Phylogeny of Limia (Teleostei: Poeciliidae) Based

Ct

mg

upptbaaLsftto

Molecular Phylogenetics and EvolutionVol. 19, No. 2, May, pp. 277–289, 2001doi:10.1006/mpev.2000.0919, available online at http://www.idealibrary.com on

on NADH Dehydrogenase Subunit 2 SequencesAaron Hamilton

Biology Department, University of California, Riverside, California 92521

Received August 2, 2000; revised December 1, 2000

INTRODUCTION

tttbBswGgcl11esdpi

Complete sequences for the mitochondrial geneNADH Dehydrogenase 2 (ND2) and partial sequencesfor the tRNA-Met and tRNA-Trp genes were obtainedfor 11 populations of the poeciliid fish genus (orsubgenus) Limia, including species from Hispaniola,

uba, Jamaica, and Grand Cayman Islands. Addi-ional sequences for Limia (2 species), Pamphorich-

thys (3), Poecilia (16), and Xiphophorus (1), all fromthe tribe Poeciliini, were extracted from GenBank,as was a sequence for Heterandria formosa, from thetribe Heterandriini. Phylogenetic analyses includedparsimony, distance methods, and maximum-likeli-hood. Several Limia species groups that had beenproposed based on morphological data were evalu-ated. The versicolor species group was strongly sup-ported, as was the close relationship between theCuban Limia vittata and the Grand Cayman Limiacaymanensis. However, the proposed vittata speciesgroup was not upheld by the ND2 data. The phylo-genetic position of Limia melanogaster, from Ja-

aica, was either sister to the versicolor speciesroup or basal to all other included Limia species.

Limia was found to be monophyletic; however,Limia species from the island of Hispaniola were notmonophyletic. There was little support for any pro-posed sister group to Limia. The phylogeny was

sed to reconsider a previous comparative study ofoeciliid courtship behavior and sexual dimor-hism. The data indicated that there may have beenwo independent appearances of courtship displayehavior in Limia; considering the tribe Poeciliinis a whole, there is evidence for as many as fiveppearances of display (including two withinimia), or a complex pattern of gains and losses ofuch behavior. The application of phylogenetic in-ormation to the comparative study did not refutehe previously hypothesized correlation betweenhe presence of a courtship display and the presencef sexual dimorphism in poeciliid fish. © 2001 Academic

Press

Key Words: molecular phylogeny; Limia; poeciliidfish; comparative methods; ND2.

277

The poeciliid fish genus Limia from the Greater An-illes is an excellent target for the comparative study ofhe evolutionary effects of sexual selection becausehere is substantial interspecies diversity in matingehavior and sexual dimorphism (Farr, 1984, 1989;isazza, 1993). Limia is also the focus of biogeographytudies of the Caribbean islands because these fresh-ater fish occur on Cuba, Jamaica, Hispaniola, andrand Cayman Islands (Rauchenberger, 1988; Bur-ess and Franz, 1989). Current methods for cross-spe-ies comparative analyses require a well-resolved phy-ogeny based on independent characters (Felsenstein,985a; Harvey and Pagel, 1991; Martins and Hansen,996). Phylogenetic comparisons are also necessary forvaluation of vicariance-based biogeography hypothe-es (Rosen, 1978; Rauchenberger, 1988; Page and Ly-eard, 1994). The absence of a robust phylogeny hasrecluded rigorous investigations into both the histor-cal biogeography of Limia and the process of sexual

selection in this genus.The subfamily Poeciliinae (family Poeciliidae) is a

group of Central American and Caribbean freshwaterfish in the order Cyprinodontiformes. All poeciliidshave internal fertilization and all but one are livebear-ers (Rosen and Bailey, 1963; Meffe and Snelson, 1989;Parenti and Rauchenberger, 1989). Limia is in thetribe Poeciliini; this tribe includes the better-studiedsailfin mollies and guppy (both in genus Poecilia) andthe swordtails and platies (genus Xiphophorus). Manypoeciliid species are model organisms used in experi-ments on sexual selection, especially in the study offemale choice (Endler, 1983; Basolo, 1990; Houde,1997; Schluter et al., 1998).

Limia, originally a genus, was later listed as a sub-genus of Poecilia by Rosen and Bailey (1963) and Pa-renti and Rauchenberger (1989). Rivas (1978, 1980)reelevated Limia to the status of a separate genus,which he intended to split into subgenera and speciesgroups (Burgess and Franz, 1989). Table 1 lists the 20named species of Limia and the proposed speciesgroups (as reported by Burgess and Franz, 1989). Un-

1055-7903/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

tAcvGfcfssttgLL(r

fOd

by researchers to recover samples of the OdontolimiassctD

1

TABLE 1

278 AARON HAMILTON

fortunately, Rivas was unable to complete his study ofLimia morphology before his death, so little publishedinformation is available about the morphological char-acters used to define the species groups. The relation-ships between the species groups remain unresolved.

Rivas’ vittata species group includes the Cuban L.vittata, the Hispaniolan L. perugiae and L. melanono-ata, and L. caymanensis from Grand Cayman Island.dditionally, a sister group relationship between L.aymanensis and L. vittata has been hypothesized (Ri-as and Fink, 1970; Burgess and Franz, 1989), with therand Cayman Limia perhaps derived from the Cuban

orm. The versicolor species group consists of L. versi-olor from central/southern Hispaniola and L. zonatarom north/northeastern Hispaniola. Six Hispaniolanpecies (L. dominicensis, L. pauciradiata, L. tridens, L.ulfurophila, L. rivasi, and L. yaguajali) are listed inhe dominicensis species group. Finally, Rivas placedhe Jamaican L. melanogaster and the Haitian L. ni-rofasciata into monotypic species groups. Two namedimia species recognized by Rosen and Bailey (1963),. caudofasciata and L. nicholsi, were deleted by Rivas

1980) as synonyms of L. melanogaster and L. zonata,espectively.

Rivas also classified six Limia species (five new), allrom one small lake in southern Haiti, as subgenusdontolimia (Rivas, 1980). Some of these species wereescribed from few specimens, of one sex only. Efforts

Taxonomy of Limia as Listed in Burgess and Franz(1989), Based on Work by Rivas

Genus Limia (20 named species)Subgenus Limia (14 species)

Nigrofasciata species group [1]:Limia nigrofasciata

Melanogaster species group [1]:Limia melanogaster

Vittata species group [4]:Limia vittataL. caymanensisL. melanonotataL. perugiae

Versicolor species group [2]:Limia versicolorL. zonata

Dominicensis species group [6]:Limia dominicensisL. pauciradiataL. rivasiL. sulfurophilaL. tridensL. yaguajali

Subgenus Odontolimia (6 species)Grossidens species group [1]: L. grossidensOrnata species group [5]: L. ornata, L. fuscomaculata,

L. garnieri, L. immaculata, L. miragoanensis

Note. Rivas (1980) considered Limia a genus; others have classifiedit as a subgenus of Poecilia (Rosen and Bailey, 1963).

pecies and L. yaguajali and L. rivasi have been un-uccessful (C. Rodruguez, pers. comm.; D. Isla, pers.omm.). Burgess and Franz (1989) proposed four addi-ional undescribed Limia species, termed A, B, C, and. These species and the Odontolimia species were not

included in this study, which therefore covers whatRivas defined as the subgenus Limia.

Chambers (1987) surveyed the structural variationof the gonopodia (the male intromittent organ) of allthe available Limia species. Although the gonopodiumand related structures are considered useful for classi-fying poeciliids (Rosen and Bailey, 1963), Chambers(1987) did not use these morphological data to definerelationships between species or species groups anddeferred to Rivas’ judgment on most issues of taxon-omy. One remark about the potential use of the mor-phology of the gonopodium for phylogeny reconstruc-tion is that such characters may not evolveindependently of sexual behavior or dimorphism, areasof interest to Limia researchers.

