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Molecular Phylogeny of the Live-Bearing Fish GenusPoecilia

(Cyprinodontiformes: Poeciliidae)Felix Breden,* Margaret B. Ptacek, Michael Rashed,* Donald Taphor n,

and CarlosAugusto Figueiredo

* Behavioural Ecology Research Group and Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby,Brit ish Columbia, Canada V5A 1S6; Department of Biological Sciences, Idaho State University, Pocatello, Idaho 83209-8007;

Museo de Ciencias Naturales, UNELLEZ, Guanare, Edo. Portuguesa, Venezuela 3310; andDepartamento de Zoologia, Laboratorio deIctiologia Geral e Aplicada, Universidade Federal do Rio de Janeiro, Rio de Janeiro, CEP21944-970 Brazil

Received December 2, 1997; revised September 14, 1998

Me m be rs o f t h e g e n u s P o e c i l i a e x h i bi t e x t e n s iv e

m o r p h o l o g i ca l , b e h a v i o r a l, a n d l i fe h i s t o r y v a r i a t i o n

w i t h i n a n d b e t w e e n s p e c i e s . T h i s n a t u r a l v a r i a t i o n ,

c o u p l e d w i t h s h o r t g e n e r a t i o n t i m e s a n d t h e e a s e w i t h

w h i c h m e m b e r s o f t h i s g e n u s c a n b e c u l t u r e d i n t h e

ab , h a v e m a d e s e v er al s p e ci e s m o d el s y s te m s fo r

s t u d y i n g t h e e f fe c t s o f s e x u a l a n d n a t u r a l s e l e c t i o n o n

t h e e v o l u t i o n o f n a t u r a l p o p u l a t i o n s . Gi v e n t h a t t h e r e

s n o c l e a r u n d e r sta n d i n g o f th e p h y l o g e n e ti c r e l a ti o n -

s h i p s w i t h i n t h e g e n u s , th e s e s t u d i e s h a v e n o t b e e n p u t

n to a h i sto r i c a l c o n te x t, a n d b e tw e e n -sp e c i e s c o m p a r i -

s o n s h a v e b e e n l i m i t e d . We s e q u e n c e d t h e c o m p le t e

N AD H D e h y d r o g e n a s e S u b u n i t 2 ( N D 2) m i t o c h o n d r i a lg e n e ( 1 0 4 7 b p ) i n r e p r e s e n t a t i v e s o f t h e m a j o r d i v i -

s i o n s o f t h e g e n u s i n o r d e r t o e x a m i n e t h e s e r e l a t i o n -

s h i ps . Th e s u bg e n e ri c g ro u p s o f R os e n a n d B a il ey

19 63 ) a r e , f o r t h e m o s t p a rt , s u p p o r te d , w i t h s o m e

a d ju s t m e n t w i t h i n t h e s u b g e n e ra P o e c i l i a a n d P a m -

p h o r i c h t h y s . T h e m o r ph o l o gi c a l d i s t i n c tn e s s o f t h e

g ro u p s w i t h i n P o e c i l i a s u g g e s t t h a t t h e o r i g i n a l g e -

n e ri c d e s ig n a ti o ns b e r ei n st a te d , b u t t h is a w a it s a

m o r e t h o ro u g h a n a ly s i s . Tw o i m p li c a ti o n s f ro m t h e

p h y l o g e n y a r e p a r ti c u l a r l y re l e v a n t to se x u a l se l e c ti o n

s t u d i e s : w i t h i n t h e N o r t h a n d C e n t ra l Am e r i c a n m o l -

i e s, th e th r e e sp e c i e s o f sa i l fi n m o l l i e s fo rm a m o n o p h y -

e ti c g ro u p , a n d w i t h in t h e s u bg e n u s L e b i s t e s , th es i s t e r t a x o n t o t h e g u p p y , P. r e t i c u l a t a , i s m o s t l i k e l y

t he g ro u p o f s pe c i es p re v i ou s ly d e s ig na te d a s

M i c r o p o e c i l i a . 1 9 9 9 Ac a d e m i c P r e s s

INTRODUCTION

As part of their revision of the family Poeciliidae,Rosen and Bailey (1963) placed several well-estab-

is h e d g en e r a , i n cl u d in g Mollienesia, Allopoecilia,Lim ia, Pamph orichthys, Lebistes, a n d Micropoecilia,

nto one genus, Poecilia, reta ining four subgenera. Theresulting genus, Poecilia, is a complicated and widelyd is t r ib u t ed gr ou p , r a n g in g fr om t h e s ou t h e a s t er n

Un ited Sta tes to Bolivia an d sout her n Br azil. Species ofPoecilia are found in a wide range of habitats, exhibitmorphological an d behaviora l different iation withina n d b et w ee n s p ecie s, a n d h a v e b ee n s t u d ie d e xt e n -sively for th e effects of na tu ra l an d sexua l selection. Wepresent a phylogeny of this genus based on NADHDehydrogenase Su bun it 2 (ND2) sequen ce var iation, inorder to resolve some of the relationships within Poe-cilia and to provide a phylogenetic context for studies ofsexua l selection in th is group.

