Copeia, 1997(2). pp. 298-305

Phylogenetic Relationships of Species of the Brachyrhaphis () Inferred from Partial Mitochondria1 DNA Sequences

C.4~11~1,~L. MOJICA,AXEL MEYER,AND GEORGEW. BARLOW

Phylogenetic relationships were reconstructed using partial mitochondrial DNA sequence data for the cytochrome b gene among all nine described species of Bra- &flaphis and several members of the tribe Gambusiini. We included three of the more than 40 members of the genus Gambusia and Belonesox belimnus, the third, monotypic genus of the tribe Gambusiini. Xiphophorus malinche served as the out- group. Phylogenetic trees were generated using maximum-parsimony, maximum- likelihood, and neighbor-joining analyses. Hypotheses of relationships withii the tribe and genus Brachyrhaphis made by previous authors were tested. The existence of a slim- and a deep-bodied clade within the monophyletic genus BraFhflaphis was supported. Bmchyrhaphis hariwegi is the basal member of the deep-bodied clade, whereas B. punctifer is the basal member of the slim-bodied clade.

PECIES of the genus Brachyrhaphis are found unique condition within the family. Anal fins of S in freshwater streams, rivers, and swamps of females of some species are as colorful as the Central America. The geographic distribution male gonopodia. Additionally, the black pig- of Brachyrhaphis is disjunct; seven of the eight ment is placed in such a way in the females of species are found in Costa Rica and Panama, certain species that it traces the first three rays whereas the eighth species, B. hartwegi, is found of the anal fin from their origin to the tip. This in Guatemala (Rosen and Bailey, 1963). These highlighted, lengthened region is about the fishes are found at the shallow edges of bodies same width as a male gonopodium. The fourth of water where they are able to feed on terres- ray is lengthened in some species relative to the trial insects that fall into the water (Bussing, last rays of the anal fin, giving the fin a falcate 1988). The geographic ranges of individual spe- shape. cies may overlap, but segregation seems to oc- The result of this combination of unique col- cur both by microhabitat and elevation. When or patterns and morphology possessed by these found in the same area, one species usually oc- females is that the anal fins bear a striking re- cupies open streams, whereas the other inhabits semblance to the gonopodia of males of their swamps or backwater areas. Similarly, one spe- own species. Both males and females use these cies may replace another with increasing eleva- fins during social interactions. The black area tion in a given drainage. appears to change in intensity depending on so- Male poeciliid fishes are well known for their cial status (pers. obs.). This suggests that these sexually dimorphic traits; these usually involve structures function in social communication. unique color patterns and elaborate morphol- The hypothesis, then, is that selection for com- ogy. Sex-specific differences range from vertical munication has produced elaborate, colorful fe- bars (Morris et al., 1995) and elongated male anal fins that mimic the gonopodia of "swords" (Basolo, 1995) to elaborate coloration males. A phylogenetic tree is necessary to test in male guppies, Poecilia reticulata (Houde and this hypothesis because we will want to compare Endler, 1990). Compelling evidence indicates closely related species that vary in the expres- that these traits have evolved via sexual selection sion of derived traits (Brooks and McLennan, (Houde and Endler, 1990; Endler, 1995). But in 1991). Such a comparative study requires a phy- the genus Brachyrhaphis (and some Gambusia, logeny if it is to accommodate the potential e.g., G. sexradiata), these traits are found in both nonindependence among data points. The the males and females, making sexual selection morphology and color patterns will be more via female choice unlikely explanations for the thoroughly described, and hypotheses concern- evolution of the female traits: ing the adaptive significance of these traits will The novel morphology possessed by both sex- be tested elsewhere (CLM, unpubl.) using the es varies dramatically from the colorless and typ- phylogeny constructed here. ical anal fin or gonopodium of Brachyrhaphis This investigation used mitochondrial DNA hartwegi, to the largely black anal fin/gonopo- sequence data to resolve the phylogenetic rela- dium of B. rhabdophora. Ornamentation of both tionships of the taxa mentioned. Such data have male gonopodia and female anal fins are a proven to be phylogenetically informative in MOJICA ET AL.-BRACHYRHAPHZS PHYLOGENY 299 many taxa (Meyer, 1994). Sequence data from support this combination: "With the exception the cytochrome b gene have been particularly of B. parismina (including B. cascajalensis) the useful in resolving poeciliid systematics (Meyer species of the genus also have a pattern of ver- and Lydeard, 1993; Lockhart et al., 1995; Ly- tical black bars . . ." (Bussing, 1988:81). deard et al., 1995). Brachyrhaphis holdridgei, B. roseni, B. rhabdopho- ra, and B. punctifer resemble the deepbodied B. .