Rodriguez (1997) agreed with Rivas that Limiashould be recognized as a genus separated from twoother genera, Pamphorichthys and Poecilia, based onmorphological characters. The names in this paper re-flect the Rivas/Rodriguez arrangement. The alterna-tive would be to retain the generic name Poecilia forLimia and the other groups combined into Rosen andBailey’s genus Poecilia.

Rodriguez did not determine the sister taxon toLimia with the morphological data. Hypothesized sis-ter taxa include the genus Pamphorichthys, one speciesusually placed in Pamphorichthys (P. heterandria), Po-ecilia, or P. caucana (Rosen and Bailey, 1963; Rosen,976; Meyer, 1993; Rodriguez, 1997; Breden et al.,

1999). There were no clear indications of a sister groupto the two samples from Limia included in the molec-ular phylogeny of Breden et al. (1999), which coveredthe entire genus Poecilia (as defined by Rosen andBailey).

Farr’s (1984) survey of courtship display behaviorand sexual dimorphism in Limia illustrates the virtueof having a resolved phylogeny of the group. Farr eval-uated the mating behavior and coloration of six Limiaspecies. Of these, three (L. perugiae, L. nigrofasciata,and L. melanogaster) have male courtship displays andsexual dimorphism in secondary sexual characteristicssuch as color patterns or certain aspects of body shapelike the “humpback” of L. nigrofasciata. The otherthree (L. vittata, L. dominicensis, and L. zonata) lackmajor sexual color differences and have no observedcourtship display; males use the alternate tactic calledgonopodial thrusting which is seen in nearly all poeci-liids. Farr (1984, 1989) reasoned that the ability ofpoeciliid fish to mate with this alternate tactic demon-strated that a display is not required for the mechanicsof mating or for species recognition in poeciliid fish,

therefore leaving mate choice as the best explanation

bsgtislemttLptgftF(paebacstc

r1cgptaIpcdtboa

D

iLDF

dominicensis from Pont Salomon, River Picot, in Haiti;LMPlpsEwrvtqnpPwgocts

vcaiTgBmgw5fIh

defddirefPkq

Antds(

279PHYLOGENY OF Limia

for the appearance of courtship in some species. Ex-panding on the Limia results, Farr found a correlationetween the presence of display and the presence ofexual dimorphism when he surveyed various otherroups of poeciliid fish (Farr, 1989). Farr hypothesizedhat female choice is the primary selective factor driv-ng the evolution of both courtship displays and maleecondary sexual characters in Limia and other poeci-iids. Farr also proposed that female choice is mostffective at shaping behavior and morphology in ani-als that occupy seasonally stable environments or

hat are specialized ecologically or to extreme condi-ions, an hypothesis supported by preliminary data onimia habitats (Farr, 1984, 1989). However, withouthylogenetic information, any correlation found be-ween display and sexual dimorphism may be due to aroup of species inheriting behavioral and color traitsrom a common ancestor, a factor not accounted for byhe simple species-count contingency table used byarr (1989) to demonstrate the correlation. Bisazza

1993) also mentioned this concern, although he sup-orted Farr’s conclusion. Farr and Bisazza did not haveccess to phylogenetic data for species within the Po-ciliini or other tribes. Given a phylogenetic tree, itecomes possible to examine the pattern of appear-nces of display and sexual dimorphism to evaluate theorrelation while correcting for the nonindependence ofpecies and to look for multiple independent deriva-ions of these traits that would indicate examples ofonvergence.

In the present study, I examine the evolutionaryelationships of Limia species using sequences from a-kb fragment of the mitochondrial genome that in-ludes the NADH Dehydrogenase subunit 2 (ND2)ene. I test the validity of several species groups pro-osed by Rivas and resolve the relationships betweenhe species groups. I also evaluate Limia monophylynd the level of support for the proposed sister groups.then look at the existing data on behavior and mor-hology in Limia and its relatives in a phylogeneticontext to address the possibility of multiple indepen-ent origins of courtship and sexual dimorphism in theribe Poeciliini and to test the proposed correlationetween display and sexual dimorphism. An analysisf Limia biogeography is beyond the scope of this papernd will be addressed in a future publication.

MATERIALS AND METHODS

NA Extraction, Amplification, and Sequencing

The populations that were sampled for this studyncluded L. caymanensis from Grand Cayman Island;. versicolor from Cano Madrigal, Rio Haina, in theominican Republic; L. zonata from La Jaquisa, Sanernando de Marcois, in the Dominican Republic; L.

. pauciradiata from Pont Gleo, east of Quartierarin, in Haiti; L. melanonotata from Canal Cristobal,rovincia de Independencia, in the Dominican Repub-

ic; Limia sp. from Las Salinas, in the Dominican Re-ublic, which had been identified as L. tridens; and twoamples from Balneario La Zurza and La Zufrada,nriquillo National Park, in the Dominican Republic,hich represented the original locality and a second

eported locality for L. sulfurophila. The Cuban L.ittata and the Jamaican L. melanogaster were ob-ained from aquarium stocks. The two GenBank se-uences for Limia from Breden et al. (1999) are a L.igrofasciata from Lake Miragoane, Haiti and a L.erugiae from Lake Enriquillo, Dominican Republic.artial sequences of up to a few hundred base pairsere obtained for two additional individuals of L. ni-rofasciata from Lake Miragoane, another individualf L. perugiae from Lake Enriquillo, and a second L.aymanensis from Grand Cayman Island. These par-ial sequences were identical to the same regions of theequences already available for those populations.DNA was extracted from tail muscle of each sample

ia proteinase-K digestions followed by phenol andhloroform extractions (Fajen and Breden, 1992). Thenterior section of each fish (including the gonopodiumn mature males) was preserved for future reference.he primers GLN and ASN in those respective tRNAenes (Kocher et al., 1995; Ptacek and Breden, 1998;reden et al., 1999) were used to amplify a region of theitochondrial DNA genome including the entire ND2

ene and flanking tRNAs. PCRs (Saiki et al., 1988)ere done using 35 cycles with 1 min at 94°C, 2 min at2°C, and 3 min at 72°C. PCR products were trans-ormed into competent Escherichia coli cells using thenvitrogen PCR 2.1 vector and T4 DNA ligase (Hana-an, 1983).The cloned DNA was sequenced using the

ideoxynucleotide chain-termination method (Sangert al., 1977). Both strands were completely sequencedor each sample, with new sequencing primers beingesigned for Limia (available from the author) in ad-ition to primers used by Breden et al. (1999). A min-mum of two clones were used, with a third clone toesolve any conflict in the sequences obtained. For sev-ral samples additional sequencing was done directlyrom Qiagen-purified PCR products using Amershamharmacia Cy5 and Cy5.5 dye-terminator sequencingits and a Visible Genetics Opengene Long-Read se-uencing tower.Accession numbers for the new sequences areF353192–AF353202. Sequences for L. perugiae, L.igrofasciata, Poecilia, and Pamphorichthys were ob-ained from GenBank (Ptacek and Breden, 1998; Bre-en et al., 1999). The outgroups represent all of theubgenera of Poecilia as listed by Rosen and Bailey1963) and include the taxa that have been hypothe-

sized to be the sister groups to Limia. The available P.c9iTBtt

S

p(sMedm(fdsa1

Ns4swnsmaecwfwaHv1ltfhrptmFsmw1p

sion ratio. I divided the mitochondrial sequence intotpmmmt

btstta

KwpipcTavLdcpR[ht

C

“(aCelwppt

odli11t1fTd

280 AARON HAMILTON

aucana sequence is incomplete, comprising the first25 bp of the 1105 bp in the aligned data set; thencluded Poecilia petenensis sequence is 760 bp long.he two more distant outgroups obtained from Gen-ank are Xiphophorus nigrensis, a swordtail from the

ribe Poeciliini, and Heterandria formosa, from theribe Heterandriini.