Taxonomy

The following description of four subgenera followsRosen an d Bailey (1963), with n otes from Ra uchenber g-er s (1989) an notated list of species in th e subfam ilyPoecili inae. Table 1 outl ines these major divisionswithin the genus and the specimens examined in t hisstudy. We also review proposed t axonomies th at havereinstated or adopted the genus rank for some of theassemblages within Poecilia (e.g., Costa, 1991; Meyer,1993; Rodr iquez, 1997).

Subgenus Poecilia includes at least 20 named speciesfr om N or t h a n d C en t r a l A me r ica a n d n or t h e a s t e r nSouth America that are commonly referred to as mol-

l ies. This subgenus includes the previously n amedgenera, Mollienesia L eS u e u r, 1 82 1 a n d AllopoeciliaHu bbs, 1924. The North and Centra l American m ollies(referred to as group Mollienesia within the subgenusPoecilia by Rauchenberger (1989)) have been dividedinto two species complexes: P. latipinna a n d P. sphenops(Hubbs, 1933; Miller, 1983). Th e P. lat ipinn a complex,or sa ilfinm ollies, includ es th ree species, P. latipin na,P. petenensis, a n d P. velifera, a n d ma l e s o f a l l t h r e especies are sexually dimorphic, having a n enlargeddorsal fin used in a courtship display directed towardfemales (Ptacek and Travis, 1997). These three species

are confined t o the Atlantic slope a nd ran ge from t hesouthern United States southeastward into northernG u a t e ma la a n d n or t h e r n B el iz e. P os s es s ion of t h e

Molecular Ph ylogenetics an d E volution

Vol. 12, No. 2, July, pp. 95104, 1999

Article ID mpev.1998.0600, available online at http://www.idealibrary.com on

951055-7903/99 $30.00Copyright 1999 by Academic PressAll rights of reproduction in any form reserved.

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possible sister taxon relationship between P. reticulataa n d t h e Micropoecilia group.

In summa ry, this complex group includes severalspecies that have been u sed as model systems for thes t u d y of n a t u r a l a n d s ex u a l s el ect i on . S u ch s t u d ie swould benefit from knowledge of sister taxon relation-

ships, particularly to polarize changes observed withinspecies. The genus also includes several species com-plexes whose uti li ty in studies of speciation wouldgreatly benefit from a more resolved phylogeny. Finally,her e are m an y unr esolved taxonomic issues within th e

g en u s . F or t h e s e r e a s on s , w e s eq u en ce d N D 2 fr omrepresentatives of the four subgenera in an attempt toexamine th e cohesiveness of th e subgeneric classifica-

i on s of R os en a n d B a il ey (1 96 3) a n d t o e lu cid a t erelationships within an d am ong th ese groups.

MATERIALS AND METHODS

DN A S ources

Ta b le 1 l is t s t h e col le ct i on s it e s for t h e 1 8 t a x astudied. We compared sequences of 15 species in thegenus Poecilia (16 specimens, in cludin g 1 from ea ch ofwo major divisions within P. reticulata) a n d 2 o t h e r

species: Xiphophorus n igrensis, in the same tr ibe as thegenus Poecilia, tribe Poeciliini, and Heterandria for-mosa, in the tr ibe Hetera ndr iini, all with in the subfam-ly Poeciliinae (Parenti, 1981).

ND2 S equences

The sequencing stra tegy is displayed in Fig. 1 andp r im e r s eq ue n ce s a r e give n in Ta b le 2 . F or m os tspecies, the ND2 gene was first a mplified using primer sdirected at th e meth ionine an d tr yptophan tRNAs. ForH. form osa, amplification primers were directed atgluta mine a nd a spar agine tRNAs (Kocher et al., 1995).Typical r eaction conditions wer e a pproximat ely 100 ngof genomic DNA used a s templat e for a 50-l reactionsolution containing each dNTP at 1 mM, each primer at0.5 M, 3 mM MgCl 2, 5 l of 10 PCR buffer (200 mMTris, pH 8.4, 500 m M KCl), an d 2.5 u nits of Taq DNAPolymerase (Gibco-BRL, Life Technologies Inc.). Reac-

ions were amplified for 35 cycles at 94C for 70 s, 50Cfor 90 s, and 72C for 150 s. This set of cycles waspreceded by heating to 94C for 100 s and followed byextension a t 72C for 240 s. An appr oxima tely 1150-bpPCR product was acrylamide gel-purified and clonedusing the pT7Blue T-Vector system (Novagen) for allbut thr ee of th e species listed in Table 1. Cloned DNAwas sequen ced by th e Un iversit y Core DN A Services ofh e Un iversity of Calgar y, Alberta using cycle sequ enc-ng and an ABI automated sequencer Model 377. Thehree species that were not cloned (P. orri, P. velifera,

a n d P. petenensis) were sequenced directly using the

forwar d and r everse am plificat ion primer s and inter na lprimers. Complete ND2 sequence and flank ing regions(1105 bp) were obtained for all taxa. Sequences were

confirm ed in two ways. First, t he forwar d primer s wereclose enough such that all regions, but the first, weresequen ced t wo times. Second, sequ ences pr oduced fromtwo reverse primers, vector primer U19 and internalprim er In t-1R, confir med 40% of th e gene, including th e

first region, for wh ich t her e was n o overlap ping forwa rds e qu e n ce . A s a ch e ck on t h e fr e qu e n cy of clon i n gart ifacts, par t ial sequences from cloning and fromdirect sequencing of a crylamide-purified PCR productwere compared for four species; there was exact agree-men t between th e two techn iques for over 2000 bp.