-Regan (191 3) described the mono- +iscopi and B. terrabensis in body depth and col- typic genus Brachyrhaphis (B. rhabdophora) based or pattern. All have black, orange, yellow, on the possession of a shorter gonopodium and/or red pigments accenting their fins; oli- than that of Gambusia. The most thorough sys- vaceous bodies; and vertical bars along the tematic study to date of the genus was based on flank. Brachyrhaphis cascajalensis and B. parismina morphology and is part of the revision of the lack these pigments, whereas B. hartwegi is yel- family Poeciliidae by Rosen and Bailey (1963). low throughout. They used similarities in male gonopodia to des- Bussing (1988) stated that, when examining ignate species. They included in the genus Bra- specimens of B. rhabdophora, he found addition- chyrhaphis several species formerly assigned to al differencesbetween those occurring in north- Gambusia ( G. cascajalensis, [Meek and Hilde- ern Costa Rica and those in the south. The geo- brand, 19131 ; G. +iscopi [Steindachner, 18781 ; graphic boundaries thought to delimit the G. parismina [Meek, 19121; G. rhabdophma, Re- northern forms include Guanacaste and the gan, 1908; G. terrabensis, Regan, 1907b). They Tarcoles drainage in central Costa Rica. The also added B. punctifer (Trigonophallus punctifer southern form is distributed from Punta Mala [Hubbs, 19261) to the genus and described B. on the Nicoya Peninsula down to the Terraba hartwegi, a new species. Bussing later described drainage, where it is replaced by B. roseni (W.A. two additional species to the genus (B. holdridg- Bussing, pers. comm.). Bussing does not com- ei, 1966, and B. roseni, 1988). ment on whether these species are each others' Rosen and Bailey (1963) stated that a "partic- closest relatives. ularly close relationship" between B. hartwegi Eight species currently make up the genus and B. terrabasis existed based on the posses- Brachyrhaphis that is part of the tribe Gambusi- sion of a "bean-shaped fleshy pad near the tip ini. Members of the tribe also include the genus of the first pelvic ray." Other close relationships Gambusia, which contains more than 40 de- were hypothesized when certain species (B. +- scribed species and the monotypic genus Belo- iscopi and B. terrabensis) were referred to as nesox. With respect to the tribe, Rosen and Bai- deep-bodied species and others (B. hartwegi, B. ley (1963) stated that Gambusia and Belonesoxare cascajalensis, and B. parismina) as slender-bodied most closely related to one another and that a species. They stated that B. cascajalensis "is most form resembling B. cascajahsis gave rise to the closely related to B. parismina." ancestor of these two genera. The tribe was re- The classification based on body depth was vised by Rauchenberger (1989). Her cladistic not used consistently, however, because they analysis of morphological data supported the also referred to B. hartwegi as a slender-bodied previous notion that Gambusia and Belonesox are species even though it was presumed to be sister taxa. closely related to the deep-bodied B. terrabensis. Having seen live specimens of every species, we Hypothesis testing-The purpose of this study is describe B. hartwegi as a deep-bodied species. to elucidate the relationships within the genus Rosen and Bailey (1963) did not discuss body Brachyrhaphis and within the tribe Gambusiini so depth when treating B. rhabdophma or B. punc- that the resultant phylogenetic tree can be used tver, but we describe the former as a deepbod- in addressing the evolution of novel morphol- ied fish and the latter as a slender-bodied spe- ogy and behavior patterns. In producing a phy- cies. Brachyrhaphis holdridgei and B. roseni had logenetic tree, we tested the hypotheses put not been described when Rosen and Bailey forth or implied by previous investigators. These (1963) made their revisions. We believe both of include the following: (1) Brachyrhaphis is a these to be deep-bodied as well. An informal monophyletic genus with respect to other gam- comparison of the body-depth data given in the busiin fishes (Rauchenberger, 1989); (2) a original descriptions of all Brachyrhaphis species deep-bodied clade consists of B. giscopi, B. hold- supports these ideas (unpubl. data). ridgei, B. rhabdophma, and B. terrabasis (Rosen Bussing (1988) hypothesized a close relation- and Bailey, 1963); (3) B. hartwegi and B. terra- ship between B. roseni and B. rhabdophora. Ad- bensis are closely related (Rosen and Bailey, ditionally, he included B. cascajalensis in B. par- 1963); (4)B. cascajalensis and B. parismina com- ismina without providing an analysis of data to prise a slim-bodied clade, and/or the two latter 300 COPEIA, 1997, NO. 2 described forms represent one species (Rosen tomated sequencing protocols. The sequences and Bailey, 1963; Bussing, 1988); (5) B. holdridg- obtained were deposited in GenBank. ei is more closely related to the slim-bodied clade than to the other species of the genus Phylogenetic analysis.