Phylogenetic Relationships of the Creek Chubs and the Spine-Fins: an Enigmatic Group of North American Cyprinid Fishes (Actinopterygii: Cyprinidae)

Cladistics 13, 187-205 (1997) WWW http://www.apnet.com

Andrew M. Simons1 and Richard L. Mayden Department of Biological Sciences, The University of Alabama, Box 870344, Tuscaloosa, AL 35487, U.S.A.

Accepted 23 April, 1997

Phylogenetic analyses based on the complete nucleotide INTRODUCTION sequence of the mitochondrial 12S ribosomal RNA gene were performed for representatives of major North American cyprinid clades. These analyses resolved four The North American Cyprinidae, commonly known monophyletic groups: (1) Western Clade, represented by as chubs, shiners, and minnows, is the largest family of Acrocheilus and Gila, (2) Creek Chub Clade, represented freshwater fishes on the continent with over 300 spe- by Couesius, Hemitremia, Semotilus, and Margariscus, (3) cies in about 50 genera (Burr and Mayden, 1992). This Open Posterior Myodome Clade, represented by Cam- group constitutes a major component of most freshwa- postoma, Phenacobius, Cyprinella, Notropis, Platygobio, ter ecosystems and is the focus of numerous and Rhinichthys, and (4) Plagopterin Clade, represented behavioral, ecological, biogeographical, and evolution- by Snyderichthys, Lepidomeda, and Meda. The overall results of these analyses refute previous hypotheses of ary studies. Despite the importance of these fishes, the relationships of North American Cyprinidae based on phylogenetic relationships of North American cyprin- morphological data. Our analyses support recognition of ids are poorly understood. Currently there are only Snyderichthys, formerly considered a subgenus of Gila, two explicitly phylogenetic studies that include a large and indicate that the Plagopterin Clade is sister to the proportion of the North American cyprinid fauna; Creek Chub Clade. Furthermore, this analysis indicates Mayden's (1989) analysis of relationships among taxa that hypotheses of relationship between plagopterins with an open posterior myodome and Coburn and and other Western taxa are unsupported and the Open Cavender's (1992) analysis of nearly all North Ameri- Posterior Myodome Clade is a monophyletic group. can cyprinid genera. While both were based on © 1997 The Willi Hennig Society morphological characters these studies argued different assessments of relationship, primarily due to different interpretations of some character systems. 1Correspondence to: Andrew M. Simons. Current address: The In an effort to resolve these differences, we are inves- University of Massachusetts, Department of Biology, Graduate Pro- tigating the phylogenetic relationships of North gram in Organismic and Evolutionary Biology, Morrill Science Center, Box 35810, Amherst, MA 01003, U.S.A. Fax (413)545-3243. American cyprinids utilizing DNA sequences of mito- E-mail: [email protected]. chondrial ribosomal RNA genes. In preliminary

188 Simons and Mayden

analyses we identified taxa that consistently formed a lar and short preterminal maxillary barbel, to support monophyletic group among North American cyprin- its monophyly even though a similar barbel is ids. This group includes: observed in Margariscus (McPhail and Lindsey, 1970; Scott and Crossman, 1973; Jenkins and Burkhead, 1. Creek chubs containing the genera Semotilus 1994). Rafinesque, Hemitremia Cope, Couesius Jordan, and Margariscus is monotypic, containing the pearl dace, Margariscus Cockerell, M. margarita. Previously allocated to Clinostomus, Gila, 2. plagopterins containing the genera Lepidomeda and Richardsonius (Jenkins and Burkhead, 1994), the Cope, Meda Girard, and Plagopterus Cope, and pearl dace was referred to Semotilus by Bailey and 3. the monotypic genus Snyderichthys Miller. Allum (1962) based on 2,5–4,2 pharyngeal teeth, a preterminal barbel, although it is often absent in some Close genealogical relationship among these taxa has specimens (McPhail and Lindsey, 1970; Scott and never been proposed. Here, we examine the mono- Crossman, 1973; Jenkins and Burkhead, 1994), and a phyly and relationships of this newly hypothesized physiognomy similar to Semotilus. Margariscus was ele- clade and examine its placement with respect to other vated from synonymy with Semotilus by Coburn and major groups of North American cyprinids, utilizing Cavender (1992) as it and Semotilus were not resolved the complete sequence of the 12S mitochondrial riboso- as a monophyletic group in their analysis. mal RNA gene. We present evidence to support the Couesius and seven other nominal genera were sub- recognition of the plagopterins, including Snyderich- sumed into the genus Hybopsis Agassiz by Bailey thys, as a monophyletic group sister to a Creek Chub (1951). This change was based solely on the shared Clade containing the genera Semotilus, Hemitremia, occurrence of a terminal premaxillary barbel. Couesius, Margariscus, and Couesius. These taxa form the sister however, has a barbel that, although superficially sim- group to a Western Cyprinid Clade. ilar to that of taxa in Hybopsis sensu Bailey (1951), is actually preterminal in origin (McPhail and Lindsey, 1970; Scott and Crossman, 1973). McPhail and Lindsey (1970) and Jenkins and Lachner (1971) argued for the TAXONOMIC PERSPECTIVE retention of Couesius, suggesting that it had closer affinities with Semotilus, particularly with Semotilus margarita, based on barbel position, patterns of squa- Creek Chubs mation, opercular, brancheostegal and breast tuberculation, breeding color, scale morphology, ver- Creek chubs are large insectivorous species that, as a tebral number, and other features. In addition, Jenkins group, have a complex taxonomic history. Their classi- and Lachner (1971) suggested a relationship between fication has been largely based on breeding behavior, Nocomis and the nest-building species of Semotilus. coloration, pharyngeal tooth count, and barbel mor- The genus Hemitremia contains a single species, H. phology. Semotilus as currently recognized contains flammea, that is restricted to spring habitats in Ala- four species: S. atromaculatus, S. thoreauianus, S. lumbee bama, Georgia, and Tennessee. Unlike the other Creek and S. corporalis (Mayden et al., 1992). These species all chubs, Hemitremia lacks barbels. Its pharyngeal tooth have a broad head, pharyngeal teeth 2,5–4,2, and a count, breeding coloration and tuberculation pattern small, triangular, flap-like barbel in the groove have been used to suggest a relationship with Semotilus between the maxilla and the snout. The location of this or Couesius (Etnier and Starnes, 1993). Coburn and barbel is different from most barbeled North American Cavender (1992) postulated a sister-group relationship cyprinids in that it is preterminal, rather than terminal, between Hemitremia and Semotilus based on osteologi- with respect to the posterior end of the maxilla. In all of cal data. these species, males construct large gravel nests during Although relationships between the taxa Marga- the spawning season. Johnston and Ramsey (1990) pro- riscus, Semotilus, Hemitremia, and Couesius have been posed relationships for species of Semotilus and used suggested by a variety of authors, the only explicitly two characters, nest-building behavior and a triangu- phylogenetic hypothesis involving these taxa found

Phylogeny of North American Cyprinids 189 them to form a paraphyletic grade basal to a clade con- “Resemblance is particularly close between Lepidomeda and sev- taining the Exoglossine chubs (Coburn and Cavender, eral species referred to the genus Gila, and even more strikingly with a species of the Bonneville system, copei that has been 1992; Fig. 1). referred to a monotypic genus, Snyderichthys (Miller, 1945). It is not now apparent whether such relationship extending even to details of coloration is indicative of intimate relationship.” Plagopterins Coburn and Cavender (1992) placed the plagopterins as sister to Moapa and Rhinichthys within their Western Clade, a clade composed of taxa largely restricted to The plagopterins, or spine-fins, are an unusual clade Pacific drainages, including the Colorado River of six species classified into three genera, Plagopterus, system. Lepidomeda, and Meda. These fishes are endemic to the The leatherside chub, Snyderichthys copei, is endemic lower Colorado Basin of Utah, Nevada, and Arizona to the Bonneville and Snake river systems in Utah, and their populations have decreased dramatically Idaho, and Wyoming. This species was classified in six due to alterations of stream systems and the introduc- different genera before it was referred to the mono- tion of exotic fishes. Plagopterins are characterized by typic genus Snyderichthys by Miller (1945) based on spine-like rays in the dorsal and pelvic fins and a bright pharyngeal tooth count and attachment of gill mem- silvery body coloration. Although non-phylogenetic branes. Uyeno (1961) placed S. copei back in Gila based hypotheses of relationships within plagopterins exist on similarities in external morphology and osteology. based on morphology (Miller and Hubbs, 1960) and However, this study was restricted to the genera Gila, chromosome structure (Uyeno and Miller, 1973), their Richardsonius, Snyderichthys, Clinostomus, and Pty- relationship to other North American cyprinids is chocheilus and assumed that these taxa formed a essentially unknown. Miller and Hubbs (1960: 6) raised natural group. Thus, monophyly of this clade and rela- the possibility of a relationship of plagopterins to some tionships between these taxa and other cyprinids could Gila. be neither identified nor tested. Phylogenetic relationships of the above mentioned taxa remained unstudied and unchanged until Coburn and Cavender's (1992) phylogenetic analysis of North