Phylogenetic Analysis

The data set for phylogenetic analysis consisted oft h e c o mp l e t e N D 2 g e n e a n d fl a n k i n g r e g i o n s i n t h eme t h ion i n e a n d t r y pt op h a n t R N A. S e qu e n ce s w er ealigned with Clusta lV, using mu ltiple a lignment gap

penalties of 25. Nucleotide variation and substitutionpatterns were examined using MEGA (Kumar et al.,1993; version 1.01). Aligned sequence data were ana-

TABLE 1

S p e c i e s E x a m i n e d a n d C o l le c t i o n S i t e s

SubfamilyHeterandriini

Heterandria

formosa

Savanah River, South

CarolinaSubfamily Poeciliini

Xiphophorus

nigrensis

Rio Choy, Mexico

Subgenus Poecilia

(m ollie s)P. vivipara Georgetown, Guyana South American MollyP. gilli Lake Nicaragua, N ica-

r ag u aShort fin, Atlantic

SlopeP. orri Cost a Rica Sh or t fin , At la nt ic

SlopeP. sphenops R io C au tla, Mex ico Sh or t fin , Pacific Slop eP. latipin na Ta m pi co, M exico S a ilfi nP. petenen sis Laguna Caobas, Quin-

tana Roo, MexicoSailfin

P. velifera Cenote Azul, QuintanaRoo, Mexico

Sailfin

Subgenus LebistesP. reticula ta Oropuche River,

TrinidadOropuche River (Oro)

P. reticula ta Lower Aripo River,Trinidad

Caroni Drainage (Car)

P. picta Georgetown, GuyanaP. parae Georgetown, Guyana

Subgenus P a m -

phorichthys

P. m inor Par intins, Amazonas,Brazil

P. a raguaiensis RioAraguaia, Goias,Brazil

P. h eterand ria Est ado Fa lcon, Ven-ezuela

Subgenus L i m i aP. perugiae Dominican Republic,

HispaniolaP. nigrofasciata Haiti, Hispaniola

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yzed by m aximum parsimony and neighbor joiningalgorithms as implemented in test version 4.0d64 of PAUP*, written by David L. Swofford, and maximumikelihood by DNAML in the PHYLIP 3.572 (Felsen-

stein, 1993).The first step in the analysis was to search for the

m os t p a r sim on iou s t r e e b y t h e b r a n ch a n d b ou n dmethod and optimal trees under neighbor joining and

maximum likelihood algorithms. Support for the nodesof these trees was examined by 500 bootstrap i tera-ions. Because the various optimality criteria assume

different models of evolution, we assume that agree-ment among these models indicates a robust result .Based on the results of simulation studies (Hillis andBull, 1993), we consider bootstr ap values great er tha n80% as indicating moderate support for a node andg r ea t e r t h a n 9 0% a s s t r on g s u p por t . T h e n e ig h bor

oining searches assumed the HKY85 substitution modelfor calcula ting dista nces (Swofford et al., 1996). Thebootstrap iterations of the maximum parsimony algo-

ri thm uti l ized a heurist ic search, with the followingoptions: keep minimal trees only, collapse zero-lengthbranches, random step-wise addition of taxa with 10replicat ions, and tr ee bisection an d reconn ection. Themodel of evolution employed for the likelihood analysiswas determined by the following steps. First, an opti-ma l t r e e w a s ob t a in e d u n d e r t h e n e ig h bor joi n in galgorithm, assuming uncorrected distances, equal fre-quencies of bases, and no among-site rate variation.The l ikelihood of this tree was compared, assumingequal base frequencies versus the HKY85 model of substitution. Then, this l ikelihood was compared to

h a t ca lcu la t e d for t h e s a m e t r e e, a s su m in g a 2 :1ra nsit ion to tr ansversion rat io versus estimat ing this

ratio from the data during the l ikelihood estimation.Fina lly, th e likelihood assum ing equa l ra tes of subst itu-ion a mong sites was compa red to tha t obtained assum -ng var iable ra tes appr oxima ting a gam ma distribution

with three rate categories. The likelihood of this treewas significantly improved by t he addition of t hesepar am eters to the model at ea ch step (Huelsenbeck an dCrandall, 1997). Therefore, we used the following modelof e volu t i on t o ob t a in t h e op t ima l t r e e a n d for t h ebootstrap iterations of the maximum likelihood algo-

rithm: maximum likelihood estimates of the base pairfrequencies un der th e H KY85 m odel (frequ encies of A,C, G, and T set to 0.286, 0.330, 0.110, 0.274, respec-

tively), t ra nsit ion to tra nsversion rat io of 2.79, andgamma distribution shape factor of 0.323, with threecategories of substitution rate.

