2000, No. 1COPEIA February 1

Copeia, 2000(1), pp. 1–10

Phylogenetic Relationships in the North American Cyprinid (: ) 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 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 distrib- uted in the basin and the American Southwest. The Shiner, C. callistia, was basal in the mtDNA tree, although a monophyletic Cyprinella that in- cluded C. callistia was not supported in more than 50% of bootstrap replicates. There was strong bootstrap support (89%) for a 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. lu- trensis 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 fish 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. mundsdo`ttir 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 (ϭ 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 and systematics of Cyprinella, with 1998). The second largest cyprinid genus in member species being variously placed in at North America (after ), 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 fishes in eastern and Gibbs (1956) recognized Cyprinella as a subge- central North America. Adult Cyprinella seldom nus of the large shiner genus Notropis. Mayden

᭧ 2000 by the American Society of Ichthyologists and Herpetologists 2 COPEIA, 2000, NO. 1

(1989) identified more than 34 osteological, be- havioral, coloration, and tuberculation charac- ters 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 conflicting 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 tu- bercles in rows (considered ancestral) or scat- tered (considered derived). Basal linages in- cluded 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 de- rived 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) pro- posed 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) identified two primary : 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 GOLD—MOLECULAR 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- Ϫ80 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 Amplification of the ND2 gene employed prim- ( Jenkins and Lachner, 1971; Coburn and Cav- ers (developed by T. Dowling) in the flanking ender, 1992; Dimmick, 1993). These include C. tRNAMET and tRNATRP genes: ND2B-L: 5Ј- monacha, C. labrosa, C. zanema, and the unde- aagctttcgggcccataccc-3Ј, and ND2E-H: 5Ј-ttctact- scribed thinlip chub, presently considered a taaagctttgaaggc-3Ј. 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: 5Ј-cacaacaataatccttgccgc-3Ј. from each other than do most sister-species Amplification of the ND4L gene employed ARG pairs of Cyprinella, and neither appears to be primers in tRNA (ArgB-L: 5Ј-caagaccctt- closely related to C. monacha ( Jenkins and Burk- gatttcggctca-3Ј) and the coding region of the head, 1994). Mayden (1989) placed C. monacha adjacent ND4 gene (NAP2: 5Ј-tggagcttct- Ј in the genus Erimystax and C. labrosa and C. za- acgtgrgcttt-3 ); ArgB-L was used for sequencing. ␮ nema in his modified genus Hybopsis, yet there Fifty l amplification reactions included 1X Taq buffer (Promega Inc.), 200 mM of each dNTP, remains uncertainty regarding the phylogenetic 2 mM MgCl , 0.5 ␮M each primer, approxi- and taxonomic status of these barbeled taxa. 2 mately 100 ng of template DNA, and 0.25 units In this paper, we report the phylogenetic of Taq polymerase. Reaction conditions were 25 analysis of nucleotide sequences of the mito- cycles of 95 C for 1 min, 55 C for 1 min, and chondrially encoded ND2 and ND4L genes 72 C for 2 min. Double-stranded reaction prod- from 21 nonbarbeled and three barbeled spe- ucts were purified with Prep-A-Gene (Bio-Rad cies of Cyprinella. The study included all species Laboratories). Sequences were generated via of Cyprinella occurring in the United States (six dye-terminator reactions read on an ABI 377 species of the C. lutrensis group restricted to Prism sequencer or manually with the fmol sys- Mexico were not available). The ND2 gene tem (Promega Inc.), using 32P-labeled primers. (1047 nucleotides) has been shown to be infor- Sequences of the complete protein-coding re- mative phylogenetically in studies of numerous gion of each gene were aligned by eye and com- vertebrate groups, including fishes (Mindell bined into a single data matrix. The possibility and Thacker, 1996; Zardoya and Meyer, 1996; of nucleotide compositional bias among taxa, Kocher and Carleton 1997). ND4L is smaller, including outgroups, was examined with a chi- consisting of 297 nucleotides but has been square test of nucleotide frequency homogene- shown to be useful in phylogenetic analysis of ity. Potential differences in evolutionary rate closely related taxa (Bielawski and Gold, 1996). among lineages were investigated with Tajima’s (1993) 1D test of rate homogeneity. PAUP* vers. MATERIALS AND METHODS 4.01b was used for heuristic parsimony searches with 100 random addition replicates, TBR Complete ND2 and ND4L gene sequences branch swapping, and ACCTRAN optimization. were obtained from all species and included at Equal weighting of all character state changes least two individuals of each species except for was used in initial searches. Bootstrapping C. gibbsi, C. formosa, C. labrosa, C. zanema, C. mon- (1000 replicates) was used to assess relative sup- acha, and the outgroup species. Where possible, port for clades on the most-parsimonious tree. conspecific individuals were from geographical- Congruence of phylogenetic signal among ly distant localities. Outgroup taxa, Notropis ath- genes was examined by comparison with ran- erinoides, Lythrurus roseipinnis, Luxilus cornutus, dom partitions of the data with the incongru- and Hybopsis winchelli, were selected based on ence length test (Farris et al., 1995) imple- hypothesized phylogenetic relationships to Cy- mented in PAUP*. prinella (Mayden, 1989; Coburn and Cavender, 1992). All sequences were generated in our lab- RESULTS AND DISCUSSION oratory (except L. cornutus, provided by T. Sequence variation.—Only one alignment gap, in- Dowling) and may be found in GenBank (ac- volving a deletion of a single codon in the ND2 4 COPEIA, 2000, NO. 1

TABLE 1. NUCLEOTIDE VARIATION BY GENE AND CODON POSITION.