equence Alignment and Phylogenetic Analyses

The 34 sequences, including the complete ND2 andartial tRNA genes, were aligned using CLUSTAL XThompson et al., 1994). The length of the alignedequences was 1105 bp, including the 39 end of tRNA-et (32 bp), the entire ND2 gene (1047 bp), and the 59

nd of tRNA-Trp (26 bp). There were no insertions oreletions in the complete aligned sequences. The align-ent is available from the author on request

Hamiltona@hotmail.com). Two sets of data were usedor phylogenetic analysis; first, the complete 34-taxonata set; second, a reduced taxon set that included 15pecies with complete sequences for which behavioralnd dimorphism data were available from Farr (1984,989).Parsimony, minimum-evolution (ME) (Rzhetsky andei, 1992), and maximum-likelihood (ML) (Felsen-

tein, 1981) analyses were performed using PAUP*.0b4a (Swofford, 1998). In parsimony and ML analy-es, starting trees were obtained via stepwise additionith 10 randomized input orders. Starting trees wereeighbor-joining trees for minimum-evolution. Branchwapping was tree bisection and reconnection for allethods. Character weights were equal in parsimony

nalyses; transitions and transversions were weightedqually, except in the parsimony analysis of ND2 thirdodon positions, in which transitions were given zeroeight because of the possibility of saturation effects

or more divergent taxa. Minimum-evolution searchesere performed with both logdet distances (Lockhart etl., 1994) and maximum-likelihood distances under theKY85 (Hasegawa et al., 1985) and general time re-

ersible (GTR; Lanave et al., 1984; Rodriguez et al.,990) models of sequence evolution with maximum-ikelihood estimates of the substitution parameteriza-ions. ME analyses under the HKY85 model were per-ormed with and without an allowance for rateeterogeneity in the form of a gamma distribution ofates across sites (Yang, 1996). The value of the shapearameter (alpha) for the gamma distribution was es-imated via maximum-likelihood from the most parsi-onious tree(s) (Swofford et al., 1996). Phylip 3.572 (J.elsenstein) and WAVEBOOT (D. King and C. Krajew-ki) were used to construct neighbor-joining trees withaximum-likelihood-weighted average distances. Theeighted average distance method (Krajewski et al.,997) allows each partition to have its own base com-osition, substitution rate, and transition to transver-

hree partitions as follows: tRNAs, first and secondositions of ND2, and third positions of ND2. Maxi-um-likelihood trees were obtained under the HKY85odel of sequence evolution with equal rates and aaximum-likelihood estimate of the transition to

ransversion ratio obtained from a parsimony tree.The method of random trees (Hillis and Huelsen-

eck, 1992) was used to test for phylogenetic signal inhe data. Clade support was assessed using the boot-trap procedure (Felsenstein, 1985b) with 500 replica-ions (parsimony, distance methods) or 200 replica-ions (maximum-likelihood) and other search settingss indicated above.A priori systematic hypotheses were tested using theishino and Hasegawa (1989), Templeton (1983), andinning-sites (Prager and Wilson, 1988) tests witharsimony and the Kishino–Hasegawa test with max-mum-likelihood. If the best tree did not contain theredicted clade, a tree was generated with the cladeonstrained to appear for comparison to the best tree.he a priori hypotheses tested were Rivas’ versicolornd vittata species groups, the proposed pairing of L.ittata 1 L. caymanensis, the possible monophyly ofimias from the island of Hispaniola (all species in theata set except for L. melanogaster, L. vittata, and L.aymanensis), and the monophyly of Limia. Each of theroposed sister groups to Limia (Poecilia as defined byodriguez (1997), P. caucana alone, Pamphorichthys

P. minor 1 P. araguanensis, with and without P.eterandria], and P. heterandria alone) were alsoested with the full data set.

omparative Methods

The 15-taxon tree, which was congruent to nearly allpruned” 34-taxon results, was used to map Farr’s1989) character-state data set for display behaviornd sexual dimorphism. The computer program Mac-lade (Maddison and Maddison, 1992) was used tostimate ancestral states for each of the different phy-ogenetic trees. Farr’s characters were categorical,ith two states for each character: “courtship displayresent” vs “display absent/thrusting only” and “dimor-hic” vs “monomorphic” for secondary sexual charac-ers.

In addition to the mapping of the character statesnto the trees, three different comparative methods foriscrete characters were employed to look for a corre-ation between display and sexual dimorphism. Thendependent character evolutions (ICE) test (Ridley,983; Ridley and Grafen, 1996; Grafen and Ridley,996) was done on paper, whereas MacClade was usedo perform the concentrated changes test (Maddison,990) and the computer program DISCRETE was usedor Pagel’s discrete characters test (Pagel, 1994, 1997).he purpose of these tests was to take phylogeneticata into account for a comparative analysis of Farr’s

data set, which before could be assessed only by use of This was done by giving a negligible length to the

t(mtrpaatoK

L

281PHYLOGENY OF Limia

species counting. Farr’s data set is difficult to convertinto meaningful continuous characters.

The ICE test looks for a correlation between twocharacter states using a contingency table, looking onlyat branches where a change in either character stateoccurs, collapsing all other branches, and ignoringbranch lengths. This method was performed using theestimated ancestral character states at the root as oneof the nodes in the character-state change tree. For thebest tree obtained for the 15-taxon data set, everycombination of ancestral character state reconstruc-tions for the two characters was tested. As the ICE testlooks only at branches in which a change occurs, ifthere are too few changes, the expected values in thecontingency table may be so small that the x2 test haslittle power. This method is conservative when the rootnode’s character states favor the hypothesis of correla-tion, but is too liberal in the opposite situation (Grafenand Ridley, 1996; Ridley and Grafen, 1996).

Unlike the ICE test, the concentrated changes test(Maddison, 1990) is directional and tests whether thestate of the chosen independent character (here, thepresence of a courtship display) makes it more likelythat a clade will gain a chosen character state of thedependent character (“dimorphic”). This variation onthe hypothesis presents a problem if the two characterschange states on the same branch. This test was ap-plied first under the assumption that all such brancheshad the independent character change first and thenagain under the assumption that the dependent char-acter changed first in at least one of those branches. Asin the ICE test, for the best tree obtained for the15-taxon data set, every combination of ancestral char-acter state reconstructions was tested. This test alsoignores branch lengths.

Pagel’s discrete analysis test (computer programDISCRETE) (Pagel, 1994, 1997) is less dependent onreconstructing a set of ancestral character states thanthe ICE and concentrated changes tests and, unlike theother tests, can account for differences in branchlengths. This program calculates the likelihood of theevolution of two characters over a phylogeny under twomodels, one representing independent evolution of thetwo characters and the other representing correlatedevolution. The likelihood ratio (LR) for the two modelscan be compared to a probability distribution gener-ated using Monte Carlo simulations to test whetherthere is a significant difference between the two mod-els. The test was repeated for the best 15-taxon treewith and without differing branch lengths being in-cluded. The branch lengths were maximum-likelihoodestimates (obtained using PAUP*) from a single heu-ristic tree. It was necessary to simulate a trichotomy atthe root of the tree when branch lengths were includedbecause of the difficulty of subdividing the branchlength of H. formosa without any further outgroup.

branch connecting the root node to the ingroup clade.One hundred Monte Carlo simulations were done foreach application of the test.