We included two outgroups in the data set, X. n igren-si s a n d H. form osa. However, we ana lyzed the da ta with

H. formosa as t he sole outgroup to examine the r elation-ship of X. nigrensis relative to species in the genusPoecilia.

RESULTS

ND2 Sequence Variation

Eighteen taxa in the genera Poecilia, Xiphophorus,a n d Heterandria were sequenced for N D2 and flank ingregions for a tota l of 1105 bp. Sequen ces an d tr an slat edproteins have been deposited in GenBank under Acces-sion Nos. AF031386AF031402 an d AF084973. TheND2 gene was 1047 bp and showed no indels withinthese three genera or compared to 31 cichlid species

(Kocher et al., 1995). All predicted proteins were 348am ino acids long.

We observed a high d egree of sequen ce divergence fort h e N D2 ge n e w it h in t h e ge n us Poecilia (Table 3).Percenta ge sequence divergence r an ged to 19% withinthe genus Poecilia an d from 20 to 23% between Poeciliaa n d X. n igrensis. The r an ge of divergence was 20 to 25%between H. formosa a n d X. nigrensis or between H .

form osa and species of Poecilia. Thus, variation be-tween species within Poecilia w a s a l mo s t a s g r e a t a sth at observed between P oeciliini a nd Hetera ndr iini.

The ba se composition a t t he t hr ee codon positions for

a l l t a x a s h o w e d a b i a s s i mi l a r t o t h a t f o u n d i n t w ostudies of cichlid mitochondrial genes: cytochrome b(Roe et al., 1997) and ND2 (Kocher et al., 1995). Mostnotably, there was a strong a nti-G bias a t the secondand third positions (11.6 and 3.5%, averaged across alltaxa). To examine possible saturation at the variouspositions, we plotted divergences for first, second, andth ird position tr an sitions a nd first an d second positiontransversions against third position transversion diver-gence (Fig. 2). Third position tr an sversions were usedt o e xa mi n e s a t u r a t i on , b eca u s e t h e s e t r a n s v er s ion soccur relatively ra rely in a near ly Poisson fashion an d

therefore saturate most slowly (Kocher et al., 1995),an d to provide a compar ison t o th e extensive data set onND2 sequence variation among East African cichlids

FIG. 1. Amplification and sequencing strategy for the ND2 gene. Bars depict the ND2 and flanking tRNA genes. Arrows indicate thedirections of the sets of primer s used. P rimer sequences ar e given in Table 2.

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(Kocher et al., 1995; Kocher a nd Ca rleton, 1997). Thereappears to be saturation of third posit ion transit ionsstart ing at approximat ely 5% th ird posit ion tra nsver-

sion divergence, while tra nsit ions and tra nsversiondivergences at other positions appear to increase lin-early with third position transversion divergence (Fig.

2 ). I n fa ct , on l y t h e r e la t i on s h ip of t h i r d p os it i ontr an sitions to th ird position tra nsversion divergences h ow ed a s ign ifi ca n t q u a dr a t i c r e gr e s sion t e r m(P 0.001).

Phylogenetic Analysis

We employed three approaches in our phylogenetican alysis: neighbor joining, ma ximu m likelihood, an dmaximum parsimony. The maximum parsimony crite-r i on ga v e t w o m os t p a r s imon i ou s t r e e s. T h e s t r ictconsensus of these two most parsimonious trees andthe optimal trees obtained from neighbor joining andma ximu m parsimony is presented in Fig. 3, along withth e proport ion of the 500 bootstr ap iterat ions support-ing each node of this consensus tree. Using H. form osaas the outgroup, species in t he genus Poecilia form aclade relative to X. nigrensis in 100% of th e bootstr apiterat ions for all thr ee algorithm s.

Several groups within the genus Poecilia a r e s u p-ported at the 99100% level in all three analyses: thetwo species of the subgenus Limia, the two species ofthe subgenus Pamphorichthys from Bra zil, and t he sixspecies of Centra l a nd Nort h American mollies (group

Mollienesia; Rauchenberger, 1989). Within this Mollien-esia group, the sailfin mollies, P. latipinna, P. petenen-sis, a n d P. velifera, form a strongly supported group(94100%), and the species P. orri a n d P. gilli cluster in100% of th e bootst ra p it era tions. The th ree short fins p eci es for m a mod e r a t el y s u p p or t e d cl a de in t h eneighbor joining an d ma ximum likelihood an alyses,

but not with the ma ximu m par simony ana lysis.The subgenus Lebistes for ms a cl a de w it h a h i gh

degree of support (9093%). With in t he su bgenus Lebi-stes, the two individuals of the Trinidad guppy, P.reticulata, ar e clear ly associat ed (100%). However, th ereis only weak support for a clade composed of the twospecies previously classified as Micropoecilia (6468%).