Shiner Clade American cyprinid relationships based primarily on Western Clade osteological and lepidological characters. These Phoxinus authors included nearly all North American cyprinid Margariscus genera in their study, and identified three major clades Semotilus Hemitremia of North American cyprinids: a Western Clade, a Chub Couesius Clade, and a Shiner Clade (Fig. 1). The genera Marga- Hybognathus riscus, Semotilus, Hemitremia, and Couesius were placed Dionda in the Chub Clade. The plagopterins were placed in the Campostoma Western Clade as sister to Moapa plus Rhinichthys. Phenacobius Exoglossum Although the leatherside chub, Snyderichthys copei, was Macrhybopsis mentioned in the text of their paper, it was not Erimystax included in any phylogenetic analysis. Nocomis Platygobio

MATERIALS AND METHODS FIG. 1. Phylogeny of North American Cyprinidae proposed by Coburn and Cavender (1992). The Chub Clade has been expanded to show relationships among genera. The Creek chubs Margariscus, We have sequenced the entire 12S mitochondrial Semotilus, Hemitremia, and Couesius form a paraphyletic grade at the base of a monophyletic group containing the Exoglossine chubs: rRNA gene in representatives of all Creek chub genera, Hybognathus, Dionda, Campostoma, Phenacobius, Exoglossum, two plagopterin species, and Snyderichthys copei. In our Macrhybopsis, Erimystax, Nocomis, and Platygobio. analysis we include other North American cyprinid

190 Simons and Mayden

taxa to test the monophyly of the Creek Chub–Plagop- 4. Couesius plumbeus Mill Creek, 100 miles north of terin Clade and to determine its phylogenetic position Ft. Nelson, B.C. Canada, UAIC 11366.01. with respect to other North American cyprinids. Taxa 5. Cyprinella venusta Cahaba River, Bibb Co. AL. were chosen to represent the three major clades pro- 6. Gila cypha Colorado River, Coconino Co. AZ. posed by Coburn and Cavender (1992) and the Open 7. Hemitremia flammea Unnamed spring, Madison Posterior Myodome Clade as proposed by Mayden Co. AL, UAIC 10687.01. (1989). Acrocheilus, Gila, and Rhinichthys are all mem- 8. Hemitremia flammea King (Buffler) Spring, Lauder- bers of the Western Clade sensu Coburn and Cavender dale Co. AL, UAIC 10697.01. (1992). Campostoma, Phenacobius, and Platygobio are 9. Lepidomeda vittata Rudd Creek, Apache Co. AZ. members of their Chub Clade. In Coburn and Caven- 10. Margariscus margarita Plover River, Marathon der's analysis their Chub Clade, which included the Co. WI, UAIC 10241.07. 11. Meda fulgida Aravaipa Creek, Graham Co. AZ. Creek chubs, was sister to their Western Clade. In May- 12. Notemigonus crysoleucas Mill Creek, Portage Co. den's (1989) analysis, a Chub Clade, not including the WI, UAIC 10527.02. Creek chubs, was sister to a Shiner Clade (Fig. 2). The 13. Notropis atherinoides Waupaca River, Waupaca Shiner Clade of both Coburn and Cavender (1992) and Co. WI, UAIC 10485.06. Mayden (1989) is represented by Cyprinella and 14. Phenacobius catostomus Cahaba River, Bibb Co. Notropis. AL, UAIC 7884.07. 15. Platygobio gracilis Mississippi River, Jackson Co. Specimens Examined IL, UAIC 11558.01. 16. Rhinichthys atratulus Russell Creek, Clairborne Co. TN, UAIC 9850.01. 1. Acrocheilus alutaceus Kettle River, B.C. Canada, 17. Semotilus atromaculatus Masterson Creek, UAIC 11365.01. Lawrence Co. AL, UAIC 10875.13. 2. Campostoma anomalum Rolling Fork River, Nelson 18. Semotilus thoreauianus Snake Creek, Russell, Co. Co. KY, UAIC 10147.02. AL, UAIC 10858.05. 3. Couesius plumbeus Lizard Hot Springs, Ft. Nelson, 19. Snyderichthys copei Sevier River, Garfield Co. UT. B.C. Canada, UAIC 11367.01.

DNA Amplification and Sequencing Shiner Clade

Hybognathus Genomic DNA was extracted using standard phe- nol/chloroform extraction procedures (Hillis et al., Exoglossum 1990) and with the QIAGEN QIamp tissue kit Dionda (Catalogue No. 29304) following manufacturer's Snyderichthys copei Gila cypha Campostoma instructions. Four taxa, , , Lepidomeda vittata, and Meda fulgida, were obtained as Nocomis pure MtDNA preps. The 12S gene was amplified in Phenacobius two overlapping fragments with Tfl DNA polymerase (Epicentre products Catalogue No. F10250) under Erimystax conditions recommended by the manufacturer. Ampli- Platygobio fication primers used were PHEa and 12Sb for the first fragment and 12Sa and 16Sd for the second fragment Macrhybopsis (Table 1). Initial amplification was from genomic DNA in a volume of 25µl. The amplification profile included ° ° FIG. 2. Phylogeny of the Open Posterior Myodome Clade pro- denaturation at 94 C for 1min, annealing at 50 C for posed by Mayden (1989). The Chub Clade has been expanded to 1min and extension at 72°C for 1min 30s for 30 cycles. show relationships among genera. Amplification products were gel-purified on 2.5%

TABLE 1 Primer Used in Ampliﬁcation and Sequencing of the 12S rRNA Gene for North American Cyprinids Name Sequence (5’→3’) Strand Reference

12Sa AAACTGGGATTAGATACCCCACTA L Kocher et al., 1989 12Sb AGGAGGGTGACGGGCGGTGTGT H Modified from Kocher et al., 1989 12Sc GGAAAGAAATGGGCTACA L Titus, pers. comm. 12Sd GGGTTGGTAAATCTCGTGC L Titus and Larson, 1995 16Sd CAAGAGGCGATGTTTTT H Titus, pers. comm. PHEa AAAGCACAGCACTGAAGATG L Titus and Frost, 1996

NuSieve agarose. Single-stranded DNA amplification phylogenetic structure was due to congeneric taxa (Gyllensten and Erlich, 1988) was performed in a vol- (Titus and Larson, 1995). PTP tests (Archie, 1989; Faith ume of 50µl. The amplification profile was identical to and Cranston, 1991) were performed using PAUP double-stranded DNA amplification but was run for 4.0.0d51 (provided by D. L. Swofford). PTP tests were 35 cycles. Primer concentrations were generally used at run for all taxa and for a reduced data set containing a ratio of 10:1, although concentrations were varied only one species from each genus. Each PTP test was depending upon the template. Asymmetric amplifica- based on 1000 replicates. tions were purified in Millipore filter units (Millipore Phylogenetic trees were generated using PAUP 3.1 Ultrafree MC polysulfone 30,000 NMWL Catalogue (Swofford, 1993). Two subsets of the complete No. UFC3TTK00) and resuspended in 30µl dH2O. sequence data were used in a series of analyses, differ- Sequencing was performed using Sequenase 2.0 ing in the treatment of areas of ambiguous alignment (United States Biochemical) following manufacturer's and gaps. Subset 1 excluded ambiguous alignments instructions. Sequencing primers used were PHEa, with gaps treated as missing characters (variable 12Sd, 12Sa, and 12Sc (Table 1). Termination products characters=203; informative characters=119). Subset 2 were electrophoresed on 8% long-ranger gels (AT Bio- excluded ambiguous alignments with gaps coded as chem). Sequences were deposited under Genbank binary characters (variable characters=211; informa- accession numbers AF023183–AF023201. tive characters=127). For each subset, four weighting schemes were used: (1) equal weighting of transitions and transversions (unweighted parsimony), (2) trans- Analyses versions weighted 2:1, (3) transversions weighted 3:1, and (4) only transversions (transversion parsimony). Multiple alignment was performed using CLUSTAL

W (Thompson et al., 1994). The gap opening penalty 9 was varied from 5 to 20 and the gap extension penalty 8 was varied from 2.5 to 10 to maximize the number of 7 possible alignments found. 6 5

Pairwise comparisons of all taxa were generated ransversions using MEGA (Kumar et al., 1993). Saturation of 4 transitions was assessed by plotting the TS:TV ratio for 3 all pairwise comparisons versus genetic distance 2

ransitions/T 1 (Fig. 3.) T The g-statistic (Hillis, 1991) was calculated based on 0 2 4 6 8 10 12 10,000 randomly generated trees using PAUP 3.1.1 Divergence (%) (Swofford, 1993). The g -statistic was generated for all 1 FIG. 3. Ratio of transitions to transversions versus percentage taxa, and for a reduced data set containing only one divergence for all pairwise comparisons except those between iden- species from each genus to exclude the possibility that tical taxa.