Number of variable sitesb Parsimony infor- All Total sites mativea positions 1st 2nd 3rd ND2 1047 460 560 161 (0.461) 60 (0.172) 339 (0.971) ND4L 297 113 161 39 (0.394) 29 (0.293) 93 (0.939) Total 1344 573 721 200 89 432

a Sites with alternative nucleotide state in more than one individual. b Parentheses represent proportion of variable sites. gene in C. caerulea, was observed. Mean nucle- change consistent with the tree (or homoplasy, otide frequencies for all taxa were A ϭ 0.254, C taken as 1 Ϫ ci). The ci values were lower for ϭ 0.303, G ϭ 0.167, and T ϭ 0.276. There was the two transition changes (T ↔ C, A ↔ G) no significant difference in nucleotide compo- than for transversion changes (all others) and 2 sition among the taxa [␹ (87) ϭ 69.8, P ϭ 0.91]. third-codon positions exhibited lower ci values The pronounced bias against G nucleotides is relative to first- and second-codon positions. similar to that observed in mtDNA coding genes ND4L exhibited slightly higher ci values than in other vertebrate taxa (e.g., Collins et al., ND2, although ND4L provided substantially 1994). Uncorrected sequence difference varied fewer useful characters. Despite variable levels among ingroup taxa, from 2.08% (C. callitaenia of homoplasy across genes, phylogenetic signal vs C. zanema) to 24.26% (C. proserpina vs C. spi- of each gene did not differ significantly from loptera and C. proserpina vs C. formosa). Among random data partitions (incongruence length ingroup taxa, 721 of 1344 sites were variable test, P ϭ 0.366). with 573 sites informative phylogenetically (Ta- ble 1). Nearly all third-codon positions were var- Phylogenetic analysis.—Maximum-parsimony anal- iable in both genes. More second-codon posi- ysis, employing equal weights for all characters, tions were variable in the ND4L gene, whereas recovered a single most-parsimonious tree for ND2 exhibited more variation at first codon po- Cyprinella (Fig. 2). However, because of the high sitions. This is consistent with Bielawski and proportion of changes at third-codon positions Gold’s (1996) observation that among cyprinids in both genes (Table 1), and low ci values for ND4L appears to evolve rapidly at the amino transitions relative to transversions (Table 2), we acid level. We examined relative amounts of ho- considered the possibility that unweighted char- moplasy in the data with a modification of the acters might support spurious phylogenetic hy- consistency index (ci; Kluge and Farris, 1969) potheses. Although differential weighting of for each of the six classes of nucleotide trans- characters (by codon position) and character- formation (Table 2). All changes on the tree state changes (transitions vs transversions) (output as a ‘‘change list’’ by PAUP) were sorted might overcome potentially misleading phylo- by codon position and by class of change. For genetic signal at rapidly evolving sites (e.g., Si- each group, the minimum number of changes mon et al., 1994; Kocher and Carleton, 1997; (ϭ the number of characters exhibiting a Martin and Bermingham, 1998), it is not clear change of that type) was divided by the total when multiple nucleotide changes generate number of changes observed. Resulting values spurious or misleading hypotheses of relation- provide a useful measure of the amount of ships. Where homoplasy is evident, the goal is to extract true phylogenetic signal from back- ground noise. To this end, it may be useful to TABLE 2. CONSISTENCY INDICES FOR CLASSES OF NU- identify a priori characters that are misleading CLEOTIDE CHANGE BY GENE AND CODON POSITION. (and to what extent), and impose a weighting ND2 ND4L scheme that penalizes homoplastic changes Change 1st 2nd 3rd 1st 2nd 3rd without losing historically consistent informa- tion. A ↔ G 0.428 0.313 0.327 0.643 0.813 0.462 ↔ The successive approximations approach of C T 0.404 0.508 0.346 0.379 0.846 0.412 Farris (1969) imposes weights that are based on A ↔ C 0.776 0.889 0.568 0.889 0.909 0.759 the relative consistency of characters on a tree. A ↔ T 0.730 1.000 0.683 0.800 0.750 0.810 C ↔ G 0.680 0.800 0.633 0.833 0.833 0.727 An advantage of this approach is that it provides T ↔ G 0.750 0.800 0.741 0.833 0.714 0.750 an objective criterion for determining the rela- tive weight for a character; a disadvantage is that BROUGHTON AND GOLD—MOLECULAR SYSTEMATICS OF CYPRINELLA 5

1B). Topological constraints consistent with each of the morphological studies (but includ- ing additional unresolved taxa in the case of Gibbs’ hypothesis) were used to obtain starting trees. After one round of reweighting, each analysis converged on the same topology, that of the equally weighted mtDNA tree. Thus, re- gardless of the initial tree or index used for weights, the equal weights mtDNA tree was re- covered. Furthermore, employing subjective a priori character-state weights (transversions vs transitions: 5:1, 10:1, and 1:0) resulted in tree topologies that differed little from the topology derived from equal weighting of characters. These results support the hypothesis that even when there have been numerous changes per nucleotide site leading to apparent ‘‘saturation’’ for some types of changes, substantial phyloge- netic signal may be retained (Fitch and Ye, 1991; Yang, 1998).

