic, suggesting further study and revision within this group are warranted. In general, the most-parsimonious mtDNA tree was similar in terms of relationships among species to those proposed more than 40 years ago by R. H. Gibbs. HE largest number of freshwater ®sh spe- reach more than about 12 cm (SL), but breed- T cies in North America belong to the family ing males often exhibit striking colors and elab- Cyprinidae, and because of their diversity, cyp- orate tuberculation. The native distribution of rinids have played an important role in studies the genus is streams and rivers from the Atlantic of speciation and North American biogeogra- coast to the Great Plains and from southern phy (Mayden, 1988; Buth et al., 1991; Krist- Canada to the Gulf Coast and northern Mexico. mundsdoÁttir and Gold, 1996). However, North Girard (1856) originally described the genus American cyprinids have received limited atten- Cyprinella, with Leuciscus bubalinus Baird and Gi- tion with respect to phylogenetic relationships rard (5 C. lutrensis) as the type species. The among genera and species (Mayden, 1989; Co- next 100 years saw extensive confusion regard- burn and Cavender, 1992; Simons and Mayden, ing taxonomy and systematics of Cyprinella, with 1998). The second largest cyprinid genus in member species being variously placed in at North America (after Notropis), Cyprinella pres- least 15 genera or subgenera (Gibbs, 1957). ently comprises of 30 species (Mayden, 1989; However, many workers noted that species of Robins et al., 1991) and includes some of the Cyprinella were a natural group, and Bailey and most abundant freshwater ®shes in eastern and Gibbs (1956) recognized Cyprinella as a subge- central North America. Adult Cyprinella seldom nus of the large shiner genus Notropis. Mayden q 2000 by the American Society of Ichthyologists and Herpetologists 2 COPEIA, 2000, NO. 1 (1989) identi®ed more than 34 osteological, behavioral, coloration, and tuberculation characters supporting monophyly of Cyprinella; conse- quently, he elevated Cyprinella to generic status (along with the related groups Luxilus and Lyth- rurus). Species relationships in Luxilus (Gilbert, 1964; Buth, 1979; Dowling and Naylor, 1997) and Lythrurus (Snelson, 1972; Mayden, 1989; Schmidt et al., 1998) have been studied exten- sively using both morphological and molecular characters. However, morphologically based as- sessments of relationships in Cyprinella (Gibbs, 1957; Mayden, 1989) yielded con¯icting results that have contributed to uncertainty regarding phylogenetic relationships of several species and/or species groups. Gibbs (1957) proposed relationships of Cypri- nella (Fig. 1A), based primarily on external mor- phology and pharyngeal tooth patterns. Al- though his analysis was essentially phenetic, Gibbs distinguished two groups of species, based largely on the presence of breeding tubercles in rows (considered ancestral) or scattered (considered derived). Basal linages included species endemic to drainages of either the Atlantic (C. callisema, C. nivea, C. pyrrhomelas, and C. xaenura) or Gulf of Mexico (C. callistia, and C. callitaenia), or both (C. leedsi). The derived group included six species (C. camura, C. galactura, C. lutrensis, C. spiloptera, C. venusta, and C. whipplei) broadly distributed in Mississip- pi River drainages along with two from the At- lantic slope (C. analostana and C. chloristia) and one from the Gulf slope (C. caerulea). Species of Cyprinella from the southwestern United States and Mexico (the C. lutrensis group) were not included beyond the suggestion that this Fig. 1. Phylogenetic hypotheses (redrawn for com- group is sister to C. spiloptera. Contreras-Bald- parison) for genus Cyprinella: (A) Gibbs (1957) and eras (1975) examined members of the C. lutren- (B) Mayden (1989). Only species included in the pre- sis group, noting the existence of additional, un- sent study are shown. Gibbs excluded species in the C. lutrensis group and C. gibbsi had not been de- described species, but did not propose explicit scribed. phylogenetic relationships. Mayden (1989) proposed relationships within Cyprinella based on a cladistic analysis of numerous osteological and morphological characters. This study incorpo- bercles-linear group). An exception was C. ca- rated all species historically recognized as Cypri- erulea, a species with scattered tubercles but that nella including all of the nominal species in the was sister to a clade of C. gibbsi and C. trichroistia C. lutrensis group. Mayden's phylogenetic hy- (both with linear tubercles). Both Gibbs (1957) pothesis (Fig. 1B) identi®ed two primary clades: and Mayden (1989) placed C. analostana and C. a C. lutrensis clade, comprised of 10 species pri- chloristia (primarily Atlantic slope endemics with marily inhabiting the American Southwest and scattered tubercles) as sister to C. whipplei, a cos- Mexico, and a C. whipplei clade that included mopolitan, tubercles-scattered species distribut- the remaining 17 species. Within Mayden's C. ed in Mississippi River and Gulf slope drainages. whipplei clade, species from the Mississippi basin Thus, Gibbs (1957) and Mayden (1989) agreed, (corresponding largely to Gibbs' tubercles-scat- in large part, on the distinction between two tered group) were basal relative to a clade com- groups of species, one of Atlantic and/or Gulf posed primarily of Atlantic and/or Gulf slope slope endemics with linearly arranged tubercles endemics (corresponding largely to Gibbs' tu- and one of more widespread species primarily BROUGHTON AND GOLDÐMOLECULAR SYSTEMATICS OF CYPRINELLA 3 from the Mississippi basin and southwest with cession numbers AF111205±AF111262). Speci- tubercles scattered. However, a trenchant differ- mens were collected by seine, placed in liquid ence between their hypotheses is the relative nitrogen or dry ice for transport, and stored at position of the two groups as basal versus de- 280 C until DNA extraction. Collection locali- rived. ties and voucher information are given in Ma- In addition to the 27 species included in Cy- terial Examined. prinella by Mayden (1989), three or four bar- Total DNAs were obtained by grinding tissues beled species, formerly placed in the genus Hy- in liquid nitrogen, extracting with phenol and bopsis Bailey (1951), possess morphological and chloroform, and precipitating with ethanol. behavioral similarities to nonbarbeled Cyprinella Ampli®cation of the ND2 gene employed prim- ( Jenkins and Lachner, 1971; Coburn and Cav- ers (developed by T. Dowling) in the ¯anking ender, 1992; Dimmick, 1993). These include C. tRNAMET and tRNATRP genes: ND2B-L: 59- monacha, C. labrosa, C. zanema, and the unde- aagctttcgggcccataccc-39, and ND2E-H: 59-ttctact- scribed thinlip chub, presently considered a taaagctttgaaggc-39. The gene was sequenced in subspecies of C. zanema ( Jenkins and Lachner, three segments using external primers and an 1980). Although C. labrosa and C. zanema share internal primer, developed during the course of morphological similarities, they differ more the study: ND2G-L: 59-cacaacaataatccttgccgc-39. from each other than do most sister-species Ampli®cation of the ND4L gene employed ARG pairs of Cyprinella, and neither appears to be primers in tRNA (ArgB-L: 59-caagaccctt- closely related to C. monacha ( Jenkins and Burk- gatttcggctca-39) and the coding region of the head, 1994). Mayden (1989) placed C. monacha adjacent ND4 gene (NAP2: 59-tggagcttct- 9 in the genus Erimystax and C. labrosa and C. za- acgtgrgcttt-3

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