-All sequences were en- (Bussing, 1966); and (6) what is currently B. tered into PAUP, Vers. 3.1.1 for the Macintosh rhabdophora is a paraphyletic group of species (Bussing, 1988). (Swofford, 1991, unpubl.), and aligned by eye. The last hypothesis refers to Bussing's asser- This program was used to perform a phyloge- tion that B. rhabdophora should be split along netic analysis using the principle of maximum geographic boundaries. This study cannot estab- parsimony (MP).The weighting scheme applied lish the existence of two species. Bussing's hy- to the data followed the suggestions made by pothesis can be supported, however, if the pop- Meyer (1994) in his evaluation of cytochrome b ulations are found to be paraphyletic. This study as a molecular marker. will be inconclusive with respect to this hypoth- The shortest tree was found in a heuristic esis if monophyly is the result. Partial mitochon- search with tree bisection-reconnection (TBR) drial DNA sequences for the mitochondria1 cy- branch-swapping in 10 random-addition repli- tochrome b gene were used in a phylogenetic cates. The MULPARS option was chosen (all analysis to construct a tree with which to test minimal trees saved). ~Ghophorusmalinche was this and the other six hypotheses. designated as the outgroup (Rosen and Bailey, 1963; Meyer and Lydeard, 1993). The integrity of the tree was measured by bootstrapping (Fel- senstein, 1985) using a heuristic search as de- Spen'ms.-All specimens of Brachyrhaphis were scribed above and 100 replicates. collected in the field or obtained from other Nodes within the tree were compared using collectors and sequenced by CLM. At least one the Decay Index (DI; B. D. Mishler, M. J. Don- individual per species was sequenced. Addition- oghue, and V. A. Albert, unpubl.). The DI is the al sequences from two species were acquired to number of steps required to collapse a node in reveal inter- and intrapopulation variation. Data a parsimony analysis (B. D. Mishler, M. J. Don- from three individual B. qbiscopi were gathered: oghue, and V. A. Albert, unpubl. abstract). Suc- two from the same population (Barro Colorado cessively longer trees were saved during PAUP's Island, Panama) and one from a second popu- heuristic search. The strict consensus of the re- lation (Rio Mendoza, Pipeline Road, Panama). sultant trees revealed the nodes that had col- This study also included three representatives lapsed. from the geographic range of B. rhabdophora: a The software package PHYLIP (Felsenstein, northern type from the Rio Tenorio (Rio Beb- 1989) was used to execute a second phyloge- dedero Drainage, Guanacaste Province, Costa netic method, maximum likelihood (ML; el- Rica); a central form from Santa Ana (San Jose senstein, 1985), and a distance-based method, Province, Costa Rica) ;and a southern type from neighbor-joining (NJ; Saitou and Nei, 1987). Pa- the Rio Angel (near San Isidro del General, San rameters specified in the maximum-likelihood Jose Province, Costa Rica) . The three specimens analysis (DNAML) included one category of of Gambusia and one Belonesox were obtained substitution, default parameters, and the desig- and sequenced in AM'S laboratory (Meyer and Lydeard, 1993; Lydeard et al., 1995). Voucher nation of X. malinche as the outgroup. Tree ro- specimens of Brachyrhaphis were deposited at bustness was inferred from P-v~lues~calculated the California Academy of Sciences. for length of branches. A Kimura two-parameter model (Kimura, Sequence collection.-Conventional methods were 1980) was used in calculating a corrected dis- used to obtain 12 of the 17 sequences via man- tance matrix in the DNADIST subroutine and ual sequencing from a 350-base-pair (bp) re- served as the input for the subroutine NEIGH- gion of the cytochrome b gene of the mitochon- BOR in PHYLIP. Tree robustness was assessed drial genome (Kocher et al., 1989; Walsh et al., by bootstrapping. One hundred data sets were 1991; Smith and Patton, 1993). Five sequences generated in SEQBOOT, distance matrices were were obtained via automated sequencing (Ly- calculated for each data set in DNADIST, the deard et al., 1995). Double-stranded products shortest tree for each was found in NEIGHBOR, for five of the specimens (B. episcopi [B2], B. and the consensus tree was generated by CON- rhabdophora [S] , Gambusia afjnis, G. nicaraguen- SENSE. These three sets of analyses were per- sis, Belonesox belizanus, and Xiphophorus malinche) formed to test whether different results were were used to acquire sequences via standard au- produced by different phylogenetic methods. MOJICA ET AL.-BRACHYRHAPHIS PHYLOGENY 301