RESULTS

Sequence divergence between the Limia sequencesranged ,1–10%, compared to 11–18% in comparisonsbetween Limia and outgroups in Poecilia and Pam-phorichthys, 19–22% between Limia and X. nigrensis,and 20–23% between Limia and H. formosa. A distri-bution of 10,000 random trees indicated significantphylogenetic signal in the 34-taxon data set includingoutgroups (g1 5 20.533814, P , 0.01), in the data setrestricted to Limia (g1 5 20.616427, P , 0.01), and inhe 15-taxon data set used in the comparative analysisg1 5 20.715381, P , 0.01.) There were 417 parsi-

ony-informative sites in the full data set. The dis-ances between pairs of Limia species fall into theegion where substitutions accumulate linearly inlots of ND2 third codon position transitions and firstnd second position transitions and transversionsgainst third position transversions (Fig. 1), indicatinghat there may still be useful phylogenetic informationn Limia relationships in all of the codon positions (seeocher et al., 1995; Breden et al., 1999).All bootstrap trees for the full data set using parsi-

mony, minimum-evolution, and maximum-likelihoodresulted in the same relationships for Limia (Fig. 2),except that ME trees resolved the polytomy (labeledclade I) including L. perugiae and four other samples,and the third-position transversion parsimony treeplaced L. melanogaster as sister to the other Limiaspecies. Most relationships within Limia have highbootstrap support (Table 2). Limia monophyly (labeledclade A, Fig. 2) was well supported (95–100% for the34-taxon data set).

The Canal Cristobal sample (L. melanonotata), theLas Salinas fish, and the samples from Balneario LaZurza and La Zufrada all had sequences very similar tothat of L. perugiae (a sequence generated indepen-dently by Breden et al., 1999). The Canal Cristobal andLa Zurza fish had identical sequences, the Las Salinassample and L. perugiae each differed from these by oneunique transversion, and the La Zufrada sample dif-fered from the others by two transitions. Minimum-evolution trees did resolve the polytomy, but there waslittle bootstrap support for any particular arrangementof these populations.

The sister group to the L. perugiae complex (L. pe-rugiae and the other four near-identical sequences)was L. nigrofasciata (100% bootstrap support for thisclade, H). L. dominicensis and L. pauciradiata formeda clade (G) with 93–100% support; this clade was sistergroup to the clade containing L. nigrofasciata and the

. perugiae complex (F, 73–92%). L. vittata (from Cuba)

282 AARON HAMILTON

and L. caymanensis (from Grand Cayman Island) werepaired (E) with 100% bootstrap support and formed thesister group to the above Hispaniolan fish (D, 98–100%support).

L. versicolor and L. zonata also formed a clade (C,96–100% support) matching Rivas’ versicolor speciesgroup. With the exception of the third-position trans-version parsimony analysis, the bootstrap trees consis-tently placed the Jamaican species, L. melanogaster, asthe sister group to the versicolor species group but withweak support (B, 58–89% support). There was lowersupport for trees in which L. melanogaster was basal tothe other Limias (3–31%) except in the ND2 third-position transversion parsimony analysis, which gavethis arrangement 63% support. The hypothesized vit-tata species group received zero support from anymethod used. There was not strong support for any ofthe proposed sister–groups to Limia (see Table 2).

FIG. 1. Accumulation of sequence differences for the ND2 first a(Tv) and the third codon position transitions charted against the th

Systematic Hypothesis EvaluationThe “versicolor” species group appeared in all trees,

but was significantly supported only by the KH–MLtest (Table 3). The “vittata” species group did not ap-pear in any trees and was rejected by all tests. Thesister group relationship between L. vittata and L.caymanensis (Rivas and Fink, 1970) was present in alltrees and was significantly supported in the tests. Themonophyly of Hispaniolan Limias (all included speciesexcept L. melanogaster, L. vittata, and L. caymanensis)was not indicated by any of the trees. This arrange-ment was rejected by all but the winning-sites test. Themonophyly of Limia was indicated by all trees, but thiswas not significantly supported by the tests.

Each of the proposed sister groups to Limia wastested (Table 3). The P. caucana trees were signifi-cantly worse in all tests; neither Poecilia-, Pam-phorichthys- (P. minor 1 P. araguanensis, tested with

second codon position combined transitions (Ts) and transversionsposition transversions.

ndird

ssPa

283PHYLOGENY OF Limia

and without P. heterandria as part of the clade), or P.heterandria-constrained trees were significantly differ-ent from the best tree.

Comparative Methods Analysis on 15 Taxa

The best tree obtained by most methods for the 15-taxon data set showed an arrangement of clades con-gruent to that of the pruned 34-taxon data set. Thesame single best reduced-sampling tree was obtainedby all methods except for parsimony and third-positiontransversion parsimony (which gave three trees), forwhich the points of uncertainty were the placement ofL. melanogaster within Limia, the position of P.vivipara, and the relationships within Poecilia (Lebi-stes). Without most of the proposed sister groupspresent, the sister group of Limia was the Poecilia(Mollienesia) clade. Farr’s (1989) character states weremapped onto the 15-taxon phylogeny which matchedthe pruned 34-taxon results (Fig. 3). The displayingtaxa include L. perugiae, L. nigrofasciata, and L. mela-nogaster within Limia, two sailfin mollies for whichcomplete ND2 sequences were available (P. latipinnaand P. vellifera), the Lebistes clade (P. picta, P. parae,and the guppy, P. reticulata), and the single included

FIG. 2. Single heuristic maximum-likelihood tree for ND2 nucelected branches for which bootstrap values are reported in Table 2.upport on average for all the methods used; clades designatedamphorichthys are named as genera separate from Poecilia (Rodrignd “Mollienesia” are indicated in parentheses.

swordtail, X. nigrensis. For the best tree shown therewere three alternate reconstructions of the ancestralcharacter states; because display and sexual dimor-phism cooccur in the same set of species, each recon-struction can be applied to both characters. For testingthe characters, all three possibilities for the pattern ofevolution of display in the 15-taxon tree were pairedwith all three possibilities for the evolution of sexualdimorphism, for a total of nine arrangements of thecharacter states. The ICE (Ridley, 1983) and concen-trated changes (Maddison, 1990) tests were both ap-plied to each arrangement. The ICE test supported thehypothesis of a correlation between display and sexualdimorphism at a significant level (P , 0.05) for sevenof nine arrangements. Of the various ancestral charac-ter state arrangements tested, in five of the nine casesthe root character states supported the hypothesis ofcorrelation, meaning that the ICE test was very con-servative; in the other four cases (including those thatwere nonsignificant) the ancestral character states didnot support the hypothesis, possibly indicating that thetest is liberal for those arrangements (Grafen and Rid-ley, 1996; Ridley and Grafen, 1996).

tide sequences of Limia and outgroups. Capital letters designatethe outgroup taxa, branches designated “x” had over 95% bootstraphad an average of 85–95% bootstrap support. Here Limia and, 1997) and the Poecilia species that comprise the groups “Lebistes”

leoFor“y”uez

TABLE 2

LE

K

284 AARON HAMILTON

The concentrated changes test (Maddison, 1990) isdirectional, which presented a difficulty because in thedifferent ancestral character state reconstructionsthere are between three and five branches where thetwo characters go from 0 to 1 (thrust to display, or

Bootstrap Support for Selected C

Clade

T

Parsimony(500)