These weighting schemes were used to account for transition bias among some taxa at low genetic dis- increasing saturation of TS with genetic distance tances. This bias decreased to a value of approximately (Fig. 3) and to assess the stability of the data at various 2.5 with increasing genetic distance (Fig. 3), suggesting weighting schemes. 1000 bootstrap replicates were that saturation is occurring at some sites (Kraus and performed on each of the four subsets of data. The Miyamoto, 1991). We note, however, that many complete data set is presented in Appendix 1. pairwise comparisons exhibit a low TS:TV ratio at low genetic distance. Therefore, in addition to an unweighted analysis we chose weights to bracket the RESULTS TS:TV ratio at saturation and transversion-only parsimony to explore the effect of weighting upon the analysis. It has been argued that downweighting tran- Sequence alignment resulted in three different sitions with respect to transversions may eliminate hypotheses, differing only in the placement of gaps in phylogenetic information provided by transitions in the sequences of Semotilus atromaculatus and S. tho- conserved regions of the gene (Reeder, 1995). Our data reauianus between positions 300 and 317. This region is exhibit more transition bias than Reeder's data with a contained within a loop between stems 23 and 24 in the mean ratio of 3.5 versus 2.2 for comparisons of all pairs secondary structure model for mitochondrial SSU of taxa exhibiting <4.0% genetic distance. The lack of rRNA of the cyprinid Cyprinus carpio (Van de Peer et resolution produced by transversion parsimony indi- al., 1994). This region of ambiguous alignment was not cates that the transitions are contributing substantially included in the phylogenetic analyses. to the resolution of the Creek Chub Clade in the Källersjö et al. (1992) criticize the use of skew- remaining analyses. ness-based significance tests, as critical values for Equally weighted phylogenetic analyses of subsets 1 significance were based on simulated data where all and 2 resulted in four or five equally parsimonious states had the same frequency (Hillis, 1991; Hillis and trees respectively (Fig. 4A–E). In both analyses equally Huelsenbeck, 1992). Real data are likely to exhibit parsimonious topologies resulted from differential variation in the frequency of character states (Källersjö placement of the Plagopterin Clade and alternative et al., 1992). To address this problem we calculated relationships of taxa in this clade. The Plagopterin critical values of g1 for our data by randomizing Clade was resolved as either sister to the Western character states between taxa using MacClade 3.0 Clade, sister to the Creek Chub Clade, or nested within (Maddison and Maddison, 1992). One hundred ran- the Creek Chub Clade, sister to Margariscus. The Creek domized data sets were produced and g1 values were chubs, Western cyprinids, and plagopterins formed a obtained for each random data set based on 1000 ran- monophyletic group, sister to the Open Posterior Myo- domly generated trees for each data set (Reeder, 1995). dome Clade inclusive of Rhinichthys. Weighted Estimated g1 values for analysed sequences indicated analyses (2:1, 3:1) all produced a single topology that these data were significantly more structured than (Fig. 4A). In this tree plagopterins formed the sister − − − random (g1 = 0.807, P0.05= 0.271, P0.01= 0.354) group to Creek chubs, together forming the sister to the − even after the exclusion of congeneric taxa (g1= 0.626, Western Clade, and this group was sister to the Open P0.05=−0.300, P0.01=−0.388). The PTP test also indi- Posterior Myodome Clade. Relationships of the four cated that the data were significantly more structured clades were unchanged in transversion parsimony, than random (all taxa, PTP= 0.001; congenerics although intraclade relationships were largely unre- excluded, PTP=0.001). solved (Fig. 4F). Several studies have demonstrated a higher number In all analyses, four groups were consistently of transitions with respect to transversions among resolved: (1) the Western Clade, containing Acrocheilus closely related taxa. As the genetic distance increases, and Gila, (2) the Creek Chub Clade, containing Coue- however, the transitions become saturated (Brown, sius, Hemitremia, Semotilus, and Margariscus, (3) the 1985; Mindell and Honeycutt, 1990; Kraus and Miya- Open Posterior Myodome Clade, containing Campos- moto, 1991; Alves- Gomes et al., 1995). In our analyses, toma, Phenacobius, Cyprinella, Notropis, Platygobio, and pairwise comparisons of TS:TV ratios indicated a Rhinichthys, and (4) the Plagopterin Clade, containing

A. alutaceus A. alutaceus G. cypha G. cypha C. plumbeus S. copei C. plumbeus L. vittata H. flammea M. fulgida H. flammea C. plumbeus S. atromaculatus C. plumbeus S. thoreauianus H. flammea M. margariscus H. flammea S. copei S. atromaculatus L. vittata S. thoreauianus M. fulgida M. margariscus C. anomalum C. anomalum P. catostomus P. catostomus C. venusta C. venusta N. atherinoides N. atherinoides P. gracilis P. gracilis R. atratulus R. atratulus (A) (B)

A. alutaceus A. alutaceus G. cypha G. cypha C. plumbeus C. plumbeus C. plumbeus C. plumbeus S. copei S. copei L. vittata L. vittata M. fulgida M. fulgida M. margariscus M. margariscus H. flammea H. flammea H. flammea H. flammea S. atromaculatus S. atromaculatus S. thoreauianus S. thoreauianus C. anomalum C. anomalum P. catostomus P. catostomus C. venusta C. venusta N. atherinoides N. atherinoides P. gracilis P. gracilis R. atratulus R. atratulus (C) (D)

A. alutaceus A. alutaceus G. cypha G. cypha C. plumbeus C. plumbeus C. plumbeus C. plumbeus S. copei H. flammea M. fulgida H. flammea L. vittata S. atromaculatus M. margariscus S. thoreauianus H. flammea M. margariscus H. flammea S. copei S. atromaculatus L. vittata S. thoreauianus M. fulgida C. anomalum C. anomalum P. catostomus C. venusta C. venusta N. atherinoides N. atherinoides P. catostomus P. gracilis P. gracilis R. atratulus R. atratulus (E) (F)

FIG. 4. Topologies produced in analyses. (A–E) TV:TS 1:1, (A) TV:TS 2:1, TV:TS 3:1, (F) transversions only.

Snyderichthys, Lepidomeda, and Meda. The Western hypotheses of relationship regardless of weighting Clade, the Open Posterior Myodome Clade, and the schemes or alignments. Four clades were recognized in Plagopterin Clade were monophyletic in all analyses all analyses, with inconsistencies occurring only for regardless of weighting scheme. Unweighted analyses relationships within the plagopterins and the phyloge- produced multiple topologies either through alterna- netic position of the plagopterins with respect to the tive placements of the Plagopterin Clade or differences Creek Chub and Western Clades. in relationships of taxa within this clade. The Plagop- terin Clade was resolved as either sister to the Western Clade, sister to the Creek chubs, or sister to Marga- Relationships of the Plagopterins riscus, within the Creek Chub Clade. Bootstrap values were similar for all runs. The boot- Plagopterin cyprinids have traditionally been con- strap values were high for the nodes supporting the sidered a monophyletic group with six species Open Posterior Myodome Clade (92%) and the West- classified in three genera, Plagopterus, Meda, and Lepi- ern Clade (99%). The bootstrap values for the Creek domeda (Miller and Hubbs, 1960). These fishes were Chub Clade plus the Plagopterin Clade were lower once considered so distinctive that they were classified (53%) as were the values for the Creek Chub Clade by Cope (1874) in a separate subfamily, Plagopterinae, (53%) and the Plagopterin Clade (58%). These lower and a separate family, Medidae, by Jordan et al. (1930). bootstrap values were not unexpected given the rela- Traditionally, plagopterins have been thought to be tively low numbers of characters supporting these allied to western North American cyprinids (Miller nodes. This also explains the various equally parsimo- and Hubbs, 1960; Coburn and Cavender, 1992). Our nious topologies found in some analyses (Fig. 4A–E). analysis unequivocally supports the monophyly of the Values for the analysis excluding gaps, with TV:TS plagopterins, plus the leatherside chub, Snyderichthys weighted 2:1, are presented in Figure 5. copei, a relationship once considered by Miller and Hubbs (1960). In a footnote to their paper, however, Miller and Hubbs recommended recognition of Sny- DISCUSSION derichthys as a subgenus of Gila based on the then unpublished work of Uyeno (1961). The relationship of Snyderichthys to other plagopterins remains equivocal Cyprinid fishes constitute nearly 30% of the North using 12S DNA sequences. It is clear, however, that American ichthyofauna. Ironically, even though they Snyderichthys copei is not related to members of the constitute a major component of most aquatic ecosys- genus Gila. Hence, we elevate Snyderichthys from syn- tems, they have received minimal systematic attention onymy with Gila where it is currently classified. and many systematic problems remain. Lack of histor- Furthermore, we argue for a sister-group relationship ical information provided through explicit of the Plagopterin Clade to the Creek Chub Clade, a phylogenetic hypotheses continues to limit progress relationship resolved in weighted parsimony analyses. with evolutionary and ecological studies (Mayden, 1992) in which these and other species serve as the pri- mary focus. Recent morphological studies have greatly Relationships of Creek Chubs advanced our current understanding of relationships of cyprinid fishes. However, comparison of phyloge- Although monophyly of this clade has been sug- nies provided by Mayden (1989) and Coburn and gested previously, the absence of unequivocal Cavender (1992) raises several important questions; shared-derived morphological features uniting Creek while some cyprinid clades are consistently recog- chubs has resulted in a complex taxonomic history. nized, others are not. The most notable differences This is especially true of features often considered to be between these analyses occur at the generic level and important at the generic level in cyprinid systematics above and involve taxa targeted in the present study. such as breeding behavior, pharyngeal tooth counts, Phylogenetic analysis of DNA sequences of cyprinid and the presence and structure of sensory barbels. The taxa included in this study resulted in consistent only phylogenetic analysis including these taxa with