Relationships among species.—Bootstrap propor- tions exceeding 50% are shown on the most- parsimonious mtDNA tree (Fig. 2). Of the spe- Fig. 2. Single, most-parsimonious tree for ND2 cies examined only C. lutrensis and C. lepida (see and ND4L nucleotide sequences in the genus Cypri- below) were not monophyletic. The tradition- nella. Branch lengths reflect number of character ally recognized Cyprinella (exclusive of barbeled state changes. Scale bar corresponds to 1% difference species) form a monophyletic group relative to in nucleotide sequence. Numbers indicate proportion outgroup taxa in the shortest mtDNA tree (Fig. of bootstrap replicates supporting a node (branch). 2). With respect to barbeled species, C. labrosa Tree length ϭ 3320 steps, CI ϭ 0.353, CI (excluding and C. zanema were deeply embedded within uninformative) ϭ 0.318, and RI ϭ 0.455. C. lepida-a, Cyprinella as sister species to C. pyrrhomelas and Frio River, Texas; C. lepida-b, Nueces River, Texas; C. lutrensis-a, Brazos and Pecos rivers, Texas and New C. callitaenia, respectively, whereas C. monacha Mexico, respectively; C. lutrensis-b, Colorado River, fell outside a monophyletic Cyprinella as sister to Texas. Hybopsis winchelli. These results contrast with Dimmick’s (1993) hypothesis that C. monacha is allied with Cyprinella, whereas C. labrosa and C. it is not clear to what extent final results are zanema are related to Hybopsis, and with Jenkins dependent on the initial topology used to assess and Lachner’s (1971) morphologically based character consistency (Swofford et al., 1996). If hypothesis that C. labrosa and C. zanema are sis- the same characters are consistently misleading ter taxa. A close relationship between C. zanema based on a variety of different starting trees, one and C. callitaenia is consistent with Jenkins and can place reasonable confidence in the succes- Burkhead’s (1994) suggestion that C. zanema sive weighting approach. Its efficacy is based on and C. labrosa may be related to certain south- the notion that characters reflecting the true eastern species with an inferior mouth, for ex- phylogeny will support the same tree (i.e., will ample, C. nivea and C. leedsi. However, a close be additive), whereas homoplastic characters phyletic relationship between C. labrosa and C. support numerous, essentially random trees. pyrrhomelas was unexpected, because the two Thus, homoplastic characters, as a group, species share fewer morphological similarities. should not be consistent on any tree, making it Because C. labrosa and C. pyrrhomelas are sym- possible to extract phylogenetic signal from patric where they were collected [Green River noise even when historically consistent charac- (Santee River drainage), Polk County, North ters are in the minority (Farris, 1983). Carolina] hybridization may have generated a We applied character weights implied by the C. labrosa-like population with mitochondrial ge- ci or the retention index (ri; see Archie, 1996) nomes derived from C. pyrrhomelas. Cyprinella on three different starting trees: (1) the equally pyrrhomelas to C. labrosa would be the inferred weighted, mtDNA tree; (2) Gibbs’ hypothesis direction of introgression as the mtDNA se- (Fig. 1A); and (3) Mayden’s hypothesis (Fig. quences of these species were included in a 6 COPEIA, 2000, NO. 1 clade with C. xaenura (Fig. 2), a species hypoth- one (C. caerulea) with scattered tubercles, and esized previously (Gibbs, 1957; Mayden, 1989) one barbeled species (C. zanema). Bootstrap to be sister to C. pyrrhomelas. Hybridization support in the mtDNA tree for the hierarchical among taxa (e.g., Harrison, 1993), and arrangement ((C. callisema, C. nivea)(C. leedsi specifically among North American cyprinids, is (C. callitaenia, C. zanema))) was 100% for all common (Hubbs, 1955; Smith, 1992) and may branches. Gibbs (1957) and Mayden (1989) result in introgression of mtDNA genomes be- concurred on monophyly of the four nonbar- tween species (Dowling et al., 1989). A potential beled species and on the hypothesis C. nivea (C. hybridization event between C. labrosa and C. leedsi (C. callisema, C. callitaenia)). Inclusion of pyrrhomelas would appear to be of some antiq- C. caerulea in the mtDNA tree as the basal mem- uity, given the 14.4% nucleotide sequence dif- ber of this assemblage was not strongly sup- ference between them. ported. Gibbs (1957) placed C. caerulea as basal Among historically recognized, nonbarbeled in a group that contained C. venusta, C. spilop- Cyprinella, the mtDNA tree (Fig. 2) indicated tera, and the C. lutrensis group, in large part be- that basal lineages in Cyprinella are composed cause C. caerulea has scattered tubercles and largely of Gibbs’ (1957) tubercles-linear species. (along with C. venusta and C. spiloptera) yellow These include all the Atlantic and/or Gulf slope pigmentation on fins of breeding males and re- endemics except C. analostana and C. chloristia, duced dorsal fin pigmentation. Mayden (1989), both of which have scattered