The relationships among these strongly supportedgroups are much less clear. The optimal tree obtainedunder neighbor joining cri terion showed a spli t be-tween the subgenus Lebistes an d the oth er thr ee subgen-e r a , Poecilia, Lim ia, a n d Pamphorichthys, with P.

vivipara basal to these two divisions. The maximumlikelihood optimal tree showed a similar split, but inth is case included P. vivipara within the clade formedby subgenera Poecilia, Lim ia, a n d Pamphorichthys.H ow ev er , on e of t h e t w o mos t p a r s imon i ou s t r e e ssuggested a very different topology, linking subgenusPamphorichthys a n d P. vivipara w it h t h e s u bg en u s

Lebistes. None of th ese th ree t ree t opologies repres ent asignificantly more likely tree than the others (Likeli-hood Ratio Tests P 0.7; Huelsenbeck and Crandall,1997). Fur thermore, bootstra p support for any of th epossible relationships among the monophyletic groups

in Fig. 3 was less th an 80%.The inferred position of P. heterand ria, placed by

Rosen and Bailey (1963) in the subgenus Pamphorich-

TABLE 2

P r i m e r s U se d fo r A m p l i fi c a ti o n a n d S e q u e n c i n g o f

N A D H D e h y d r o g e n a se S u b u n i t 2 (N D 2 )

Primary amplification and sequencing of PCR product

Primer

pair

Primer sequence

(5 t o 3 )

Species

amplified

-Met a AAG CTA TCG GGC CCA TAC CC All except P. reticu-lata Car, P. m in or,

a n d H. formosa

C-Trp a CTG AGG GCT TTG AAG GCC C-MetCT b ACC CTG AAC ATG ACS GYT AAA A P. reticula ta C ar an d

P. m inor

C-Trp-P a m c

GTC TAA GGA ATT ATC CTA AG

GL N d CTA CCT GAA GAG ATC AAA AC H. formosa

ASN d CGC GTT TAG CTG TTA ACT AA

Interna l sequencing primers

PrimerPrimer sequence

(5 t o 3)Species

sequenced

ND2A TGA AGC YAC CAC TAA AT All except belowND2AM TGA AGC CGC CAC TAA AT P. p arae, P. m in or,

a n d P. ara-guaiensis

ND2AG TGA GGC CAC CAC TAA AT P. reticula ta Ca r

NT-1 TGA ATR CCA GAA GTA AT All except belowNT-1X TGA ATG CCA GAA GTA CT X. nigrensis

NT-1M TGA ATR CCA GAA GTT AT P. p arae, P. m in or,

a n d P. ara-guaiensis

NT-1R ATT ACT TCT GGY ATT CA All species (reverse

direct ion of INT-1)ND2B CAA CTC CGA AAA ATC CTA GC All except belowND2BX CAA CTC CGA AAA ATT CTT GC X. nigrensis

ND2BM CAA CTW CGA AAA ATC CTA GC P. latipinn a a n d P.sphenops

ND2DX GCT GCC CTT TCA TCC CT X. nigrensis

ND2D1 GTA GCA CTT TCA TCT TT P. picta

ND2D2 GCT GCA CTC TCA TCC CT P. gilli, P. latipinna,P. m in or, P. ara-

guaiensis, a n d P.heterandria

ND2D3 GCC GCA CTA TCA TCC CT P. nigrofasciata a n dP. perugiae

ND2D4 GCT GCA CTA TCA TCC CT P. sphenops

ND2D5 GCA GCA CTT TCA TCC CT P. vivipara

ND2D6 GCC GCA CTT TCA TCC CT P. reticula ta a n d P.parae

Note. R, A/G; S, C/G; W, A/T; Y, C/T.a P a r k et al. (1993).b Designed from methionine tRNA sequence, downstrea m from

-Met.c Designed from tryptophan tRNAsequence, upstream from C-Trp.d Kocher et al. (1995).

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thys, also changes depending on the approach taken.Only th e maximum likelihood cri terion places thiss p eci es in a gr ou p w it h t h e ot h e r Pamphorichthysspecies, and this relationship is upheld by very lowbootstrap support (47%). The most parsimonious treeobtained when constraining the three species of subge-n u s Pamphorichthys t o o n e c l a d e i s t h e s a me a s t h eoptimal t ree obtained un der m aximum likelihood, andas stated above, none of these trees is significantlymore likely tha n th e other s.

Because of saturation in third position transitions,we conducted three further types of analyses on theND2 region: maximu m pa rsim ony an d neighbor joiningemploying third position transversions only or exclud-ing the third position entirely and protein parsimony asimplemented in PHYLIP 3.572 (Felsenstein, 1993).None of these approaches produced results that dif-fered from t he a na lysis of th e full dat a set ; i.e., all of themonophyletic clades in Fig. 3 were su pported by thesea n a l ys e s, b u t t h e r e la t i on s h ip s a mon g t h e s e cl a de swere not further resolved.