Acrocheilus alutaceus 5 Western Clade (99) Gila cypha

Couesius plumbeus 10 (100) Couesius plumbeus

7 Hemitremia flammea (60) 4 13 (100) (53) Hemitremia flammea Creek Chub Clade 18 (100) Semotilus atromaculatus 13 6 (99) (58) Semotilus thoreauianus 4 (53) 14 Margariscus margarita

8 Snyderichthys copei 3 (64) 8 6 Lepidomeda vittata Plagopterin Clade (58) 29 Meda fulgida

26 Campostoma anomalum 11 (54) 47 Phenacobius catostomus

8 38 (59) Cyprinella venusta 8 Open Posterior (53) 11 Myodome Clade 8 Notropis atherinoides 11 (77) (92) 6 Platygobio gracilis

14 Rhinichthys atratulus

FIG. 5. Phylogenetic hypothesis of the spine-fins and Creek chubs. Branch lengths and bootstrap values (in parentheses) are based on analysis of data subset 1 with transversions weighted two times transitions. other North American cyprinids was that of Coburn Cavender (1992) wherein the above clade formed a and Cavender (1992). However, their analysis pro- basal grade in a larger clade of chubs referred to as vided no evidence for a Creek Chub Clade as identified Exoglossine chubs (Fig. 1). herein. In the present analysis, Semotilus and Hemitre- mia consistently formed a monophyletic group, a relationship also supported by morphological data Open Posterior Myodome Clade (Coburn and Cavender, 1992). Herein Margariscus formed the sister group to Semotilus plus Hemitremia, Creek chubs and plagopterins consistently form the and Couesius formed the sister to the whole clade. This sister group to the Western Clade and together form is in contrast to the hypothesis of Coburn and the sister group to a clade consisting of Rhinichthys,

Campostoma, Phenacobius, Platygobio, Cyprinella, and ACKNOWLEDGEMENTS Notropis. Interestingly, the five latter taxa were included in Mayden's (1989) Open Posterior Myodome We are grateful for help with this project from the following people and Clade (Fig. 2) but were distributed in all three of institutions. T. Dowling provided mtDNA preparations of Gila cypha, Lepi- Coburn and Cavender's (1992) major clades (Western, domeda vittata, Meda fulgida, and Snyderichthys copei. G. R. Haas and J. D. Shiner, and Chub Clades). In the latter study Rhinich- McPhail provided specimens of Acrocheilus alutaceus and Couesius plumbeus. W. W. Dimmick provided Platygobio gracilis tissue. B. R. Kuhajda, E. B. thys was argued to be member of the Western Clade as Jones, R. M. Wood and S. R. Layman provided assistance in the field. T. A. a close relative of Moapa; this clade was sister to the Titus and E. O. Wiley provided the primers used in this study. K. J. Clam- plagopterins. Campostoma, Phenacobius and Platygobio boy, C. L. Lydeard, and R. M. Wood provided comments on the manuscript. were included in the Exoglossine Clade, along with We are especially grateful for the help provided by K. E. Knott and B. R. Kuhajda. This research was supported by the Department of Biological Sci- creek chubs in a Chub Clade. Notropis and Cyprinella ences at the University of Alabama and by National Science Foundation were placed in the Shiner Clade (Fig. 1). grant 5-31943 to RLM. Rhinichthys unambiguously forms the basal member of the Open Posterior Myodome Clade in our analyses. The remaining members of this clade are all members REFERENCES of the Open Posterior Myodome Clade sensu Mayden (1989). This clade was dismembered by Coburn and

Cavender (1992) based, in part, on their reinterpreta- Alves-Gomes, J. A., Ortí, G., Haygood, M., Heiligenberg, W., and tion of Mayden's proposed synapomorphy involving Meyer, A. (1995). Phylogenetic analysis of South American electric the open posterior myodome. The open posterior myo- fishes (Order Gymnotiformes) and the evolution of their electrogenic system: A synthesis based on morphology, dome is an opening in the base of the neurocranium electrophysiology, and mitochondrial sequence data. Mol. Biol. bounded by the parasphenoid and basioccipital. Evol. 12, 298-318. Archie, J. W. (1989). A randomization test for phylogenetic informa- Coburn and Cavender (1992) considered this character tion in systematic data. Syst. Zool. 38, 239–252. derived only in taxa where the opening extended pos- Bailey, R. M. (1951). A check-list of the fishes of Iowa, with keys for teriorly beyond the parasphenoid. This interpretation identification. In “Iowa Fish and Fishing” (J. R. Harland, and E. B. Speaker, Eds), pp.187–238. State Conservation Commission, State excluded taxa such as Campostoma, with a relatively of Iowa. small opening that closes ontogenetically. Bailey, R. M., and Allum, M. O. (1962). “Fishes of South Dakota”. Miscellaneous Publications of the Museum of Zoology, University Based on congruence with sequence data, it is clear of Michigan, No. 93. that the open posterior myodome should be consid- Brown, W. M. (1985). The mitochondrial genome of animals. In ered a synapomorphy for the Open Posterior “Molecular Evolutionary Genetics” (R. J. MacIntyre, Ed.), pp.95– 130. Plenum Press, New York. Myodome Clade sensu Mayden (1989). This interpreta- Burr, B. M., and Mayden, R. L. (1992). Phylogenetics and North tion suggests that the closed condition observed in American freshwater fishes. In “Systematics, Historical Ecology, and North American Freshwater Fishes” (R. L. Mayden, Ed). Campostoma and other chubs is secondarily derived, pp.18–75. Stanford University Press, Stanford, California. and does not require hypothesized convergence to Coburn, M. M., and Cavender, T. M. (1992). Interrelationships of explain the open posterior myodome within other North American cyprinid fishes. In “Systematics, Historical Ecol- ogy, and North American Freshwater Fishes” (R. L. Mayden, Ed), chub genera such as Macrhybopsis, Erimystax, and pp.328–373. Stanford University Press, Stanford, California. Dionda. Cope, E. D. (1874). On the Plagopterinae and the ichthyology of Utah. Proc. Am. Phil. Soc. 14, 129–140. Within the Open Posterior Myodome Clade, Phen- Etnier, D. A., and Starnes, W. C. (1993). “The Fishes of Tennessee”. acobius and Campostoma form a monophyletic group University of Tennessee Press, Knoxville, Tennessee. sister to Platygobio plus Notropis and Cyprinella. These Faith, D. P., and Cranston, P. S. (1991). Could a cladogram this short have arisen by chance alone?: On permutation tests for cladistic relationships are discordant with Mayden's (1989) structure. Cladistics 7, 1–28. hypothesis based on morphological characters, in Gyllensten, U. B., and Erlich, H. A. (1988). Generation of sin- which a monophyletic Chub Clade was proposed gle-stranded DNA by the polymerase reaction and its application to direct sequencing of the HLA-DQA locus. Proc. Nat. Acad. Sci. within the Open Posterior Myodome Clade. U.S.A. 85, 7652–7656.