tubercles. Includ- alternatively, placed C. caerulea as sister to the ed also in a basal position is C. caerulea, a Gulf C. gibbsi/C. trichroistia species pair. Because of slope endemic with scattered tubercles. Some- low bootstrap support, C. caerulea is likely best what surprisingly, C. callistia occupied the basal considered for now as a single lineage within position in the mtDNA tree. Both Gibbs (1957) the large clade that is sister to the C. gibbsi/C. and Mayden (1989) placed C. callistia as sister trichroistia species pair. However, the strong to a clade that included C. nivea, C. leedsi, C. bootstrap support in the mtDNA tree for clades callitaenia, and C. callisema. These latter four spe- that include C. venusta, C. spiloptera, and the C. cies (and C. zanema) also formed a clade in the lutrensis group (see below) argue against Gibbs’ mtDNA tree, but in the mtDNA tree, this clade hypothesis regarding placement of C. caerulea. was sister to C. caerulea, not C. callistia. However, A second clade (i.e., sister to the one contain- the basal position of C. callistia in the mtDNA ing C. caerulea, C. nivea, and others) contained tree is consistent with Gibbs’ (1957) observa- a number of mtDNA lineages with fairly strong tions that (1) retention of linear tubercles on bootstrap support. Basal in this large clade was the head and notal-ridge was the only character an assemblage with C. xaenura as sister to the (he found) linking C. callistia to Cyprinella, and species pair C. pyrrhomelas and C. labrosa. All (2) C. callistia was ‘‘. . . by far the most divergent three are Atlantic slope endemics: C. pyrrhomelas form in the [sub]genus.’’ Relative to bootstrap and C. xaenura are tubercles-linear species at the support, a monophyletic Cyprinella that includ- base of Gibbs’ (1957) tree; whereas C. labrosa is ed C. callistia was not supported by more than one of the three barbeled Cyprinella. Mayden 50% of replicates. (1989) also found C. pyrrhomelas and C. xaenura There was strong bootstrap support (89%) to be sister species with this group sister to a for a clade of all species of Cyprinella except C. clade that included C. caerulea, C. gibbsi, and C. callistia (and C. monacha). The sister species C. trichroistia. Two additional assemblages within gibbsi and C. trichroistia are the basal lineage this second clade were (1) the species pair C. within this clade and this sister relationship is analostana and C. chloristia, and (2) a clade that consistent with their prior status as conspecifics included C. proserpina as sister to the species (Howell and Williams 1971). However, place- pair C. camura and C. galactura. Both Gibbs ment of the two as the base of Cyprinella is in- (1957) and Mayden (1989) placed C. analostana consistent with Gibbs’ (1957) and Mayden’s as sister to C. chloristia. Based on morphological (1989) hypotheses in which C. trichroistia (and similarities, Gibbs (1957, 1963) hypothesized C. C. gibbsi in Mayden’s study) was embedded well whipplei as sister to this species pair, placing this within Cyprinella (Fig. 1). larger clade within his derived, tubercles-scat- The remaining species of Cyprinella fell into a tered group. Mayden (1989) also found C. whip- series of fairly well-supported lineages, however, plei to be the sister of the C. analostana/C. chlor- bootstrap support for relationships among some istia species pair. The sister relationship be- of these lineages was not high. In one group tween C. camura and C. galactura in the mtDNA were six Atlantic and/or Gulf slope endemics, tree (supported by 100% of bootstrap repli- including four species (C. callisema, C. callitaen- cates) is consistent with Gibbs (1957, 1961) but ia, C. leedsi, and C. nivea) with linear tubercles, not with Mayden (1989) who placed C. camura BROUGHTON AND GOLD—MOLECULAR SYSTEMATICS OF CYPRINELLA 7 as basal to the C. whipplei (C. analostana, C. chlor- opportunity for homoplasy to obscure historical istia) clade and C. galactura as sister to C. ven- phylogenetic signal (Kuhner and Felsenstein, usta. The relationship of C. proserpina with the 1994; Lockhart et al., 1994), we view the phy- C. camura/C. galactura species pair in the logenetic placement of C. proserpina in the mtDNA tree was unexpected and is discussed mtDNA tree as tentative. However, a maximum more fully below. likelihood analysis accounting for rate hetero- A derived clade in the mtDNA tree included geneity (PAUP*, HKY model, 4-class discrete many of the cosmopolitan, tubercles-scattered gamma model of among-site rate variation) re- species distributed primarily in the Mississippi covered a tree (not shown) consistent with the River basin and the American Southwest. Rela- parsimony analysis, including the position of C. tionships of species within this clade generally proserpina. Moreover, a uniquely shared chro- had strong support and are similar to Gibbs’ mosomal nucleolus organizer region (NOR) (1957) hypothesis where C. venusta is sister to a character state supports