B. roseni I LB. terrabensis r- B. terrabensis B. rhabdophora(S) B. rhabdophora (S)

B. rhabdophora (C) B. rhabdophora (C) LB rhabdophora (N) 'B, rhabdophora (N) I 1 B. episcopi (Bl) Il r- B. episcopi (Bl) 4 LB episcopi (82) ri '+B. episcopi (B2) LB episcopi (MI L B. episcopi (M) 49 I I fl- B. holdridgei B. holdridgei 4 B. hartwegi B. hartwegi 8.parismina 9. parismina

I 1 46 ]'*LB. cascajalensis B. cascajalensis ri IB. punctifer L B punctifer affinis 4 r G. affinis sexradiata G. sexradiata

nicaraguensis G. nicaraguensis

Belonesox )-"I IBelonesox

IX. malinche IX malinche Fig. 1. Maximum-parsimony result. Consensus Fig. 2. Maximum-likelihood result. Tree gener- tree (50% majority-rule) generated in PAUP 3.1.1 ated in PHKIP (Felsenstein, 1989). Nonsignificant (Swofford, 1991, unpubl.). Decay indices are shown branch lengths are collapsed. See Materials and Meth- below, and bootstrap values (100 replicates) are ods for parameters chosen. Specimens of Brachyrha- shown above each internode. Specimens of Brachy- phis +iscopi from Barro Colorado Island, Panama, are rhaphis episcopi from Barro Colorado Island, Panama, denoted B1 and B2. The specimen from the Rio Men- are denoted B1 and B2. The specimen from the Rio doza is labeled M. Specimens of B. rhabdophora from Mendoza is labeled M. Specimens of B. rhabdophora the southern, central, and northern parts of Costa from the southern, central, and northern parts of Rica are labeled S, C, and N, respectively. Costa Rica are labeled S, C, and N, respectively.