Hillis, D. M. (1991). Discriminating between phylogenetic signal and Mayden, R. L., Burr, B. M., Page, L. M., and Miller, R. R. (1992). The random noise in DNA sequences. In “Phylogenetic Analysis of native freshwater fishes of North America. In “Systematics, His- DNA Sequences” (M. M. Miyamoto, and J. Cracraft, Eds.), pp. torical Ecology, and North American Freshwater Fishes” (R. L. 278–294. Oxford University Press, New York. Mayden, Ed.), pp. 827–863. Stanford University Press, Stanford, Hillis, D. M., and Huelsenbeck, J. P. (1992). Signal, noise and reliabil- California. ity in molecular phylogenetic analyses. J. Heredity 83, 189–195. Miller, R. R. (1945). Snyderichthys, a new generic name for the leath- Hillis, D. M., Larson, A., Davis, S. K., and Zimmer, E. A. (1990). erside chub of the Bonneville and Upper Snake drainages in Nucleic acids III: Sequencing. In “Molecular Systematics” (D. M. Western United States. J. Acad. Sci. 35, –28. Hillis and C. Moritz, Eds), pp. 318–370. 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APPENDIX 1 PAUP Input File #NEXUS BEGIN DATA; DIMENSIONS NTAX=19 NCHAR=970 ; FORMAT MISSING=? INTERLEAVE DATATYPE=DNA SYMBOLS="012" GAP=-;

[ 10 20 30 40 50 60] ac_alut TAAAGGCATGGTCCTGACCTTATTATCAGCTCTAACCCAACTTACACATGCAAGTCTCCG ca_anom TAAAGGCATGGTCCTGACCTTATTATCAGCTTTAACCTAACTTACACATGCAAGTCTCCG co_plumb1 TAAAGGCATGGTCCTGACCTTATTATCAGCTCTAACCCAACTTACACATGCAAGTCTCCG co_plumb2 TAAAGGCATGGTCCTGACCTTATTATCAGCTCTAACCCAACTTACACATGCAAGTCTCCG cy_venust CAAAGGCATGGTCCCGACCTTACCATCAGCTCTAACCCAACTTACACATGCAAGTCTCCG gi_cope TAAAGGCATGGTCCTGACCTTATTATCAGCTCTAACCCAACTTACACATGCAAGTCTCCG gi_cyph TAAAGGCATGGTCCTGACCTTATTATCAGCTCTAACCCAACTTACACATGCAAGTCTCCG he_fla50 TAAAGGCATGGTCCCGACCTTATTATCAGCTTTAACCCGACTTACACATGCAAGTCTCCG he_fla59 TAAAGGCATGGTCCCGACCTTATTATCAGCTTTAACCCGACTTACACATGCAAGTCTCCG le_vitt TAAAGGCATGGTCCTGACCTTATTATCAGCTCTAACCCGACTTACACATGCAAGTCTCCG mar_marg TAAAGGCATGGTCCTGACCTTATTATCAGCTCTAGCCCAACTTACACATGCAAGTCTCCG me_fulg TAAAGGCATGGTCCTGACCTTATTATCAGCTATAACCTAACTTACACATGCAAGTCTCCG not_chry50 CAAAGGCATGGTCCTGACCTTATTATTAGCTTTAACCCAACTTACACATGCAAGTCTCCG not_ather CAAAGGCATGGTCCCGACCTTACTATCAGCTCTAGCCCAACTTACACATGCAAGTCTCCG phe_catos TAAAGGCATGGTCCCGACCTTCCTGTCAGCTTTAGCCTAATTTACACATGCAAGTCTCCG pl_grac CAAAGGCATGGTCCCGACCTTACTATCAGCTCTAGCCCAACTTACACATGCAAGTCTCCG rh_atrat TAAAGGCATGGTCCTGACCTTATTATCAGCTCTAACCCAACTTACACATGCAAGTCTCCG se_atro CAAAGGCATGGTCCCGACCTTATTATCAGCTTTAACCCAACTTACACATGCAAGTCTCCG se_thor TAAAGGCATGGTCCCGTCCTTATTATCAGCTCTAACCCGACTTACACATGCAAGTCTCCG

[ 70 80 90 100 110 120] ac_alut CAGTCCTGTGAGTACGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACA ca_anom CAGCCCCGTGAGTACGCCCTCAATCCCCTGCCCGGGGACGAGGAGCAGGTATCAGGCACA co_plumb1 CAACCCCGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACG co_plumb2 CAACCCCGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACG cy_venust CAACCCCGTGAGTAGGCCCTTAATCCCCTGCCCGGGGACGAGGAGCAGGCATCAGGCACA gi_cope CAGCCCCGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACA gi_cyph CAGTCCTGTGAGTACGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACA he_fla50 CAGCCCTGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACA he_fla59 CAGCCCTGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACA le_vitt CAGCCCCGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACA mar_marg CACCCCCGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACA me_fulg CAACCCTGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACA not_chry50 CAGCCCCGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGCATCAGGCACA not_ather CAGTCCCGTGAGTAAGCCCTTAATCCCCTGCCCGGGGACGAGGAGCAGGCATCAGGCACA phe_catos CACCCCTGTGAGTACGCCCTCAACCCCCCGCCCGGGÐÐTCAGGAGCAGGTATCAGGCGCA pl_grac CAGCCCCGTGAGTAAGCCCTTAATCCCCCGCCCGGGÐACGAGGAGCAGGCATCAGGCACA rh_atrat CAGCCCCGTGAGTACGCCCTCAATCCCCTGCCCGGGGACGAGGAGCAGGCATCAGGCACA se_atro CAGCCCTGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGTATCAGGCACA se_thor CAGCCCTGTGAGTATGCCCTTAATCCCCTGCCCGGGGACGAGGAGCGGGTATCAGGCACA

[ 130 140 150 160 170 180] ac_alut AATTTTTAGCCCAAGACGCCTGGCCAAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA ca_anom AATTTTTAGCCCAAGACGCCTGGCCAAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA co_plumb1 AATTTTTAGCCCAAGACGCCTGGCCTAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA co_plumb2 AATTTTTAGCCCAAGACGCCTGGCCTAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA cy_venust AATCTTTGGCCCAAGACGCCGAGCCCAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA

Copyright © 1997 by The Willi Hennig Society All rights of reproduction in any form reserved Phylogeny of North American Cyprinids 199 gi_cope AATTTTTAGCCCAAGACGCCTGGCCTAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA gi_cyph AATTTTTAGCCCAAGACGCCTGGCCAAGCCACACCCCCAAGGÐAATTCAGCAGTGATAAA he_fla50 AATTTTTAGCCCAAGACGCCTAGCCTTGCCACACCCCCAAGGGAATTCAGCAGTGATAAA he_fla59 AATTTTTAGCCCAAGACGCCTAGCCTTGCCACACCCCCAAGGGAATTCAGCAGTGATAAA le_vitt AATTTTTAGCCCAAGACGCCTGGCCTAGCCACACCCCCAAGGÐAATTCAGCAGTGATAAA mar_marg AATTTTTAGCCCAAGACGCCTGGCCTAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA me_fulg AATTTTTAGCCCAAGACGCCTGGCCTAGCCACACCCCCAAGGÐAATTCAGCAGTGATAGA not_chry50 AATTTTTAGCCCAAGACGCCTGGCCAAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA not_ather AATTTTÐAGCCCAAGACGCCAGGCCTAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA phe_catos AACCTAÐAGCCCAAGACGCCAAGCAAAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA pl_grac AACCTTTAGCCCAAGACGCCAGGCCTAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA rh_atrat AACATTTAGCCCAAGACGCCTGGCCTAGCCACACCCCCAAGGGAATTCAGCAGTGATAAA se_atro AATTTTTAGCCCAAGACGCCTAGCCTTGCCACACCCCCAAGGGAATTCAGCAGTGATAAA se_thor AATTTTÐAGCCCAAGACACCTAGCCTTGCCACACCCCCAAGGGAATTCAGCAGTGATAAA

[ 190 200 210 220 230 240] ac_alut TATTAAGCCATAAGTGAAAGCTTGACTCAGTCAGGGTTAAGAGGGCCGGTAAAACTCGTG ca_anom TATTAAGCCATAAGTGAAAACTTGACTCAGTTAAGGTTAAAAGGGCCGGTAAAACTCGTG co_plumb1 TATTAAGCCATAAGTGAAAACTTGACTCAGTTAGGGTTAAGAGGGCCGGTAAAACTCGTG co_plumb2 TATTAAGCCATAAGTGAAAACTTGACTCAGTTAGGGTTAAGAGGGCCGGTAAAACTCGTG cy_venust TATTAAGCCATAAGTGAAAACTTGACTTAGTCAGGGCTAAGAGGGCCGGTAAAACTCGTG gi_cope TATTAAGCCATGAGTGAAAACTTGACTCAGTCAGGGTTAAGAGGGCCGGTAAAACTCGTG gi_cyph TATTAAGCCATGAGTGAAAACTTGACTCAGTCAGGGTTAAGAGGGCCGGTAAAACTCGTG he_fla50 TATTAAGCCATGAGTGAAAACTTGACTCAGTTAGGGTTAAGAGGGCCGGTAAAACTCGTG he_fla59 TATTAAGCCATGAGTGAAAACTTGACTCAGTTAGGGTTAAGAGGGCCGGTAAAACTCGTG le_vitt TATTAAGCCATGAGTGAAAACTTGACTCAGTCAGGGTTAAGAGGGCCGGTAAAACTCGTG mar_marg TATTAAGCCATAAGTGAAAACTTGACTCAGTTAGAGCTAAGAGGGCCGGTAAAACTCGTG me_fulg TATTGAGCTATAAGCGAAAGCTTGACTCAGTCAGGGTTAAGAGGGCCGGTAAAACTCGTG not_chry50 TATTAAGCCATAAGTGAAAACTTGACTCAGTCAGGGTTAAAAGGGCCGGTAAAACTCGTG not_ather TATTAAGCCATAAGTGAAAACTTGACTCAGTTAGAGCTAAGAGGGCCGGTAAAACTCGTG phe_catos TATTTAGCCATAAGTGAAAACTTGACTCAGTCACGGCTAAGAGGGCCGGTAAAACTCGTG pl_grac TATTAAGCCATAAGTGAAAACTTGACTCAGTCAGAGCTAAGAGGGCCGGTAAAACTCGTG rh_atrat TATTAAGCCATAAGTGAAAACTTGACTCAGACAGGGTTAAGAGGGCCGGTAAAACTCGTG se_atro TATTAAGCCATGAGTGAAAACTTGACTCAGTTAGGGTTAAGAGGGCCGGTAAAACTCGTG se_thor TATTAAGCCATAAGTGAAAGCTTGACTTAGTTAAGGTTAAGAGGGCCGGTAAAACTCGTG