the mtDNA hypothesis clade of C. spiloptera and the C. lutrensis group. that C. proserpina is related to C. camura and C. Alternatively, according to Mayden (1989), the galactura (Amemiya and Gold, 1990; Amemiya C. lutrensis group (including C. proserpina) is sis- et al., 1992). ter to all other Cyprinella, with C. spiloptera as the Cyprinella from the American Southwest (ex- basal lineage in his C. whipplei clade. As noted cept C. proserpina) formed a monophyletic above, both Gibbs (1957) and Mayden (1989) group in the mtDNA tree, however, within this placed C. whipplei as sister to the C. analostana/ group, neither C. lepida nor C. lutrensis were C. chloristia species pair. However the sister re- monophyletic (Fig. 2). These results are consis- lationship of C. whipplei and C. spiloptera in the tent with the complex and confusing taxonomic mtDNA tree is consistent with their earlier con- history of the species group (Chernoff and Mill- sideration as conspecifics (Hubbs and Greene, er, 1982; Matthews, 1987). As an example, both 1928). Furthermore, analysis of over 700 bp C. formosa (Contreras-Balderas, 1975; Chernoff from the mtDNA control region from several and Miller, 1982) and C. lepida (Hubbs, 1956, allopatric populations did not find these species 1972: Matthews 1987) have at times been con- to be reciprocally monophyletic (Broughton, sidered to be distinct species or subspecies of C. 1995). lutrensis. Previous mtDNA studies (Richardson Placement of C. proserpina outside of the C. and Gold, 1995, 1999) indicated that C. lepida lutrensis group is consistent with Gold and Rich- in the Frio and Nueces Rivers were specifically ardson (1999) who estimated 17.2% sequence distinct and that C. lepida from the Frio River divergence between C. proserpina and the C. lu- was more closely related to C. formosa than to C. trensis group but 10.5% divergence within the lepida from the Nueces River. Hybridization C. lutrensis group. Morphological similarity and among these taxa may have generated the con- geographic distribution have been used to sup- flicting mtDNA haplotype relations, but the ab- port the hypothesis that C. proserpina belongs to sence of a clear geographic pattern relating the C. lutrensis group (Hubbs and Miller, 1978), mtDNA haplotypes could suggest multiple (and where it was hypothesized by Mayden (1989) to perhaps undecipherable) hybridization events. be sister to C. lepida. In general, the most-parsimonious mtDNA The terminal branch leading to C. proserpina tree (Fig. 2) appears to be more similar to the in the mtDNA tree (Fig. 2) is atypically long hypothesis of Gibbs (1957; Fig. 1A) than to the (177 inferred nucleotide changes), suggesting hypothesis of Mayden (1989; Fig. 1B). Much of that mtDNA in C. proserpina may have evolved the apparent similarity derives from placement unusually rapidly. We evaluated evolutionary of the tubercles-linear, Atlantic and/or Gulf rate variation with Tajima’s (1993) relative rate slope endemics as basal in the Cyprinella tree. test on 10 species representing major ingroup We evaluated the degree of similarity among clades in the mtDNA phylogeny. The included phylogenetic hypotheses by considering only species were C. callistia, C. trichroistia, C. nivea, the 17 species common to all three studies and C. xaenura, C. analostana, C. proserpina, C. ca- estimating the number of steps required for the mura, C. venusta, C. whipplei, and C. formosa. All most-parsimonious, unrooted network for nine pairwise comparisons involving C. proserpi- mtDNA (1843 steps), and for Gibbs’ hypothesis na differed significantly (Bonferroni corrected (2098 steps) and Mayden’s hypothesis (2225 for multiple tests) from rate homogeneity. By steps) when topological constraints correspond- comparison, only two of the other 36 compari- ing to the two morphological studies were en- sons (C. whipplei vs C. callistia and C. trichroistia) forced on the mtDNA data. Gibbs’ and May- were significant. Because large differences in den’s hypotheses were 13.8% and 20.7% longer, the number of changes per branch increase the respectively, than the shortest mtDNA tree. A 8 COPEIA, 2000, NO. 1 nonparametric Wilcoxon signed-ranks test TCWC: 8294.01; C. callisema, Middle Oconee (Templeton, 1983) indicated that both of the River, Oconee/Clarke Co., Georgia (Altamaha morphologically based topologies were signifi- River), TCWC: 8294.02; C. callitaenia, Flint Riv- cantly longer than the most-parsimonious er, Meriwether Co., Georgia (Apalachicola Riv- mtDNA tree (P Ͻ 0.0001, each). However, the er), TCWC: 7986.02; C. trichroistia, Little Cahaba greater similarity of Gibbs’ hypothesis to the River, Bibb Co., Alabama (Cahaba River), Con- mtDNA tree appears to be based on the polarity asaugua River, Bradley Co., Tennessee (Coosa of the tree (southeastern endemics basal and River); C. caerulea, Conasaugua River, Bradley Mississippi basin/Southwestern forms derived). Co., Tennessee (Coosa River), Little River, Differences among the three