A (Limia monophyly) 96B (L. melanogaster 1 versicolor species group) 62C (versicolor species group) 99D 98E (L. caymanensis 1 L. vittata) 100F 90G (L. dominicensis 1 L. pauciradiata) 97H (L. nigrofasciata 1 L. perugiae complex) 100I (L. perugiae complex) 100L. melanogaster as sister to other Limias 30.8Vittata species group 0Monophyly of Hispaniolan species 0Sister group to Limia: P. heterandria 7.2Sister group to Limia: P. caucana 0Sister group: Pamphorichthys without P. heterandria 12.3Sister group: Pamphorichthys with P. heterandria 7Sister group: Poecilia (including mollies and

Lebistes) 0.6

Note. ME-logdet, minimum-evolution with logdet-paralinear dista(HKY85) distance correction; ME-ml 1 gamma, same with an estimawith a GTR-model maximum-likelihood distance correction; Tv3parsjoining with weighted average distance analysis; ML, maximum-lik

TAB

Significance Levels of Templeton, Win

Constraint No. of trees Leng

Best trees 4 176With vittata species group 2 178Without L. caymanensis 1 L. vittata 2 178Without versicolor species group 2 177With Hispaniolan monophyly 6 177Without Limia monophyly 20 177Possible sister groups to Limia:

P. heterandria 2 176P. caucana 2 177Pamphorichthys (without P.heterandria) 4 176Pamphorichthys (including P.heterandria) 6 176Poecilia (including mollies 1 Lebistes) 2 176

Note. Asterisks denote significant differences at P , 0.05. For paishino–Hasegawa test.

monomorphic to dimorphic) on the same branch. Thistest was first performed under the assumption that allsuch combinations had the “independent” characterchange first (here, gain of display) and then againunder the assumption that the dependent character

es, Using the 34-Taxon Data Set

estimation method and number of bootstrap replicates

-logdet(500)

ME-ml(500)

ME-ml1

gamma(500)

ME-mlGTR(500)

TV3 pars(500)

NJ-WAVE(500)

ML(100)

99 100 99 100 95 97 9889 83 74 82 21 58 64

100 100 100 100 96 100 9998 99 99 99 100 98 100

100 100 100 100 100 100 10081 81 73 79 80 87 92

100 100 100 100 93 100 99100 100 100 100 100 100 100100 100 99 100 86 100 100

3.4 6 12.2 7 63 14.4 290 0 0 0 0 0 00.2 0 0 0 0 0.2 0

12.8 19 8.8 17.8 1 5.6 90 0 0 0 0 0 08 6 4.6 8.6 1.9 9.8 192 1.2 3.6 1.8 29.8 1.6 13

0 0 0 0 3.2 3.8 0

correction; ME-ml, minimum-evolution with a maximum-likelihoodvalue of gamma from a single tree; ME-ml GTR, minimum-evolutionansversion parsimony for ND2 3rd positions; NJ-WAVE, neighbor-ood.

3

g-Sites, and Kishino–Hasegawa Tests

ParsimonyMaximum-Likelihood

Templeton WS KH 2ln L KH

N/A N/A N/A 9909.1 N/A0.0351* 0.0472* 0.0349* 10041 0.0065*0.0002* 0.0004* 0.0002* 9990.8 0.0001*0.0633 0.0865 0.0633 9939.8 0.0085*0.0425* 0.0814 0.0427* 9943.8 0.0117*0.1038 0.133 0.1038 9933.5 0.1024

0.7009 0.7979 0.7011 9915.1 0.53930.0007* 0.0009* 0.0007* 9969.6 0.0003*

0.5258 0.6341 0.526 9914.2 0.6085

0.5414 0.7605 0.5417 9910.2 0.67650.0881 0.1338 0.0881 9924 0.1683

ony tests the average P score is given. WS, winning-sites test; KH,

lad

ree

ME

nceted, tr

elih

nin

th

166432

49

5

39

rsim

Limia, with the main uncertainty pertaining to the

mtF

acma

CorotpSywacbgfccsLcqgtpbnosidsapga

grg

o1acwwrtta(

285PHYLOGENY OF Limia

changed first in at least one of those branches (gain ofdimorphism before display). The concentrated changestest is significant in nearly every case (eight of nine; Pvalues ranging between 0.0039 and 0.0733) when it isassumed that each branch with “simultaneous”changes supports the hypothesis that display promotesthe evolution of sexual dimorphism, but is significantin only two cases when one fewer such branches arecounted for the directional hypothesis.

Pagel’s discrete test produced a likelihood ratio near19, regardless of whether differing branch lengthswere included (LR values for repeated trials were be-tween 19.597812 and 19.872425). Comparing these LRvalues to the probability distributions produced byMonte Carlo simulations for these trees, the LR washigher than 100% of the simulated values for all trees(Cotgreave and Pagel, 1997), supporting the model ofcorrelated evolution of the two characters.

DISCUSSION

Phylogenetic Relationships

The phylogenetic trees generated by parsimony, ME,and ML techniques agree on the relationships of

FIG. 3. MacClade trees representing the estimated distributionf character states for both display and sexual dimorphism over the5-taxon data set. Nine of the included species were reported to havecourtship display and were rated as dimorphic in secondary sexual

haracters such as color patterns; six species lacked a display andere rated as monomorphic (Farr, 1989). It should be noted thathen alternate versions of the 15-taxon tree were considered, the

esults of the analyses were similar to those reported here becausehe ancestral character state reconstructions were congruent, excepthat placing Limia melanogaster as sister to the other Limia speciesdded the additional possibility that display or sexual dimorphismor both) were lost four times.

placement of L. melanogaster. The monophyly of Limiais supported, with the caveat that no species assignedto “Odontolimia” by Rivas were included in the study.

A close relationship between L. versicolor and L.zonata was strongly supported. This clade representsone of the species groups suggested by Rivas (Burgessand Franz, 1989). Also strongly supported was therelationship between L. caymanensis and L. vittata;such a grouping has been hypothesized, with L. cay-

anensis diverging from L. vittata after dispersal tohe Grand Cayman Islands from Cuba (Rivas andink, 1970; Burgess and Franz, 1989).Another species group suggested by Rivas (Burgess

nd Franz, 1989) was not upheld. L. vittata and L.aymanensis had been grouped with L. perugiae and L.elanonotata in Rivas’ “vittata species group.” This

rrangement was rejected by the ND2 phylogeny.The ND2 data link L. perugiae with the Las Salinas,anal Cristobal, La Zurza, and La Zufrada samples, allf which have nearly identical sequences. This resultaises the possibility that these populations representne widespread species or species complex with varia-ion in size or morphology that had resulted in theroposal of more than one name. Farr (1984) andchroder et al. (1996) mentioned the probable synon-my of the names L. perugiae and L. melanonotata,hich they claimed were distinguishable mainly due todifference in size. L. tridens is more difficult to make

onclusions about, because some populations that haveeen identified as this species are similar to L. peru-iae/L. melanonotata and other populations have beenound to be nearly indistinguishable from L. domini-ensis (C. Rodriguez, pers. comm.; S. Walsh, pers.omm.). L. tridens has previously also been listed as aynonym of L. versicolor (Rosen and Bailey, 1963). Theas Salinas population included here appears to belosely related to L. perugiae based on the ND2 se-uences. It is also possible that there has been intro-ression of the mitochondrial genome from one specieso another due to hybridization. L. sulfurophila, re-orted from only a few localities, may also be vulnera-le to such hybridization. The Limia sample from Bal-eario La Zurza had an ND2 sequence identical to thatf the Canal Cristobal L. melanonotata, whereas theequence of the La Zufrada sample was also very sim-lar to the other L. perugiae-type fish. ND2 sequenceso not show enough divergence to resolve the relation-hips between these populations. These results presentn opportunity to use more rapidly evolving markers,referably including nuclear markers, to examine geo-raphic differences between populations in morphologynd possibly behavior.Rivas had assigned L. nigrofasciata and L. melano-

aster to monotypic species groups. The phylogeneticelationships between these and the other speciesroups were undefined (Burgess and Franz, 1989). The

ND2 data places L. nigrofasciata as the sister species

tspctPMtt(

fttiMa(bcnlMt(sBertto(d

gr

third position transversions per million years); how-ebbfigt

B

FaToruadsaddwiibt

mcfrrbgiHhttrcpsicoudtopt

tia

286 AARON HAMILTON

to the L. perugiae complex. The relationships of L.melanogaster are not as well resolved, as this speciescan be supported either as the sister species to theversicolor species group or as basal to all other in-cluded Limia species.