DISCUSSION

Pattern of Substitution in ND2

T h e p a t t e r n of s u bs t it u t i on a t t h e v a r iou s cod onpositions is similar to that reported for East Africancichlids (Kocher et al., 1995; Kocher and Carleton,1997). The ra te of first an d second position tr ansitionsis much lower th an tha t for t he t hird position, indicat -ing selective constr aint s at th e first a nd second position(Kocher and Carleton, 1997). Divergence between taxa

i n t h i r d p os it i on t r a n s i t ion s s a t u r a t e s a t a le ve l o f appr oxima tely 25% divergence (Fig. 2), while diver-

F IG . 2 . Accumulation of sequence divergence between taxa atfirst, second, an d t hird position tra nsitions (A) and first and second

position tra nsversions (B) of the ND2 gene, plotted against thirdposition t ra nsversion divergence.

TABLE 3

U n c o r re c t e d P a i r w i s e D i ff e re n c e s i n N D 2 S e q u e n c e s

1 2 3 4 5 6 7 8 9 10 11 12 1 3 1 4 1 5 1 6 1 7

1. H. formosa

2. X. nigrensis 0.2083. P. vivipara 0.203 0.1994. P. gilli 0.240 0.216 0.1405. P. latipin na 0.233 0.199 0.127 0.0986. P. sphenops 0 .2 32 0 .2 13 0 .1 3 4 0 .1 04 0 .0 9 97. P. orri 0 .2 34 0 .2 08 0 .1 3 5 0 .1 36 0 .0 8 9 0 .0 93

8. P. petenen sis 0 .2 26 0 .2 02 0 .1 2 0 0 .1 06 0 .0 5 3 0 .0 98 0 .0 979. P. velifera 0 .2 29 0 .1 97 0 .11 6 0 .0 99 0 .0 3 8 0 .0 93 0 .0 92 0 .0 48

10 . P. nigrofasciata 0 .2 21 0 .2 07 0 .11 3 0 .1 36 0 .1 3 3 0 .1 31 0 .1 29 0 .1 24 0 .1 2611. P. perugiae 0 .2 20 0 .2 15 0 .1 2 0 0 .1 40 0 .1 3 0 0 .1 30 0 .1 33 0 .1 27 0 .1 22 0 .0 2 012 . P. reticula ta Or o 0 .2 36 0 .2 11 0 .1 3 5 0 .1 63 0 .1 5 8 0 .1 68 0 .1 59 0 .1 62 0 .1 58 0 .1 50 0 .1 5413 . P. reticula ta C ar 0 .2 35 0 .2 20 0 .1 4 6 0 .1 70 0 .1 7 2 0 .1 72 0 .1 63 0 .1 66 0 .1 67 0 .1 5 0 0 .1 56 0 .0 5 3

14 . P. picta 0 .2 41 0 .2 16 0 .1 4 8 0 .1 74 0 .1 6 9 0 .1 82 0 .1 68 0 .1 73 0 .1 64 0 .1 7 1 0 .1 73 0 .1 4 3 0 .1 5815 . P. parae 0 .2 37 0 .2 16 0 .1 6 4 0 .1 89 0 .1 7 8 0 .1 87 0 .1 79 0 .1 88 0 .1 72 0 .1 6 8 0 .1 68 0 .1 5 4 0 .1 60 0 .1 5716 . P. m inor 0 .2 53 0 .2 27 0 .1 5 7 0 .1 67 0 .1 6 6 0 .1 72 0 .1 59 0 .1 58 0 .1 62 0 .1 5 5 0 .1 61 0 .1 7 7 0 .1 76 0 .1 82 0 .1 89

17 . P. ara guaiensis 0 .2 42 0 .2 17 0 .1 4 3 0 .1 55 0 .1 6 1 0 .1 59 0 .1 47 0 .1 51 0 .1 53 0 .1 4 3 0 .1 50 0 .1 6 5 0 .1 71 0 .1 77 0 .1 81 0 .0 3418 . P. h eterand ria 0 .2 25 0 .2 12 0 .1 2 6 0 .1 50 0 .1 3 2 0 .1 33 0 .1 41 0 .1 31 0 .1 29 0 .1 2 6 0 .1 30 0 .1 6 2 0 .1 66 0 .1 67 0 .1 75 0 .1 62 0 .1 50

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gence in the other substitution classes increases lin-early with divergence in third position transversionsover the range observed in our study. Over a greaterran ge of divergence in third posit ion tra nsversions,Kocher and Carleton (1997) observed that differencesat t he first position rea ched a platea u at appr oxima tely8%, while second position differen ces leveled a t a ppr oxi-ma tely 3%. The pat tern of substitution within Poeciliamay be approaching similar saturation levels (Fig. 2).