B. episcopi included in this study. The strictcon- sensus tree shown (Fig. 1) is 415 steps long. RESULTS Bootstrap values and the DI are given at each node. Seventy-two phylogenetically informative When these data were analyzed (GenBank ac- characters were found out of the possible 381. cession U68300-12), three shortest trees were The consistency index for this tree is 0.567. The found via MP. The three trees differed only in log of the consistency index is negatively cor- the relationships among the three specimens of related with the number of taxa (Sanderson and 302 COPEIA, 1997, NO. 2

TABLE1. LOWER-TRIANGLEDISTANCE MATKIXGENERATEDBY DNADIST IN PHYLIP (FELSENSTEIN1989). See Materials and Methods for parameters chosen. Specimens of (Brachyrhaphisqiscopi from Barro Colorado Island, Panama, are denoted B1 and B2. The specimen from the Rio Mendoza is labeled M. Specimens of B. rhab dophora from the southern, central, and northern parts of Costa Rica are labeled S, C, and N, respectively.

1. H, rosvn1 2. R, rplscopz (BI) 3. R. @ISC@JI(B2) 4. H. @sropI (M) 5. H, holdndgFI 6. R, pnri~m~no 7. H, l~rrnboisls 8. H, rhnbdophorn (S) 9. H, rhnbdophorn (C) 10. R. rhnbdophmf~ (h') 1 1. R. cnsrn]~~luns~s 12. R. punclqk 13. H. hnrtwqi 14. G. f~fin~s 15. G. s~xmd~nlo 16. G, nlrnrnffupnsrc 17. R~lonrsox 18. X. mnhnrhe

Donoghue, 1989; Klassen et al., 1991). The log loo), a Brachyrhaphis clade (MP: bootstrap = 49, of 0.567 falls above the regression fitted to num- DI = 3; ML: P < 0.01; NJ: bootstrap = 80), and ber of taxa suggesting that our data set is phy- a Gambusia clade (MP: bootstrap = 99, DI = 5+; logenetically informative. ML: P < 0.01; NJ: bootstrap = 100). Branches of nonsignificant lengths are col- lapsed in the resultant ML tree (Fig. 2). One polytomy includes the three specimens of B. ep- iscopi. The other unresolved node is at the base of the tree. The relationships among Xiphophc- Monophyly of the genera Brachyrhaphis and rus, the genus Brachyrhaphis, and Belonesox + Gambusia is well supported by our data. The Gambusia remain unclear in this analysis. The monophyly of the tribe Gambusiini with respect topology of the rest of the tree is in agreement to the outgroup taxon, X. malinche, is weakly with that of the MP analysis. supported and remains somewhat uncertain A Kimura tw~-~arameterdistance matrix was (Figs. 1-3). This finding is consistent with re- used in the NJ analysis (Table 1).The resultant cent evaluations of cytochrome b as a molecular tree (Fig. 3) differs from the MP and ML anal- marker (Graybeal, 1993; Meyer, 1994). Graybeal yses in that the relationship between the desig- (1993), in particular, found that cytochrome b nated outgroup and Belonesox to Gambusia + was useful as a phylogenetic marker at the level Brachyrhaphis was not resolved; the relationships of species divergences in bufonid frogs. Boot- strap values decrease the more internal the between the three specimens of B. episcopi- - are supported (bootstrap = loo), but the two spec- node. This is evidence that, for these fish as for imens from Barro Colorado Island do not fall other taxa, cytochrome b is most helpful in re- out as each other's closest relative; and G. ni- solving shallow divergences. A more slowly caraguensis is most closely related to G. sexradiata evolving gene, such as the 16s ribosomal gene, instead of G. afinis as ii both of the other anal- may allow more robust resolution of the inter- yses. Otherwise, the topology of the NJ tree nal nodes in future studies (Meyer and Lydeard, matches those of the other two analyses. 1993; Meyer, 1994). The central and southern populations of B. All results support the monophyly of Brachy- rhabdophura were more closely related to each rhaphis and Gambusia. The MP and ML trees are other than either was to the northern Guana- consistent with Rauchenberger's (1989) place- caste population in all three analyses (MP: boot- ment of G. afjnis and G. sexradiata as sister taxa strap = 42, DI = 1; ML: P < 0.01; NJ: bootstrap with respect to G. nicaraguensis. Lydeard et al. = 78). All three analyses support a B. roseni + (1995) recently reconstructed the phylogeny of B. terrabasis clade (MP: bootstrap = 69, DI = 24 species of Gambusia using the same part of 2; ML: P < 0.01; NJ: bootstrap = 78), a B. par- the gene as used in this study. Their analysis also ismina + B. cascajahsis clade (MP: bootstrap = failed to resolve the relationships among these 100, DI = 5+; ML: P < 0.01; NJ: bootstrap = three distantly related species. Our data weakly MOJICA ET AL.-BRACHYRHAPHIS PHYLOGENY