[ 250 260 270 280 290 300] ac_alut CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATAGTACAACGGCGTTAAAGGGTGGTT ca_anom CCAGCCACCGCGGTTAGACGAGAGGCCCTAGTTGATAGTATAACGGCGTTAAAGGGTGGTT co_plumb1 CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATAGTACAACGGCGTTAAAGGGTGGTT co_plumb2 CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATAGTACAACGGCGTTAAAGGGTGGTT cy_venust CCAGCCGCCGCGGTTAAACGAGAGGCCCTAGTTGATGGAACAACGGCGTTAAAGGGTGGTT gi_cope CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATGCTACAACGGCGTTAAAGGGTGGTT gi_cyph CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATAGTACAACGGCGTTAAAGGGTGGTT he_fla50 CCAGCCACCGCGGTTAGACGAGAGGCCCTAGTTGATAÐTGTAACGGCGTTAAAGGGTGGTT he_fla59 CCAGCCACCGCGGTTAGACGAGAGGCCCTAGTTGATAÐTGTAACGGCGTTAAAGGGTGGTT le_vitt CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATGTTACAACGGCGTTAAAGGGTGGTT mar_marg CCAGCCACCGCGGTTAGACGAGAGGCCCTAGTTGATAGTACAACGGCGTTAAAGGGTGGTT me_fulg CCAGCCACCGCGGTTAGACGAGAGGCCCCAGTTGATTCTACAACGGCGTTAAAGAGTGGTT not_chry50 CCAGCCACCGCGGTTAAACGAGAGGCCCCAGTTAATAATACAÐCGGCGTTAAAGGGTGGTT not_ather CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATAGTATAACGGCGTTAAAGGGTGGTT phe_catos CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATAGCGTAACGGCGTTAAAGGGTGGTT pl_grac CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATAGTGCAACGGCGTTAAAGGGTGGTT rh_atrat CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATAGTACAACGGCGTTAAAGGGTGGTT se_atro CCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATAGTACAACGGCGTTAAAGGGTGGTT se_thor CCAGCCACCGCGGTTAAACGAGAGGCCCCAGTTGATAGTATAACGGCGTTAAAGGGTGGTT

[ 310 320 330 340 350 360] ac_alut AAGGAAAGCAÐAACAATAAAGCCAAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGCCC ca_anom AAGGATACTGAGACAATAAAGCCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGTCC co_plumb1 AAGGAACGCATAACAATAAAGCCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGTCC co_plumb2 AAGGAACGCATAACAATAAAGCCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGTCC cy_venust AAGGATAGTGAAACAATAAAGTCGAATGGCCCTTTGGCTGTCATACGCTTCTGGGAGCCT gi_cope AAGGAGCGCAAAACAATAAAGCCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGCCC gi_cyph AAGGAAAGCAÐAACAATAAAGTCAAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGTCC he_fla50 AAGGGATGCAÐAACAATAAAGTCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGCCT he_fla59 AAGGGATGCAÐAACAATAAAGTCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGCCT le_vitt AAGGAACGCAAGATAATAAAGCCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGTCC mar_marg AAGGAÐCGCACAATAATAAAGCCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGTCC me_fulg AAGGAGCGCATAATAATAAAGCCGAACGGCCCTTTGGCTGTCATACGCTTCTAGGCGCCC not_chry50 AAGGAAAGCATGGTAATAAAGCCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGTCC not_ather ATGGATAGCGAAATAATAAAGTCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGAGTCC phe_catos AAGGATAACACAACAATAAAGTCAAATGGCCCTTTGGCTGTCATACGCTTCTAGGAGTCC pl_grac AAGGATAGTGAAACAATAAAGTCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGAGTCC rh_atrat AAGGGTAATAAATTAATAAAGTCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGAGTCC se_atro AAGGGÐTGAACAAÐAATAAAGCCGAATGGCCCTTTGGCTGTCATACGCTTCTAGGTGCCT se_thor AAGGGÐTGCACGAÐAATAAAGCCAAAAGGCCCTTTGGCTGTCATACGCTTCTAGGTGCCT

[ 370 380 390 400 410 420] ac_alut GAAGCCCAATATÐÐACGAAAGTAGCTTTAGAAAAGCCCACCTGACACCACGAAAACTGAG ca_anom GAAGACCAATATÐÐACGAAAGTAGCTTTAAAGGAGTCCACCTGACCCCACGAAAACTGAG co_plumb1 GAAGTCCAATATÐÐACGAAAGTAGCTTTAGAAAAGCCCACCTGACCCCACGAAAACTGAG co_plumb2 GAAGTCCAATATÐÐACGAAAGTAGCTTTAGAAAAGCCCACCTGACCCCACGAAAACTGAG cy_venust GAAGCCCACTATÐÐACGAAAGTAGCTTTAACAGCGTCCACCTGACCCCACGAAAACTGAG gi_cope GAAGCCCAATATÐÐACGAAAGTAGCTTTAAGAGAGCCCACCTGACCCCACGAAAGCTGAG gi_cyph GAAGCCCAATATÐÐACGAAAGTAGCTTTAGAAAAGCCCACCTGACACCACGAAAGCTGAG he_fla50 GAAGCCCAGTATÐÐACGAAAGTCACTTTAAAATAGCCCACCTGACCCCACGAAAGCTGAG he_fla59 GAAGCCCAGCATÐÐACGAAAGTCACTTTAAAATAGCCCACCTGACCCCACGAAAGCTGAG le_vitt GAAGCCCAATATÐÐACGAAAGTAGCTTTAAGAAAGCCCACCTGACCCCACGAAAGCTGAG mar_marg GAAGCCCAATATÐÐACGAAAGTAGCTTTAAGAGAGCCCACCTGACCCCACGAAAGCTGAG me_fulg GAAGCCCGATATÐÐACGAAAGTAGCTTTAGTAAAGCCCACCTGACCCCACGAAAGCTGAG not_chry50 GAAGCCCAACGTÐÐACGAAAGTAACTTTAGTAAAACCCACCTGACCCCACGAAAGCTGAG not_ather GAAGCCCAGTATÐÐACGAAAGTAACTTTAAGAAAGCCCACCTGACCCCACGAAAACTGAG phe_catos GAAGCCCAGTTTCTACGAAAGTAACTTTAGAAGAGTTTACCTGACCCCACGAAAGCTGAG pl_grac GAAGCCCAATATÐÐACGAAAGTAGCTTTAGGAAAGTCCACCTGACCCCACGAAAACTGAG rh_atrat GAAGCCCAATATÐÐACGAAAGTAGCTTTAGGAAAGCCCACCTGACCCCACGAAAGCTGAG se_atro GAAGCCCAACATÐÐACGAAAGTCGCTTTAAAGTAGCCTACCTGATCCCACGAAAACTGAG se_thor GAAGCCCAACATÐÐACGAAAGTCGCTTTAAAATAGCCTGCCTGACCCCACGAAAACTGAG