phylogenetic hy- Cherokee Co., Alabama (Coosa River); C. lutren- potheses may be related, in part, to interpreta- sis, Brazos River, Milam Co., Texas, Colorado tion of morphological characters (exemplified River, Concho Co., Texas, Pecos River, De Baca by differences in the two morphological stud- Co., New Mexico; C. proserpina, Devil’s River, Val ies) and by possible differences in evolutionary Verde Co., Texas, TCWC: 6988.01; C. lepida, Frio rates among morphological and mtDNA char- River, Bandera Co., Texas, TCWC: 7264.01, Nu- acters. Ultimately, more nucleotide characters eces River, Edwards/Real Co., Texas, TCWC: from additional genes may aid in recovering 7267.01; C. formosa, Dexter National Hatch- more robust support for all clades in an hypoth- ery, TCWC: 6622.04; C. monacha, Buffalo River, esis of phylogenetic relationships of Cyprinella. Lewis Co., Tennessee (Tennessee River); C. za- nema, Second Broad River, Rutherford Co., MATERIAL EXAMINED North Carolina (Santee River), TCWC: 7983.01; C. labrosa, Green River, Polk Co., North Caroli- Voucher specimens are deposited in the Tex- na (Santee River), TCWC: 7982.01; Hybopsis win- as Cooperative Wildlife Collection (TCWC) at chelli, Cahaba River, Bibb Co., Alabama; Luxilus Texas A&M University. Collection localities cornutus, Kalamazoo River, Calhoun Co., Michi- (drainage) of material examined are as follows: gan; Lythrurus roseipinnis, Chickashay River, , Big River, Washington Co., Clarke Co., Mississippi (Pascagoula River), Missouri (Meramec River), Stony Creek, Ver- TCWC: 7658.01; Notropis atherinoides, Brazos Riv- million Co., Illinois (Wabash River), Susquehan- er, Haskell Co., Texas. na River, Tioga Co., ; C. whipplei, Big Darby Creek, Pickaway Co., Ohio (Scioto River), ACKNOWLEDGMENTS Hocking River, Athens Co., Ohio (Ohio River), Ouachita River, Montgomery Co., Arkansas; C. We thank J. Bielawski, D. Billings, T. Dowling, galactura, Choats Creek, Giles Co., Tennessee D. Etnier, B. Freeman, T. Frizzell, B. Jensen, M. (Tennessee River), Holston River, Scott Co., Vir- Howell, L. Richardson, M. Sabaj, and W. Starnes ginia (Tennessee River); C. venusta, Cahaba Riv- for providing, or assistance collecting, speci- er, Bibb Co., Alabama, Llano River, Kimble Co., mens. Helpful discussions and/or comments on Texas (Colorado River), TCWC: 7283.01; C. ca- the manuscript were provided by M. Coburn, D. mura, Thompson Creek, West Feliciana Par., Etnier, M. Howell, R. Jenkins, W. Matthews, L. Louisiana (Mississippi River), Chickaskia River, Richardson, and W. Starnes. We thank R. Ford Kingman Co., Kansas (Arkansas River); C. callis- and L. Stewart for performing some of the se- tia, Cahaba River, Bibb Co., Alabama, Conasau- quencing reactions. All collections were made ga River, Bradley Co., Tennessee (Coosa River); under valid scientific collecting permits issued C. leedsi, Alpaha River, Echolz Co., Georgia (Su- by the states where permits were required. Spec- wanee River); C. pyrrhomelas, Green River, Polk imens of Cyprinella monacha (federally threat- Co. North Carolina (Santee River), TCWC: ened) were provided by the Illinois Natural His- 7982.02; C. analostana, Eno River, Durham Co., tory Survey and collected under Tennessee North Carolina (Nuese River), White Oak Wildlife Resources Agency Scientific Study Per- Creek, Johnston Co., North Carolina (Nuese mit No. 809 issued to L. Page. Specimens of Cy- River), TCWC: 7985.01; C. gibbsi, Tallapoosa Riv- prinella caerulea were collected in 1985 and 1988, er, Randolph Co., Alabama; C. chloristia, Broad prior to being listed (federally threatened) in River, Rutherford Co., North Carolina (Santee 1992. Specimens of Cyprinella formosa were pro- River), TCWC: 7984.01; C. nivea, Broad River, vided by the Dexter National Fish Hatchery, Maddison/Elbert Co., Georgia (Savannah Riv- Dexter, New Mexico. Funding was provided in er), TCWC: 8292.01, Deep River, Lee/Chatham part by the National Science Foundation (BSR- Co., North Carolina (Cape Fear River), TCWC: 90-20217), and in part by the Texas Agricultural 7981.01; C. xaenura, Middle Oconee River, Oco- Experiment Station (H-6703). Much of the work nee/Clarke Co., Georgia (Altamaha River), was carried out in the Center for Biosystematics BROUGHTON AND GOLD—MOLECULAR SYSTEMATICS OF CYPRINELLA 9 and Biodiversity at Texas A&M University, a fa- United States (Pisces: Cyprinidae). Unpbl. Ph.D. cility funded, in part, by the National Science diss., Tulane Univ., New Orleans, LA. Foundation (DIR-89-07006). The manuscript DIMMICK, W. W. 1993. A molecular perspective on the was prepared while REB was a NSF/Sloan Post- phylogenetic relationships of the barbeled min- nows, historically assigned to the genus Hybopsis doctoral Research Fellow in Molecular Evolu- (Cyprinidae: ). Mol. Phylogenet. tion in the laboratory of R. Harrison at Cornell Evol. 2:173–184. University. This paper is contribution number DOWLING,T.E.,AND G. J. P. NAYLOR. 