T h e s e l e v e l s o f s a t u r a t i o n a r e mu c h l o w e r t h a n t h eheoretical levels determined by nucleotide composi-ion, and this would again imply that selective con-

s t r a i n t s a r e d omi n a t in g t h e s u b st i t u t ion p r oce ss a th ese positions.

Phylogenetic Relationships

Our results from neighbor joining, maximum likeli-hood, and maximum parsimony analyses for 15 speciesof Poecilia support the subgeneric categories of Rosenand Bailey (1963), with adjustments to the subgeneraPoecilia a n d Pamphorichthys. T h e s u b ge n u s L i m i a

appear s m onophyletic, alth ough it will be necessary toan alyze more species, especially in the group Odonto-

limia. The subgenus Lebistes also appears monophy-

letic. Sequen ces from th e m itochondr ial D-loop, includ-

ing individua ls from more populations of P. picta a n d

two populat ions ofMicropoecilia bifurca (sensu Meyer,

1993) support this conclusion as well (Breden et al.,

unpublished data). The six species of the Mollienesia

group within the subgenus Poecilia, previously placed

in t h e gen u s Mollienesia, form a highly supportedclade. However, it is unlikely that the subgenus Poe-

cilia is monophyletic, since none of the analyses closely

ally the South American molly, P. vivipara, w i t h t h e

Mollienesia group. In fact, the most par simonious t ree

obtained u nder the constra int of monophyly for the

subgenus Poecilia was significantly less likely than anyof the optimal trees (Likelihood Ratio Test, P 0.01.

The t wo Bra zilian s pecies of th e South American s ubge-n u s Pamphorichthys form a well-supported clade, but

the species ofPamphorichthys from Venezuela, P. het-erandria, may not be closely related to the other twospecies ofPamphorichthys. In summary, except for the

uncertain placement ofP. heterandria a n d P. vivipara,the subgeneric classifications of Rosen and Bailey

FIG. 3. Strict consensus of the optimal trees obtained from neighbor joining, maximum likelihood, and maximum parsimony analyses ofND2 sequence varia tion in 18 taxa in th e subfamily Poeciliina e (Paren ti, 1981). Bootstr ap su pport from 500 iter at ions is given at each node forneighbor joining/maximum likelihood/maximum parsimony algorithms. The length of the two most parsimonious trees was 1347 steps;

Consist ency Index 0.530; Rescaled Consistency In dex 0.290; Homoplasy In dex 0.470.

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(1963) form well-defined clades a ccording t o th e a na ly-sis of ND2 sequence varia tion.

The position of P. heterandria may be problematic,even with furt her study. Much of the t axonomy of thisgroup is based on characteristics of the gonopodium(t h e mod ifi ed a n a l fi n u s e d i n s p er m t r a n s fe r ) a n d

supporting structures. P. heterandria is unique in thegenus in not possessing a gonopodial palp, the fleshycovering of the gonopodium, and the gonopodium doesnot possess the barbs and hooks char acterist ic of th eother species in the genus. Still, a cladistic analysis ofsevera l m orphological char acters shows thr ee syna po-morphies supporting a clade formed by P. heterandriaa n d s p e c i e s i n t h e s u b g e n u s L i m i a (Figueiredo andCosta , unpublished data ). Thus, P. heterand ria ma y notbe aligned with any of the subgenera, based on the lackof a gonopodial palp and specialized barbs and hooks, ort may be linked with subgenus Limia.

The relationships among the subgenera remain un-resolved. The optimal tree obtained from maximumikelihood supports two clades within Poecilia, on e

comprising th ree su bgenera , Poecilia, Limia, a n d P a m -phorichthys, and the second comprising subgenus Lebi-stes. Neighbor joining analysis supports a similar divi-sion, except with P. vivipara a s a s i s t e r t a x o n t o a l lother species in genus Poecilia. However, maximumparsimony does not support this division within Poe-cilia, but rather l inks P. vivipara and subgenus P a m -phorichthys with subgenus Lebistes. We are obtaining

more mitochondrial and nuclear sequences to addresshis qu estion.O u r r e s u lt s a g r ee w it h s ome of t h e r e la t i on s h ip s

suggested in Rodriquezs (1997) recent revision of th egenus Poecilia, but differ from it in several importantways. His revision, based on 27 morphological charac-ers, supports the monophyly of subgenera L i m i a a n d

Pamphorichthys (although P. heterandria was not exam -ned). However, he recognized a genus Poecilia, com-

posed of members of the subgenera Lebistes a n d Poe-cilia (sensu Rosen and Bailey, 1963), including P.vivipara. None of the three analyses of the ND2 data

support this relationship; P. vivipara does not clusterwith oth er mem bers of the subgenus Poecilia, and theres no suggestion of a r elationsh ip between group Mollien-

esia a n d s u b ge n u s Lebistes. I n fa ct , t h e t wo m os tparsimonious trees obtained under the constraint of aclade comprising Lebistes, group Mollienesia, a n d P.vivipara, were significan tly less likely tha n an y of thehree optimal trees (Likelihood Ratio Test, P 0.005).