1. B. rosm 2. B, episcopl (BI) 3. B, epirc*' (82) 4. B. rpiscopl (M) 5. B. holdndgn 6. B. pnrism~nn 7. B. lmnhsis 8. B. rhnbdophmn (S) 9. B. rhnbdophmf~(C) 10. B, rhnbdophorn (h') 1 1. B, rnscnlnlpnslr 12. B, punrli/er IS. B, hnrtwa 14. G. nJJnis 15. G. s~xradlolo 16. f;, nicnrnp~nsis 17. B~lon~sox 18. X. mnlinche

support Rauchenberger's (1989) hypothesis southern and central populations, showed dif- that Gamhsia and Belonesox are sister taxa. ferences of 4.58% and 5.22%, respectively. The The relationship between B. roseni and B. ter- minimum difference between siquences that rabensis is convincingly supported. This corrob- resulted among species was 5.19% for southern orates our assessment that B. roseni is a member B. rhabdophora and B. terrabensis (Table 1). This of the deepbodied clade. The relationships be- suggests that the central and southern popula- tween B. episcopi, B. holdridgei, B. rhabdophora, tions, which exhibited a 3.02% seauence differ- and B. roseni + B. terrabensis,on the other hand, ence between one another, may be different still seem somewhat unresolved (Figs. 1-3). The with respect to the northern B. rhabdophora, as monophyly of the clade that includes all of Bussing (1988) hypothesized. A more complete these deepbodied species when compared with phylogeographic analysis is necessary to resolve other nodes within the tree has some support this issue. (Figs. 1-3). Brachyrhaphis hartwegi is basal to this The phylogenetic trees reported here will fa- clade. It is not, however, most closely related to cilitate the study of the evolution of the novel B. terrabensis as was hypothesized by Rosen and behavioral and morphological traits described Bailey (1963). The monophyly of the B. paris- above. Studies in progress, specifically, focus on mina + B. cascajalensis clade (slim-bodied) is the adaptive significance of anal-fin coloration strongly supported. These molecular phyloge- and mdrphological elaboration. The relation- netic results agree with Rosen and Bailey's ship among sister taxa having different charac- (1963) identification of a slim- and deep-bodied ter states is particularly informative to the inter- clade within this genus. pretation of these data. Preliminary results were The data do not support or refute Bussing's used in defining the most meaningful taxa for claims (1988) that B. cascajahsis and B. paris- these studies. The distribution of the resultant mina are the same species or that B. rhabdophora morphological and behavioral characters on the deserves revision at the species level. The anal- phylogeny has allowed us (CLM and GWB, un- yses would have refuted the first claim and sup- publ.) to hypothesize the function of these nov- ported the second only if the species or popu- el features. lations had turned out to be paraphyletic and Phylogenetic approaches to behavior, mor- not monophyletic. phology, and ecology can take us beyond plau- The data are, however, suggestive with respect sibility arguments by providing refutable hy- to the second claim. The percent-sequence dif- potheses of evolutionary change in the charac- ferences between the B. rhabdophora populations ters of interest (e.g., Meyer et al., 1994). Phy- is higher than those for B. episcopi; B. episcopi logenetic trees are necessary for testing these from the Rio Mendoza shows a maximum of evolutionary hypotheses (Brooks and Mc- 2.98% difference with respect to conspecifics Lennan, 1991). They can be used to guide re- from Barro Colorado Island (Table 1). The search, interpret preliminary data, reveal the northern B. rhabdophora, when compared with need for certain kinds of comparative data or COPEIA, 1997, NO. 2