1997. Evolution- 93 of the Center for Biosystematics and Biodi- ary relationships of in the genus Luxilus versity at Texas A&M University. (Teleostei: Cyprinidae) as determined from cyto- chrome b sequences. Copeia 1997:758–765. ,G.R.SMITH, AND W. M. BROWN. 1989. Re- LITERATURE CITED productive isolation and introgression between No- tropis cornutus and Notropis chrysocephalus (family Cy- AMEMIYA,C.T.,AND J. R. GOLD. 1990. Cytogenetic prinidae): comparison of morphology, allozymes, studies in North American minnows (Cyprinidae). and mitochondrial DNA. Evolution 43:620–634. XVII. Chromosomal NOR phenotypes of 12 spe- FARRIS, J. S. 1969. A successive approximations ap- cies, with comments on cytosystematic relationships proach to character weighting. Syst. Zool. 18:374– among 50 species. Hereditas 112:231–247. 385. ,P.K.POWERS, AND J. R. GOLD. 1992. Chro- . 1983. The logical basis of phylogenetic anal- mosomal evolution in the North American cypri- ysis, p. 7–36. In: Advances in cladistics. Vol. 2. N. nids, p. 534–550. In: Systematics, historical ecology, Platnick and V. Funk (eds.). Columbia Univ. Press, and North American freshwater fishes. R. L. May- New York. den (ed.). Stanford Univ. Press, Stanford, CA. ,M.KALLERSJO,A.G.KLUGE, AND C. BULT. ARCHIE, J. W. 1996. Measures of homoplasy, p. 153– 1995. Testing significance of incongruence. Cladis- 188. In: Homoplasy. M. Sanderson and L. Hufford tics 10:315–319. (eds.). Academic Press, New York. FITCH, W. M., AND J. YE. 1991. Weighted parsimony: BAILEY, R. M. 1951. A check-list of the fishes of Iowa, does it work? p. 147–154. In: Phylogenetic analysis with keys for identification, p. 327–377. In: Iowa of DNA sequences. M. Miyamoto and J. Cracraft fish and fishing. J. Harlan and E. Speaker (eds.). (eds.). Oxford Univ. Press, New York. Iowa Conservation Commission, Des Moines, IA. GIBBS, R. H. 1957. Cyprinid fishes of the subgenus , AND R. H. GIBBS JR. 1956. Notropis callistius,a Cyprinella of Notropis I. Systematic status of the sub- new cyprinid fish from Alabama, Florida, and Geor- genus Cyprinella, with a key to the species exclusive gia. Occ. Pap. Mus. Zool., Univ. Mich. 576:1–14. of the lutrensis-ornatus complex. Copeia 1957:185– BIELAWSKI,J.P.,AND J. R. GOLD. 1996. Unequal syn- 195. onymous substitution rates within and between two . 1961. Cyprinid fishes of the subgenus Cypri- protein-coding mitochondrial genes. Mol. Biol. nella of Notropis. IV. The Notropis galacturus-camurus Evol. 13:889–92. complex. Am. Midl. Nat. 66:337–354. BROUGHTON, R. E. 1995. Evolution of duplicated se- . 1963. Cyprinid fishes of the subgenus Cypri- quences in mitochondrial DNA. Unpubl. Ph.D. nella of Notropis. The Notropis whipplei-analostanus diss., Arizona State Univ., Tempe. complex. Copeia 1963:511–528. BUTH, D. G. 1979. Biochemical systematics of the cyp- GILBERT, C. R. 1964. The American cyprinid fishes of rinid genus Notropis. I. The subgenus Luxilus. the subgenus Luxilus (genus Notropis). Bull. Fla. Biochem. Syst. Ecol. 7:69–79. State Mus., Biol. Sci. 8:95–194. ,T.E.DOWLING, AND J. R. GOLD. 1991. Molec- GIRARD, C. 1856. Researches upon the cyprinoid fish- ular and cytological investigations, p. 83–126. In: es inhabiting the freshwaters of the United States The biology of cyprinid fishes. I. Winfield, and J. of America, west of the Mississippi Valley, from spec- Nelson (eds.). Chapman and Hall, London. imens in the museum of the Smithsonian Institu- CHERNOFF, B., AND R. R. MILLER. 1982. Notropis boca- tion. Proc. Acad. Nat. Sci., Phila. 8:165–213. grande, a new cyprinid fish from Chihuahua, Mexi- HARRISON, R. G. 1993. Hybrids and hybrid zones: his- co, with comments on Notropis formosus. Copeia torical perspective, p. 3–12. In: Hybrid zones and 1982:514–522. the evolutionary process. R. G. Harrison (ed.). Ox- COBURN, M. M., AND T. M. CAVENDER. 1992. Interre- ford Univ. Press, New York. lationships of North American cyprinid fishes, p. HOWELL,W.M.,AND J. D. WILLIAMS. 1971. Notropis gibb- 328–373. In: Systematics, historical ecology, and si, a new cyprinid fish from the Tallapoosa River sys- North American freshwater fishes. R. Mayden (ed.). tem in Alabama and Georgia. Copeia 1971:55–64. Stanford Univ. Press, Stanford, CA. HUBBS, C. 1956. Relative variability of hybrids be- COLLINS, T. M., P. H. WIMBERGER, AND G. J. P. NAYLOR. tween the minnows, Notropis lepidus and N. proserpi- 1994. Compositional bias, character-state bias, and nus. Tex. J. Sci. 8:463–469. character-state reconstruction using parsimony. . 1972. A checklist of Texas freshwater fishes. Syst. Biol. 43:482–496. Texas Parks Wildlife Dept., Tech. Ser. 11:1–11. CONTRERAS-BALDERAS, S. 1975. Zoogeography and HUBBS, C. L. 1955. Hybridization between fish spe- evolution of Notropis lutrensis and ‘‘Notropis’’ ornatus cies in nature. Syst. Zool. 4:1–20. in the Rio Grande basin and range, Mexico and , AND C. W. GREENE. 1928. Further notes on 10 COPEIA, 2000, NO. 1