According to the ND2 sequence data, the Limia spe-cies from the island of Hispaniola do not form a mono-phyletic group, as L. versicolor and L. zonata fromHispaniola are basal to the clade containing L. vittata(from Cuba), L. caymanensis (from Grand Cayman Is-land), and all of the other Hispaniolan samples.

There is no strong support for Pamphorichthys, orspecifically P. heterandria, as the sister group of Limia.There is also no support for Poecilia (when defined asincluding both mollies and Lebistes) as the sistergroup; Poecilia so defined does not appear as a mono-phyletic group in trees based on ND2 data (see Bredenet al., 1999). P. caucana was rejected as the closestrelative to Limia; instead this species was the sisteraxon to the mollies (see Ptacek and Breden, 1998). Theister taxon to Limia may be any combination of Pam-horichthys, P. heterandria alone, or the mollies 1 P.aucana. For the full data set, the sister group found inhe ME bootstrap trees was the mollies 1 P. caucana 1. heterandria (30–63% bootstrap support) and for theL and parsimony trees, the same taxa plus the other

wo Pamphorichthys (47%). Other genes must be usedo supplement ND2 data to resolve these relationshipsBreden et al., 1999).

The most recent discussion of the status of the dif-erent outgroup taxa based on morphological charac-ers was by Rodriguez (1997), who favors reinstatinghree genera that Rosen and Bailey (1963) had lumpednto Poecilia: Limia, Poecilia, and Pamphorichthys.

olecular data (Ptacek and Breden, 1998; Breden etl., 1999) also supported Limia and Pamphoricththysexcept for P. heterandria) as monophyletic assem-lages but indicated that Rodriguez’s genus Poeciliaould be further divided into two more previouslyamed taxa, Lebistes (including the guppy, P. reticu-ata) and the mollies (perhaps as a subgenus or genus

ollienesia). Such an arrangement would be similar tohe subgeneric arrangement of Rosen and Bailey1963) except for the uncertain placement of speciesuch as P. vivipara, P. heterandria, and P. caucana.reden et al. (1999) recommend further study of anxpanded data set before proposing a formal taxonomicearrangement. Adding the new sequences for Limia tohe available set of GenBank sequences did not affecthe conclusions about mollies, such as the monophylyf the sailfin mollies, reported in Ptacek and Breden1998), or the conclusions about Lebistes and other taxaiscussed by Breden et al. (1999).No fossil record for Limia on which to base diver-

ence times exists. Kocher et al. (1995) estimated aate of evolution for the ND2 gene in cichlid fish (1%

ver, this was itself based on an hypothesis for theiogeography of the East African lakes. The study ofiogeography in the Caribbean islands is currently aeld of debate, so I have not attempted to use anyeologic hypothesis to estimate Limia divergenceimes.

ehavior and Phylogeny: Preliminary Analyses

The addition of phylogenetic information allowsarr’s (1989) data set to be reevaluated with compar-tive methods other than the original species counting.he application of corrections for the nonindependencef species counted as data points does not cause theejection of the correlation between courtship and sex-al dimorphism; therefore, Farr’s (1989) hypothesisbout the selective link between female choice and theevelopment of courtship display and male secondaryexual characters can still be supported by the datavailable for Limia and related poeciliid fish. The evi-ence for multiple independent appearances of bothisplay and sexual dimorphism is important, becausehen such information is available experimenters can

dentify nondisplaying relatives of displaying taxa anddentify and compare species with convergent displayehaviors and secondary sexual characters to furtherest whether a common factor selected for these traits.

Within Limia, L. perugiae, L. nigrofasciata, and L.elanogaster all have visual courtship displays, which

onsist of the males swimming next to or in front of theemales, unpaired fins spread to stimulate a receptiveesponse from a female. These three species were alsoated as “dimorphic” in color and/or some aspects ofody shape (Farr, 1984). Of these species, L. melano-aster is from Jamaica; the other two are from thesland of Hispaniola. The Cuban L. vittata and theispaniolan L. dominicensis and L. zonata reportedlyave no courtship display; males use only gonopodialhrusting, a tactic also available to displaying species,o achieve matings (Farr, 1984). These species wereated as monomorphic (Farr, 1984). In Limia, the dis-rete category “dimorphic/monomorphic” summarizesotential differences such as the coloration of the dor-al fin and caudal fin and extremes in male body shape;n future studies the phylogenetic appearance of eacholor pattern will be examined separately. In the casef L. zonata, Farr (1984) judged that a reversible, sit-ation-dependent darkening of male coloration in someominant males should not be counted as a permanentype of color dimorphism. Although Farr did not focusn body size dimorphism, Bisazza (1993) addressed theossibility that size dimorphism might also be relatedo male use of thrusting tactics.

The phylogenetic relationships revealed by the ND2ree placed L. nigrofasciata as sister to a clade contain-ng L. perugiae. However, L. melanogaster either is inclade with L. versicolor and L. zonata or is basal to all

other Limias. This suggests that the two Hispaniolan

tseon(vdL

a1adm3foarmtp1etppbt

opatscw

1989). On the other hand, it would be difficult to designaaedbecAltt

btbrmmsmmciXRe

ecpthssidFacsp

287PHYLOGENY OF Limia

displaying species L. nigrofasciata and L. perugiae areclose relatives and may have obtained male displaybehavior from a common ancestor. Still, some observedvariations in behavior and coloration could be derivedfor each species, such as the orbiting display of male L.perugiae in which they circle the head of the femaleand the body shape and coloration differences betweenmale L. perugiae and the “hump-backed” males of L.nigrofasciata. The appearance of a display in L. mela-nogaster may be due to a separate evolutionary eventbecause of the more ancient split between this Jamai-can species and the clade containing both of the His-paniolan displaying species. The reported nondisplay-ing species L. zonata, L. dominicensis, and L. vittataeach occur in a separate clade in the phylogeny. Ofthese, the latter two are more closely related to L.perugiae and L. nigrofasciata than is L. melanogaster.

Although Farr’s hypothesis reflected the assumptionhat the lack of a display was the ancestral charactertate in Limia (as is postulated for poeciliids in gen-ral), the other possibility is that the common ancestorf Limia had a display which was then lost. This sce-ario would require the loss of courtship at least once

twice if L. melanogaster is the sister taxon to theersicolor species group) and the reappearance of maleisplays at least once (in the common ancestor of. perugiae and L. nigrofasciata).There are both displaying and nondisplaying species

mong the outgroups (Liley, 1966; Farr, 1989; Ptacek,998). When Farr’s (1989) survey is used to map char-cter states on the 15-taxon phylogeny, it appears thatisplay behavior and sexual dimorphism cooccur inany different lineages within the tribe Poeciliini (Fig.