[ 430 440 450 460 470 480] ac_alut AAACGAACTGGGATTAGATACCCCACTATGCTCAGCCGTAAACCTAGATGTCCAACTACA ca_anom AAACAAACTGGGATTAGATACCCCACTATGCTCAGCCGTAAACTTAGACGTCAACCTACA co_plumb1 AAACAAACTGGGATTAGATACCCCACTATGCTCAGCCATAAACCTTGACGTCCAACTACA co_plumb2 AAACAAACTGGGATTAGATACCCCACTATGCTCAGCCATAAACCTTGACGTCCAACTACA cy_venust AAACAAACTGGGATTAGATACCCCACTATGCTCAGTTATAAACCCAGACGTCCAACTACA gi_cope GAACAAACTGGGATTAGATACCCCACTATGCTCAGCCATAAACCTAGATGTCCAGCTACA gi_cyph AAACAAACTGGGATTAGATACCCCACTATGCTCAGCCATAAACCTAGATGTCCAACTACA he_fla50 AAACAAACTGGGATTAGATACCCCACTATGCTCAGTCATAAACTTAGACGTTCAACTACA he_fla59 AAACAAACTGGGATTAGATACCCCACTATGCTCAGTCATAAACTTAGACGTTCAACTACA le_vitt GAACAAACTGGGATTAGATACCCCACTATGCTCAGCCATAAACCTAGATÐTCCAGCTACA mar_marg GAACAAACTGGGATTAGATACCCCACTATGCTCAGCCATAAACCTAGACGTCCAACTACA me_fulg GAACAAACTGGGATTAGATACCCCACTATGCTCAGCCGTAAACCTAGATGTCCACCTACG not_chry50 AAACAAACTGGGATTAGATACCCCACTATGCTCAGCCGTAAACCCAGACGTCCAGCTACA

Copyright © 1997 by The Willi Hennig Society All rights of reproduction in any form reserved Phylogeny of North American Cyprinids 201 not_ather GAACAAACTGGGATTAGATACCCCACTATGCTCAGTCATAAACCCAGACGTCCAGCTACA phe_catos AAACAAACTGGGATTAGATACCCCACTATGCTCAGCCATAAATCTAGACGTCCTTCTACA pl_grac AAACAAACTGGGATTAGATACCCCACTATGCTCAGTCATAAACCCAGACGTCCGACTACA rh_atrat GAACAAACTGGGATTAGATACCCCACTATGCTCAGCCATAAACCCAGGCGTCGAACTACA se_atro AAACAAACTGGGATTAGATACCCCACTATGCTCAGTCGTAAACTTAGACGTCCAACTACA se_thor AAACAAACTGGGATTAGATACCCCACTATGCTCAGTTGTAAACTTAGACGTCCAACTACA

[ 490 500 510 520 530 540] ac_alut ATTAGACATCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC ca_anom ATAAGACGTCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC co_plumb1 ATTÐGACGTCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAAGACCTGACGGTGC co_plumb2 ATTÐGACGTCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAAGACCTGACGGTGC cy_venust ATÐAGGCGTCCGCCTGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC gi_cope GTTAGACATCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC gi_cyph ATTAGCCATCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC he_fla50 ATTAGACGTCCGCCCGGGTACTACGAGCATTAGCTTAAAACCCAAAGGACCTGACGGTGC he_fla59 ATTAGACGTCCGCCCGGGTACTACGAGCATTAGCTTAAAACCCAAAGGACCTGACGGTGC le_vitt ATTAGACATCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC mar_marg ATTAGACGTCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC me_fulg ATÐÐGACATCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC not_chry50 ATTAGACGTCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC not_ather ATCAGGCGTCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC phe_catos ATTAGGCGTCCGCCÐGGGTACTATGAGCATTAGCTTAAAACCCAAAGGACCTGACGGTGC pl_grac ATTAGACGTCCGCCÐGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC rh_atrat GTTAGACGTCCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC se_atro ATTAGACGTTCGCCCGGGTACTACGAGCATTAGCTTGAAACCCAAAGGACCTGACGGTGC se_thor ATTAGACGTCCGCCCGGGTACTACGAGCATTAGCTTAAAACCCAAAGGACCTGACGGTGC

[ 550 560 570 580 590 600] ac_alut CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT ca_anom CTCAGACCCCCCTAGAGGAGCCTATTCTAGAACCGATAACCCCCGTTAAACCTCACCACT co_plumb1 CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT co_plumb2 CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT cy_venust CTCAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCCCGTTGAACCTCACCGCT gi_cope CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT gi_cyph CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT he_fla50 CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCCTTAAACCTCACCACT he_fla59 CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT le_vitt CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT mar_marg CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT me_fulg CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT not_chry50 CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT not_ather CTCAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCCCGTTAAACCTCACCGCT phe_catos CTCAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCCCGTTAAACCTCACCACT pl_grac CTCAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCCCGTTAAACCTCACCACT rh_atrat CTCAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCCCGTTAAACCTCACCACT se_atro CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCTCGTTAAACCTCACCACT se_thor CTTAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAATCCTCGTTAAACCTCACCACT

[ 610 620 630 640 650 660] ac_alut TCTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG ca_anom TCTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG co_plumb1 TCTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG co_plumb2 TCTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG cy_venust TCTAGCCATCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTATGAAGGTAATAAAAG gi_cope CCTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGAAATAAAAG

gi_cyph TCTAGCCATCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG he_fla50 TTTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG he_fla59 TTTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG le_vitt TCTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG mar_marg TCTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG me_fulg TCTAGCCATCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG not_chry05 TCTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGAAATAAAAG not_ather TCTAGCCATCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG phe_catos TCTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGAAATAAAÐG pl_grac TCTAGCCATCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG rh_atrat TCTAGCCACCCCÐAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGCAATAAAAG se_atro TCTGGCCCCCCCCAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGTAATAGAAG se_thor TCTGGCCCCTTCCAGCCTGTATACCGCCGTCGTCAGCTTACCCTGTGAAGGTAATAAAAG

[ 670 680 690 700 710 720] ac_alut TAAGCAAAATGGGCACAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGAAGCGGGAAGA ca_anom TAAGCAAAATGGGCACAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGAAGCGGGAAGA co_plumb1 TAAGCAAAATGGGCACAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGAAGTGGGAAGA co_plumb2 TAAGCAAAATGGGCACAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGAAGTGGGAAGA cy_venust TAAGCAAGATGGGCACAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGGAGCGGGAAGA gi_cope TAAGCAAAATGGGCATAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGGAGTGGGAAGA gi_cyph TAAGCAAAATGGGCACAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGAAGCGGGAAGA he_fla50 TAAGCAAAATGGGCATAGCÐÐAGAACGTCAGGTCGAGGTGTAGCGTACGAAGTGGGAAGA he_fla59 TAAGCAAAATGGGCATAGCÐÐAGAACGTCAGGTCGAGGTGTAGCGTACGAAGTGGGAAGA le_vitt TAAGCAAAATGGGCATAGCÐÐAGAACGTCAGGTCGAGGTGTAGCGTACGAAGTGGGAAGA mar_marg TAAGCAAAATGGGCATACCCCAGAACGTCAGGTCGAGGTGTAGCGTACGAAGTGGGAAGA me_fulg TAAGCAAAATGGGCATAACCCAAAACGTCAGGTCGAGGTGTAGCGTACGAAGTGGGAAGA not_chry05 TAAGCAAGATGGGCACAGCÐÐAGAACGTCAGGTCGAGGTGTAGCGTACGAAGCGGGAAGA not_ather TAAGCAAAATGGGCACAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGAAGCGGGAAGA phe_catos TAAGCAAAACGGGTACAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGAAATGGGAAGA pl_grac TAAGCAAAATGGGCACAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGAAGCGGGAAGA rh_atrat TAAGCAAAATGGGCACAACCCAGAACGTCAGGTCGAGGTGTAGCGTACGAAGCGGGAAGA se_atro TAAGCAGAATGGGCACAACCCAAAACGTCAGGTCGAGGTGTAGCGTACGAAGTGGGAAGA se_thor TAATCAAAATGGACATAGCCCAAGACGTCAGGTCAAGGTGTAGCGTATGAAGTGGGAAGA

[ 730 740 750 760 770 780] ac_alut AATGGGCTACATTTTCTATTATAGAACACTACGGATATGCAACATGAAATAGTGCTTGAA ca_anom AATGGGCTACATTTTCTATTATAGAACACTACGGACATGCAACATGAAATAGTGCTTGAA co_plumb1 AATGGGCTACATTTTCTATTATAGAACACTACGAATATGCAACATGAAATAGTGCTTGAA co_plumb2 AATGGGCTACATTTTCTATTATAGAACACTACGAATATGCAACATGAAATAGTGCTTGAA cy_venust AATGGGCTACATTTTCTACAATAGAACACTACGGATTTGCAACATGAAATAGTGCTTGAA gi_cope AATGGGCTACATTTTCTATTATAGAATACTACGGATATGCAACATGAAATAGTGCTTGAA gi_cyph AATGGGCTACATTTTCTATTATAGAACACTACGGACATGCAACATGAAATAGTGCTTGAA he_fla50 AATGGGCTACATTTTCTATTATAGAATACTACGGATGTGCAACATGAAATAGTGCTTAAA he_fla59 AATGGGCTACATTTTCTATTATAGAATACTACGGATGTGCAACATGAAATAGTGCTTAAA le_vitt AATGGGCTACATTTTCTATTATAGAATACTACGGATATGCAACATGAAATGGTGCTTGAA mar_marg AATGGGCTACATTTTCTATTATAGAACACTACGGACATGCAACATGAAATAGTGCTTGAA me_fulg AATGGGCTACATTTTCTATTATAGAATACTACGGATATGCAACATGAAACTGTGCTTGAA not_chry05 AATGGGCTACATTTTCTACAATAGAACACTACGGACATGCAACATGAAATAGTGCTTGAA not_ather AATGGGCTACATTTTCTATTATAGAACACTACGGACATGCAACATGAAATAGTGCTTGAA phe_catos AATGGGCTACATTTTCTATTATAGAACACTACGGATTTGTAACATGAAATAGTGCTTGAA pl_grac AATGGGCTACATTTTCTATTATAGAATACTACGGACATGCAACATGAAATAGTGCTTGAA rh_atrat AATGGGCTACATTTTCTATTATAGAACACTACGGATGTGCAACATGAAATAGTGCCTGAA se_atro AATGGGCTACATTTTCTATTATAGAACACTACGGACATGCAACATGAAATAGTGCTTGAA se_thor AATGGGCTACATTTTCTATAATAGAACACTACGGATCTGCAACATGAAATAGTGCTTGAA