the fishes of the Great Lakes and tributary waters. es from the United States and Canada. American Pap. Mich. Acad. Sci. Arts Lett. 8:371–392. Fisheries Society, Spec. Publ. 20, Bethesda, MD. , AND R. R. MILLER. 1978. Notropis panarcys,n. RICHARDSON, L. R., AND J. R. GOLD. 1995. Evolution sp., and N. proserpinus, cyprinid fishes of subgenus of the Cyprinella lutrensis species-complex. II. Sys- Cyprinella, each inhabiting a discrete section of the tematics and biogeography of the Edwards Plateau Rio Grande complex. Copeia 1978:582–592. shiner, Cyprinella lepida. Copeia 1995:28–37. JENKINS,R.E.,AND N. M. BURKHEAD. 1994. Freshwa- , AND . 1999. Systematics of Cyprinella ter fishes of . American Fisheries Society, lutrensis species-group species (Cyprinidae) from Bethesda, MD. the southwestern United States, as inferred from , AND E. A. LACHNER. 1971. Criteria for analysis restriction-site variation of mitochondrial DNA. and interpretation of the American fish genera No- Southwest. Nat. 44:49–56. comis Girard and Hybopsis Agassiz. Smiths. Contrib. SCHMIDT, T. R., J. P. BIELAWSKI, AND J. R. GOLD. 1998. Zool. 90:1–15. Molecular and evolution of the cyto- , AND . 1980. Account of Hybopsis zane- chrome b gene in the cyprinid genus Lythrurus (Ac- ma, p. 197. In: Atlas of North American freshwater tinopterygii: Cypriniformes). Copeia 1998:14–22. fishes. D. S. Lee, C. R. Gilbert, C. H. Hocutt, R. E. SIMON, C., F. FRATI,A.BECKENBACH,B.CRESPI,H.LIU, Jenkins, D. E. McAllister, J. R. Stauffer Jr. (eds.). AND P. F LOOK. 1994. Evolution, weighting, and phy- North Carolina State Musuem of Natural History, logenetic utility of mitochondrial gene sequences Raleigh. and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87:651– KLUGE,A.G.,AND J. S. FARRIS. 1969. Quantitative phy- 701. letics and the evolution of anurans. Syst. Zool. 18: SIMONS,A.M.,AND R. L. MAYDEN. 1998. Phylogenetic 1–32. relationships of the western North American phox- KOCHER, T. D., AND K. L. CARLETON. 1997. Base sub- inins (Actinopterygii: Cyprinidae) as inferred from stitution in fish mitochondrial DNA: patterns and mitochondrial 12S and 16S ribosomal RNA se- rates, p. 13–24. In: Molecular systematics of fishes. quences. Mol. Phylogenet. Evol. 9:308–329. T. Kocher and C. Stepien (eds.). Academic Press, SMITH, G. R. 1992. Introgression in fishes: signifi- New York. cance for paleontology, cladistics, and evolutionary ´ ´ ´ KRISTMUNDSDOTTIR,A.Y., AND J. R. GOLD. 1996. Sys- rates. Syst. Biol. 41:41–57. tematics of blacktail shiners (Cyprinella venusta) in- SNELSON, F. F. 1972. Systematics of the subgenus Lyth- ferred from analysis of mitochondrial DNA. Copeia rurus, genus Notropis (Pisces: Cyprinidae). Bull. Fla. 1996:773–783. State Mus., Biol. Sci. 17:1–92. KUHNER,M.K.,AND J. FELSENSTEIN. 1994. A simula- SWOFFORD, D. L., G. J. OLSEN,P.J.WADDELL, AND D. tion comparison of phylogeny algorithms under M. HILLIS. 1996. Phylogenetic inference, p. 407– equal and unequal evolutionary rates. Mol. Biol. 514. In: Molecular systematics. D. M. Hillis, C. Mo- Evol. 11:459–68. ritz, and B. Mable (eds.). Sinauer Associates, Sun- LOCKHART,J.P.,M.A.STEELE ,M.D.HENDY, AND D. derland, MA. PENNY. 1994. Recovering evolutionary trees under TAJIMA, F. 1993. Simple methods for testing the mo- a more realistic model of sequence evolution. Ibid. lecular evolutionary clock hypothesis. Genetics 135: 11:605–612. 599–607. MARTIN,A.P.,AND E. BERMINGHAM. 1998. Systematics TEMPLETON, A. R. 1983. Phylogenetic inference from and evolution of lower Central American cichlids restriction endonuclease cleavage site maps with inferred from analysis of cytochrome b gene se- particular reference to the evolution of humans quences. Mol. Phylogenet. Evol. 9:192–203. and the apes. Evolution 37:221–244. MATTHEWS, W. J. 1987. Geographic variation in Cy- YANG, Z. 1998. On the best evolutionary rate for phy- prinella lutrensis (Pisces: Cyprinidae) in the United logenetic analysis. Syst. Biol. 47:125–133. States, with notes on Cyprinella lepida. Copeia 1987: ZARDOYA, R., AND A. MEYER. 1996. Phylogenetic per- 616–637. formance of mitochondrial protein-coding genes in resolving relationships among vertebrates. Mol. MAYDEN, R. L. 1988. Vicariance biogeography, parsi- Biol. Evol. 13:933–942. mony, and evolution in North American freshwater fishes. Syst. Zool. 37:329–355. CENTER FOR BIOSYSTEMATICS AND BIODIVERSITY, . 1989. Phylogenetic studies of North Ameri- TEXAS A&M UNIVERSITY,COLLEGE STATION, can minnows, with emphasis on the genus Cyprinella (Teleostei: Cypriniformes). Univ. Kans., Mus. Nat. TEXAS 77843-2258. PRESENT ADDRESS: (REB) Hist., Misc. Publ. 80:1–189. BIOLOGICAL SURVEY AND DEPART- MINDELL,D.P.,AND C. E. THACKER. 1996. Rates of MENT OF ZOOLOGY,UNIVERSITY OF OKLAHOMA, molecular evolution: phylogenetic issues and appli- NORMAN,OKLAHOMA 73019-0235. E-mail: cations. Annu. Rev. Ecol. Syst. 27:279–303. (REB) [email protected]. Send reprint re- ROBINS, C. R., R. M. BAILEY,C.E.BOND,J.R.BROOK- quests to REB. Submitted: 24 Feb. 1999. Ac- ER,E.A.LACHNER,R.N.LEA, AND W. B. SCOTT cepted: 9 July 1999. Section editor: M. E. (EDS.). 1991. Common and scientific names of fish- Douglas.