), with the possibility of up to five independent originsor such behavior (including two origins within Limia)r, perhaps, a complex pattern including both gainsnd losses of courtship. The comparative tests do noteject the correlation between display and sexual di-orphism, given the available phylogenetic informa-

ion. Pagel’s (1994) discrete comparative analysis sup-orted the correlation, as did the ICE test (Ridley,983) to the extent that it can be considered powerfulnough with the limited 15-taxon data set. The concen-rated changes test (Maddison, 1990) usually sup-orted a directional hypothesis (that sexual dimor-hism is more likely to evolve once display has evolved)ut only when all simultaneous changes were countedoward the hypothesis.

There are advantages and disadvantages in the usef discrete characters such as Farr’s categories “dimor-hic/monomorphic” (Farr, 1984, 1989) or similar char-cters (Meyer, 1997). The more general the category,he less information is conveyed on the details of eachpecies’ coloration; such categories also present a diffi-ulty when there is great variation in pigmentationithin a sex, as in male L. zonata or P. sphenops (Farr,

meaningful continuous character that would combinelternate types of sexual dimorphism such as swords,nlarged dorsal fins, or various color patterns. Farr’secision to use such a character allowed for the possi-ility that different secondary sexual traits could bexaggerated in different groups yet still share femalehoice as the factor responsible for their appearance.lso, Farr’s (1984, 1989) surveys included many of the

ess-colorful and nondisplaying taxa, which is impor-ant considering the possibility of experimenter biasoward the more behaviorally “interesting” fish.

Although the taxon set includes only species withoth display and sexual dimorphism, versus neitherrait, Farr also reported taxa in which the other com-inations of traits could occur, for instance Xiphopho-us maculatus (thrusting only, but dimorphic or poly-orphic) or Belonesox belizanus (displaying andonomorphic; Farr, 1989). Excluded from Fig. 3 are

everal molly species for which some behavioral infor-ation is available, such as the displaying Poeciliaexicana and P. petenensis and the nondisplaying P.

hica and P. orri (Farr, 1989; Ptacek, 1998). Also notncluded was Priapella, the proposed sister group toiphophorus (Meyer et al., 1994; Meyer, 1997; but seeodriguez, 1997) or fish from several of the other po-ciliid tribes.At this point the available phylogenetic data on po-

ciliid fish appear to support Farr’s hypothesis of aorrelation between display and sexual dimorphism. Aotentially more detailed comparative study to addresshe effectiveness of female choice and the impact ofabitat type will become possible as more genes areequenced and the mating behavior and coloration aretudied for additional species. My future work willnclude a compilation of behavioral and morphologicalata from the other species of Limia not examined byarr (1984) and a more detailed description of second-ry sexual characters, such as coloration of dorsal andaudal fins, allowing a comprehensive comparativetudy of the evolution of courtship and sexual dimor-hism in Limia.

ACKNOWLEDGMENTS

I thank M. Springer, D. Reznick, and M. Zuk for their advice andguidance and the use of laboratory facilities at the University ofCalifornia, Riverside. Limia samples were obtained from D. Isla, R.McKeand, A. Cruz, M. Abney (with thanks to the Cayman IslandsNational Trust and Dept. of the Environment), S. Walsh, and W.Kohler. Advice on ND2 primers was given by M. Ptacek and F.Breden, and M. Pagel and M. Ridley provided additional informationon their comparative tests. I also thank A. Cruz and V. Rush for theiradvice. Funding was provided in part by a grant from Sigma Xi, aNewell grant, and a Dean’s Dissertation Grant from the Universityof California, Riverside to Aaron Hamilton and a National ScienceFoundation grant (DEB-9419617) to Mark Springer.

REFERENCES

B

B

C

C

E

F

F

F

F

F

F

F

G

H

H

H

H

H

K

K

unit 2 in East African cichlid fish. Mol. Phylogenet. Evol. 4: 420–

K

L

L

L

M

M

M

M

M

M

M

P

P

P

P

P

P

P

R

R

R

288 AARON HAMILTON

Basolo, A. L. (1990). Female preference for male sword length in thegreen swordtail, Xiphophorus helleri (Pisces: Poeciliidae). Anim.Behav. 40: 332–338.

Bisazza, A. (1993). Male competition, female mate choice and sexualsize dimorphism in poeciliid fishes. Mar. Behav. Physiol. 23: 257–286.reden, F., Ptacek, M., Rashed, M., Taphorn, D., and Augusto deFigueiredo, C. (1999). Molecular phylogeny of the live-bearing fishgenus Poecilia (Cyprinodontiformes: Poeciliidae). Mol. Phylogenet.Evol. 12: 95–104.urgess, G. H., and Franz, R. (1989). Zoogeography of the Antilleanfreshwater fish fauna. In “Biogeography of the West Indies” (C. A.Woods, Ed.), pp. 263–304. Sandhill Crane Press, Gainesville.hambers, J. (1987). The cyprinodontiform gonopodium, with anatlas of gonopodia of the fishes of the genus Limia. J. Fish Biol. 30:389–418.otgreave, P., and Pagel, M. (1997). Predicting and understandingrarity: The comparative approach. In “The Biology of Rarity”(W. E. Kunin and K. J. Gaston, Eds.), pp. 237–261. Chapman &Hall, London.ndler, J. A. (1983). Natural selection on color patterns in poeciliidfishes. Environ. Biol. Fishes 9: 173–190.

ajen, A., and Breden, F. (1992). Mitochondrial DNA sequence vari-ation among natural populations of the Trinidad guppy, Poeciliareticulata. Evolution 46: 1457–1465.

arr, J. A. (1984). Premating behavior in the subgenus Limia (Pi-sces: Poeciliidae): Sexual selection and the evolution of courtship.Z. Tierpsychol. 65: 152–165.

arr, J. A. (1989). Sexual selection and secondary sexual differenti-ation in poeciliids: Determinants of male mating success and theevolution of female choice. In “Ecology and Evolution of Livebear-ing Fishes (Poeciliidae)” (G. K. Meffe and F. F. Snelson, Eds.), pp.91–123. Prentice Hall, Englewood Cliffs, NJ.

elsenstein, J. (1981). Evolutionary trees from DNA sequences: Amaximum likelihood approach. J. Mol. Evol. 17: 368–376.

elsenstein, J. (1985a). Phylogenies and the comparative method.Am. Nat. 125: 1–15.

elsenstein, J. (1985b). Confidence limits on phylogenies: An ap-proach using the bootstrap. Evolution 39: 783–791.

ranz, R., and Burgess, G. H. (1983). A new poeciliid killfish, Limiarivasi, from Haiti. Northeast Gulf Sci. 6: 51–54.rafen, A., and Ridley, M. (1996). Statistical tests for discrete cross-species data. J. Theor. Biol. 183: 255–267.anahan, D. (1983). Studies on transformation of Escherichia coliwith plasmida. J. Mol. Biol. 166: 557–580.arvey, P. H., and Pagel, M. K. (1991). “The Comparative Method inEvolutionary Biology,” Oxford Univ. Press, Oxford.asegawa, M., Kishino, H., and Yano, T. (1985). Dating of the hu-man–ape splitting by a molecular clock of mitochondrial DNA. J.Mol. Evol. 21: 160–174.illis, D. M., and Huelsenbeck, J. P. (1992). Signal, noise, andreliability in molecular phylogenetic analyses. J. Hered. 83: 189–195.oude, A. E. (1997). “Sex, Color, and Mate Choice in Guppies,”Princeton Univ. Press, Princeton, NJ.ishino, H., and Hasegawa, M. (1989). Evaluation of the maximumlikelihood estimate of the evolutionary tree topologies from DNAsequence data, and the branching order in Hominoidea. J. Mol.Evol. 29: 170–179.ocher, T. D., Conroy, J. A., McKaye, K. R., Stauffer, J. R., andLockwood, S. F. (1995). Evolution of NADH Dehydrogenase sub-