ed equipment, laboratory resources and space. R. McKeand, M. C. Belk, S. Sung, and A. Basolo B. terrabensis provided specimens. Collecting trips and labo- ratory work by CLM were supported by the Pa- tricia Roberts Harris Fellowship, a Smithsonian rB. rhabdophora(S) 10-week graduate student fellowship, a Raney Award from the American Society of Ichthyol- B. rhabdophora(C) ogists and Herpetologists, National Science Foundation Grant 910852 to GWB, the Gradu- B. rhabdophora(N) ate Division (UCB), the Department of Integra- tive Biology, and Museum of Vertebrate Zoolo- B. episcopi (Bl) gy. Comments on an early version of the manu- script were kindly made by E. Jockusch. This work received partial support by grants B. episcopi (M) from the National Science Foundation to AM (BSR-9107838 and BSR-9119867) and from a B. episcopi (82) collaboration grant with the Max-Planck-Institut fur Biology in Tubingen from the Max-Planck B. holdridgei Society, Germany. This publication was pre- pared during AM'S tenure as Miller Visiting Re- IB. hartwegi search Professor at the University of California at Berkeley. The support of the Miller Institute and the hospitality of the Museum of Vertebrate B. parismina Zoology and the Departments of Integrative Bi- ology and Molecular and Cell Biology are grate- B. cascajalensis fully acknowledged. D. Reznick and an anony- mous reviewer provided comments that allowed I- B. punctifer us to greatly improve the manuscript. rG. nicaraguensis 0.sexradiata BASOLO,A. L. 1995. Phylogenetic evidence for the role of a preexisting bias in sexual selection. Proc. R. Soc. Lond. B Biol. Sci. 259:307-311. BROOKS,D. R., AND D. A. MCLENNAN.1991. Phylog- eny, ecology and behavior: a research program in Belonesox comparative biology. Univ. of Chicago Press, Chi- cago. IBUSSING,W. A. 1966. New species and new records X. malinche Iof Costa Rican freshwater fishes with a tentative list Fig. 3. Neighbor-joining result. Tree generated in of species. Rev. Biol. Trop. 14205-249. PHYLIP (Felsenstein, 1989). See Materials and Meth- . 1988. A new fish, Brachyrhaphis roseni (Poe- ods section for details. Specimens of Brachyrhaphis ep ciliidae) from Costa Rica and Panama. Zbid. 36:81- iscopi from Barro Colorado Island, Panama, are de- 87. noted B1 and B2. The specimen from the Rio Men- ENDLER,J. A. 1995. Multiple-trait coevolution and en- doza is labeled M. Specimens of B. rhabdophora from vironmental gradients in guppies. Trends Ecol. the southern, central, and northern parts of Costa Evol. 10:22-29. Rica are labeled S, C, and N, respectively. FELSENSTEIN,J. 1985. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17:368-376. experiments, and test evolutionary hypotheses . 1989. PHYLIP-phylogenetic inference once these data have been gathered (Brooks package. Cladistics 5:164-166. and McLennan, 1991). GRAYBEAL,A. 1993. The phylogenetic utility of cyto- chrome b: lessons from Bufonid frogs. Mol. Phyl. Evol. 2:256-269. HOUDE,A. E., AND J. A. ENDLER.1990. Correlated evolution of female mating preferences and male C. Orrego provided training and technical color pattern in the guppy Poecilia reticulata. Science support along with K. Le and S. Pak to CLM. K. (Wash.) 248:1405-1408. Le aided in gathering data. J. L. Patton provid- HUBBS,C. L. 1926. Studies of the fishes of the order MOJICA ET AL.-BRACHYRHAPHIS PHYLOGENY 305

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