[ 790 800 810 820 830 840] ac_alut GGAGGATTTAGTAGTAAGAAGGAAACAGAGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT ca_anom GGAGGATTTAGTAGTAAAGAGGAAGCAGAGTGTCCTTTTGAACCCGGCTCTGAGACGCGT co_plumb1 GGAGGATTTAGTAGTAAAAGGGAAGCAGAGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT co_plumb2 GGAGGATTTAGTAGTAAAAGGGAAGCAGAGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT cy_venust GGAGGATTTAGAAGTAAAGAGGAAGCAGCGTGTCCTCTTGAACCCGGCTCTGAGGCGCGT gi_cope GGAGGATTTAGTAGTAAAAAGGAAGCAGAGTGTCCTTTTGAACTCGGCTCTGAGGCGCGT gi_cyph GGAGGATTTAGTAGTAAAAAGGAAACAGAGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT he_fla50 GGAGGATTTAGTAGTAAAAAGGAAGTAGCGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT he_fla59 GGAGGATTTAGTAGTAAAAAGGAAGTAGCGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT le_vitt GGAGGATTTAGTAGTAAAGAGGAAACAGAGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT mar_marg GGAGGATTTAGTAGTAAAAAGGAAGCAGAGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT me_fulg GGAGGATTTAGTAGTAAAAAGGAAGCAGCGTGTCCTTTTGAACCCGGCTCTTAGGCGCGT not_chry05 GGAGGATTTAGTAGTAAAAAGGAAGCAGAGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT not_ather GGAGGATTTAGTAGTAAAAAGGAAGCAGCGTGTCCTTTTGAACCCGGCTCTGAGACGCGT phe_catos GGAGGATTTAGTAGTAAAAAGGAAACAGCGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT pl_grac GGAGGATTTAGTAGTAAAAAGGAAGCAGAGTGTCCTTTTGAACCCGGCTCTGAGACGCGT rh_atrat GGAGGATTTAGTAGTAAAAAGGAAGCAGAGTGTCCTTTTGAACCCGGCTCTGAGACGCGT se_atro GGAGGATTTAGTAGTAAAAAGGAAGCAGCGTGTCCTTTTGAACTCGGCTCTGAGGCGCGT se_thor GGAGGATTTAGTAGTAAAAAGGAAGCAGCGTGTCCTTTTGAACCCGGCTCTGAGGCGCGT

[ 850 860 870 880 890 900] ac_alut ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCAATAAAGATTCTTAACACNAAAGCGC ca_anom ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCAACAAGATTACCTAATACTAGAGCCA co_plumb1 ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCAATATAGATACCTAACACCAACGCGC co_plumb2 ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCAATATAGATACCTAACACCAACGCGC cy_venust ACACACCGCCCGTCACTCTCCCCTGTCGAAATGCAATCAAGTTACCTAACACCAAAGCTC gi_cope ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCAACAAAGATACCTAACCCAGACGCGC gi_cyph ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCAATAAAGATACTTAACACCAAAGCGC he_fla50 ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCAATATTAATACCTAATACTAACGCGC he_fla59 ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCAATATTAATACCTAATACTAACGCGC le_vitt ACACACCGCCCGTCACTCTCCCCTGTCAACATGCAATAAAGATACCTAACCCAAACGCGC mar_marg ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCGACTTAAATATTTAATACCAACGCGC me_fulg ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCGATAAAGATACTTAACCCGAACGCGC not_chry05 ACACACCGCCCGTCACTCTCCCCTGTCAAAATGCAGTAAAGTTACCTAACACTAAAGCAC not_ather ACACACCGCCCGTCACTCTCCCCTGTCGAAATGCAATAAAGTTACCTAACGCTAAAGCTC phe_catos ACACACCGCCCGTCACTCTCCCCTGTCAAAATGTGATAAAGCTACCTAACAAGAAAGCTA pl_grac ACACACCGCCCGTCACTCTCCCCTGTCGAAATGCAATAAAGCTTCTTAACACCAAAGCTC rh_atrat ACACACCGCCCGTCACTCTCCCCTGTCAAAGTGCAATAAAGCTACCTAACATCATAGCAG se_atro ACACACCGCCCGTCACTCTCTCCTGTCTAAATGCAATATAAATACTTAATATCACTGCGC se_thor ACACACCGCCCGTCACTCTCCCCTGTCTTAATGCAATATAAATACTTAATACCAACGCGC

[ 910 920 930 940 950 959] ac_alut TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATTAAAT ca_anom TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATAAAAT co_plumb1 TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGAATAAAT co_plumb2 TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGAATAAAT cy_venust TGACAAGGGGAGGCAAGACGTAACATGGTAAGTGTACCGGAAGTGCACTTGGAÐTAAAT gi_cope CGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATTAAAT gi_cyph TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATTAAAT he_fla50 TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATTAAAT he_fla59 TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATTAAAT le_vitt CGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATTAAAT mar_marg CGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCTCTTGGATTAAAT me_fulg CGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGACTAAAT not_chry05 TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATTAAAT

not_ather TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATAAAAT phe_catos AGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATAAAAC pl_grac TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATAAAAT rh_atrat TGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGCACTTGGATTAAAT se_atro TGACGAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGTACTTGGACTAAAT se_thor TGACGAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGTGTACTTGGACTAAAT

[binary coding of insertionÐdeletion events] [ 960...1...2...3...4...5...6...7...8...9..970] 97 126 163 278 306 311 314 483 484 613 681] ac_alut 0 0 0 0 0 1 0 0 0 0 0 ca_anom 0 0 0 0 0 0 0 0 0 0 0 co_plumb1 0 0 0 0 0 0 0 0 1 0 0 co_plumb2 0 0 0 0 0 0 0 0 1 0 0 cy_venust 0 0 0 0 0 0 0 1 0 0 0 gi_cope 0 0 0 0 0 0 0 0 0 0 0 gi_cyph 0 0 1 0 0 1 0 0 0 0 0 he_fla50 0 0 0 1 0 1 0 0 0 0 1 he_fla59 0 0 0 1 0 1 0 0 0 0 1 le_vitt 0 0 1 0 0 0 0 0 0 0 1 mar_marg 0 0 0 0 1 0 0 0 0 0 0 me_fulg 0 0 1 0 0 0 0 1 2 0 0 not_chry05 0 0 0 0 0 0 0 0 0 0 1 not_ather 0 1 0 0 0 0 0 0 0 0 0 phe_catos 1 1 0 0 0 0 0 0 0 0 0 pl_grac 1 0 0 0 0 0 0 0 0 0 0 rh_atrat 0 0 0 0 0 0 0 0 0 0 0 se_atro 0 0 0 0 1 0 1 0 0 1 0 se_thor 0 1 0 0 1 0 1 0 0 1 0 ; ENDBLOCK; BEGIN ASSUMPTIONS;

exset subset_1 = 306Ð314 960 970 [excludes all problematic alignments gaps excluded]; exset subset_2 = 306Ð314 [excludes all problematic alignments, gaps included]; charset sequences = 1Ð959;

[apply the following weights only to charset "sequences"] USERTYPE 3_1 = 4 [weights transversions 3 times transitions] G A T C [G] 0 1 2 2 [A] 1 0 2 2 [T] 2 2 0 1 [C] 2 2 1 0 ;

USERTYPE 2_1 = 4 [weights transversions 2 times transitions] G A T C [G] 0 1 3 3 [A] 1 0 3 3 [T] 3 3 0 1 [C] 3 3 1 0 ;

USERTYPE 10_1 = 4 [weights transversions 10 times transitions] G A T C [G] 0 0 10 10 [A] 1 0 10 10 [T] 10 10 0 1 [C] 10 10 1 0 ; ; USERTYPE tv = 4 [transversions only] G A T C [G] 0 0 1 1 [A] 0 0 1 1 [T] 1 1 0 0 [C] 1 1 0 0 ; ;

ENDBLOCK;

OUTGROUP not_chry05;