432.

rajewski, C., Blacket, M., Buckley, L., and Westerman, M. (1997).A multigene assessment of phylogenetic relationships within thedasyurid marsupial subfamily Sminthopsinae. Mol. Phylogenet.Evol. 8: 236–248.

anave, C., Preparata, G., Saccone, C., and Serio, G. (1984). A newmethod for calculating evolutionary substitution rates. J. Mol.Evol. 20: 86–93.

iley, N. R. (1966). Ethological isolating mechanisms in four sympa-tric species of poeciliid fishes. Behaviour Suppl. XIII.

ockhart, P. J., Steel, M. A., Hendy, M. D., and Penny, D. (1994).Reconstructing evolutionary trees under a more realistic model ofsequence evolution. Mol. Biol. Evol. 11: 605–612.

addison, W. P. (1990). A method for testing the correlated evolutionof two binary characters: Are gains or losses concentrated oncertain branches of a phylogenetic tree. Evolution 44: 539–557.

addison, W. P., and Maddison, D. R. (1992). MacClade: Analysis ofphylogeny and character evolution. Version 3.0. Sinauer, Sunder-land, MA.

artins, E. P., and Hansen, T. F. (1996). The statistical analysis ofinterspecific data: A review and evaluation of phylogenetic com-parative methods. In “Phylogenies and the Comparative Method inAnimal Behavior” (E. P. Martins, Ed.) pp. 206–233. Oxford Univ.Press, London.

effe, G. K., and Snelson, F. F. (1989). An ecological overview ofpoeciliid fishes. In “Ecology and Evolution of Livebearing Fishes(Poeciliidae)” (G. K. Meffe and F. F. Snelson, Eds.), pp. 13–31.Prentice Hall, Englewood Cliffs, NJ.

eyer, A. (1997). The evolution of sexually selected traits in maleswordtail fishes (Xiphophorus: Poeciliidae). Heredity 79: 329–337.

eyer, A., Morrissey, J. M., and Schartl, M. (1994). Recurrent originof a sexually selected trait inferred from a molecular phylogeny.Nature 368: 539–542.

eyer, M. (1993). Reinstatement of Micropoecilia Hubbs, 1926, witha redescription of M. bifurca (Eigenmann, 1909) from northeastSouth America. Zool. Abh. Mus. Tierkd. Dresden 47: 121–130.

age, R. D. M., and Lydeard, C. (1994). Towards a cladistic biogeog-raphy of the Caribbean. Cladistics 10: 21–41.

agel, M. (1994). Detecting correlated evolution on phylogenies: Ageneral method for the comparative analysis of discrete charac-ters. Proc. R. Soc. Lond. B 255: 37–45.

agel, M. (1997). Inferring evolutionary processes from phylogenies.Zool. Scr. 26: 331–348.

arenti, L. R., and Rauchenberger, M. (1989). Systematic overview ofthe poeciliines. In “Ecology and Evolution of Livebearing Fishes(Poeciliidae)” (G. K. Meffe and F. F. Snelson, Eds.), pp. 3–12.Prentice Hall, Englewood Cliffs, NJ.

rager, E. M., and Wilson, A. C. (1988). Ancient origin of lactalbuminfrom lysozyme: Analysis of DNA and amino acid sequences. J. Mol.Evol. 27: 326–335.

tacek, M. B. (1998). Interspecific mate choice in sailfin and shortfinspecies of mollies. Anim. Behav. 56: 1145–1154.

tacek, M. B., and Breden, F. (1998). Phylogenetic relationshipsamong the mollies (Poeciliidae: Poecilia: Mollienesia group) basedon mitochondrial DNA sequences. J. Fish Biol. 53: 64–81.

auchenberger, M. (1988). Historical biogeography of poeciliid fishesin the Caribbean. Syst. Zool. 37: 356–365.

idley, M. (1983). “The Explanation of Organic Diversity: The Com-parative Method and Adaptations for Mating,” Oxford Univ. Press,London.

idley, M., and Grafen, A. (1996). How to study discrete comparativemethods. In “Phylogenies and the Comparative Method in Animal

Behavior” (E. P. Martins, Ed.), pp. 206–233. Oxford Univ. Press,

R

R

R

R

R

R

R

R

S

enzymatic amplification of DNA with a thermostable DNA poly-

S

S

S

T

T

Y

289PHYLOGENY OF Limia

London.Rivas, L. R. (1978). A new species of poeciliid fish of the genus

Poecilia from Hispaniola, with reinstatement and redescription ofP. dominicensis (Evermann and Clark). Northeast Gulf Sci. 2:98–112.ivas, L. R. (1980). Eight new species of poeciliid fishes of the genusLimia from Hispaniola. Northeast Gulf Sci. 4: 28–38.ivas, L. R., and Fink, W. L. (1970). A new species of poeciliid fish ofthe genus Limia from the island of Grand Cayman, B. W. I. Copeia1970: 270–274.odriguez, C. M. (1997). Phylogenetic analysis of the tribe Poeciliini(Cyprinodontiformes: Poeciliidae). Copeia 1997: 663–679.odriguez, F., Oliver, J. L., Marin, A., and Medina, J. R. (1990). Thegeneral stochastic model of nucleotide substitution. J. Theor. Biol.142: 485–501.osen, D. E. (1976). A vicariance model of Caribbean biogeography.Syst. Zool. 24: 431–464.osen, D. E. (1978). Vicariant patterns and historical explanation inbiogeography. Syst. Zool. 27: 159–188.

osen, D. E., and Bailey, R. M. (1963). The poeciliid fishes (Cyprini-dontiformes), their structure, zoogeography and systematics. Bull.Am. Mus. Natur. Hist. 126: 1–176.zhetsky, A., and Nei, M. (1992). A simple method for estimatingand testing minimum-evolution trees. Mol. Biol. Evol. 9: 945–967.

aiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R.,Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directed

merase. Science 239: 487–491.anger, F., Nicklen, S., and Coluson, A. R. (1977). DNA sequencingwith chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.

chluter, A., Parzefall, J., and Schlupp, I. (1998). Female preferencefor symmetrical vertical bars in male sailfin mollies. Anim. Behav.56: 147–153.

chroder, J. H., Rodriguez, C. M., and Siegmund, D. R. (1996). Malesize polymorphism in natural habitats of Poecilia (Limia) perugiae(Pisces: Poeciliidae) endemic to Hispaniola. Biol. Zent. Bl. 115:315–327.

Swofford, D. L. (1998). PAUP*. Phylogenetic Analysis Using Parsi-mony (*and other methods). Version 4, Sinauer, Sunderland, MA.

Swofford, D. L., Olsen, G. P., Waddell, P. J., and Hillis, D. M. (1996).Phylogenetic inference. In “Molecular Systematics” (D. M. Hillis,C. Moritz, and B. K. Mable, Eds.), pp. 407–492. Sinauer, Sunder-land, MA.

empleton, A. R. (1983). Phylogenetic inference from restrictionendonuclease cleavage site maps with particular reference to theevolution of humans and the apes. Evolution 37: 221–244.

hompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTALW: Improving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific gap pen-alties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680.

ang, Z. (1996). Among-site rate variation and its impact on phylo-genetic analyses. Trends Ecol. Evol. 11: 367–372.