One of th e sources of the variance between our r esultsand those of Rodriquez may be that he examined only asingle species in the subgenus Lebistes, P. reticulata,even th ough this su bgenus a lso includes severa l species

previously included in the genus Micropoecilia (e.g., P.picta a n d P. parae).

Taxonomy

Several authors (Rivas, 1980; Costa, 1991; Meyer,1993; Rodriquez, 1997) have r einstat ed or adopted th egenus rank for assemblages within the genus Poecilia,sensu Rosen a nd Bailey (1963), ra th er t ha n follow th esystem of Rosen and Bailey (1963), in which several

genera were placed in one genus with four subgenera.Nevertheless, none of these authors, including Rosenand Bailey (1963), provided a complete classificationbased on a n inclusive phylogenetic ana lysis. Rodriquez(1997) r evised the genus Poecilia based on derivedfeatures, recognizing three genera: Pamphorichthys,

Limia, a n d Poecilia. However, for rea sons sta ted a bove,our phylogenetic analysis of ND2 sequence variationstr ongly r ejects Rodriquezs genus Poecilia, consistingof t h e p r e vi ou s s u b ge n u s Lebistes, t h e Mollienesiagroup, a n d P. vivipara. Also, his an alysis excluded atleast one ent ire group of species within Poecilia. There-

fore, we consider the taxonomy of th is genus unr e-solved.

Poecilias su bgenera as defined by Rosen a nd Bailey(1963) ar e morphologically qu ite different from eachother, and several lines of evidence suggest that thereare monophyletic assemblages within Poecilia (e.g.,

Limia, Pamphorichthys with t he exception ofP. h eteran-dria, Mollienesia, a n d Lebistes). They justified theirdecision t o esta blish br oad genera by stating t ha t su chg r ou p in g s w ou l d b es t d e mon s t r a t e p a t t e r n s of r e -latedness among species, especially to nonsystematists(R os e n a n d B a i le y, 1 96 3:6 ). H ow ev er , g iv en t h e

number of morphologically distinct, monophyletic as-semblages within Poecilia, w e f ee l t h a t t h e ir b r oa dclassification in fact masks man y relationships, andtherefore the taxonomic statu s of th is clade, Poecilia(sensu Rosen and Bailey (1963)), should be suprage-neric. However, the exact set of monophyletic assem-blages within Poecilia a n d t h e r e l a t i o n s h i p s a mo n gth em is still not clear. Rather tha n a dvocat e th e propertaxonomic rank for any of these clades or create newna mes, we feel that a systemat ic revision of this genusshould await a more inclusive stu dy (i .e., ana lyzingmore species and more genera and combining more

types of data) to avoid polyphyletic or paraph yleticassemblages.

Implications for S exual S election S tud ies

There was strong and consistent support for a cladeformed by the members of the subgenus Lebistes t h a twe examined and moderate support for a sister taxonrelationship between the guppy and the species previ-ously classified a s Micropoecilia. Within th is subgenus,the guppy, P. reticulata, is a model organism for thestudy of sexual selection, mainly because attractivemale characters and female preferences vary between

populations (Houde, 1997). In order to evaluate thedirection of chan ge within P. reticulata and to evaluate

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he sensory exploitation model for the evolution of female pr eference, it is necessary t o infer th e an cestra lstat es for t hese preferences and ma le traits. In t erms ofcoloration, P. parae h a s t h r e e ma l e mo r p h s : o n e h a snearly the entire body covered by a pigmented stripe,one has a dorsal caudal stripe, and one is colorless

(Rosen and Bailey, 1963; Liley, 1966). Males of P. p ictah r o u g h o u t t h e r a n g e h a v e a n o r a n g e s t r i p e o n t h e

dorsum of th e ta il an d yellow an d black spots on th e ta iland body. P. bifurca a n d P. branneri have some malecolora tion, alth ough th ese ar e not well described. Givenh at ma le colora tion is widespread in both th e Micropoe-

cilia group and P. reticula ta, conspicuous coloration ismos t li ke ly a n ce st r a l i n t h e s u b ge n u s . T h is w ou l dsuggest that critical tests of the sensory exploitationmodel would have to be conducted on females fromspecies from the sister taxon to Lebistes t h a t d o n o texhibit m ale colora tion (Basolo, 1996). The s equen ce

variation in ND2 is not sufficient to determine thesister taxon relationship with the other monophyleticassemblages within Poecilia.

Results of th e phylogenetic ana lyses strongly supp orth e monophyly of the s ailfin m olly species. This findingmplies a single origin of th e sailfin phenotype a nd

subsequent speciation ofP. lat ipin na , P. velifera, a n d P.petenensis. A s i m ila r r e su lt w a s ob t a in e d fr om a na n a lys is t h a t e xa m in e d b ot h N D2 a n d D -loop s e-q u en ce s fr om mor e s p ecie s w it h i n t h e Mollienesiagroup (Ptacek and Breden, 1998). Since males of al lhr ee sailfin species possess an enlarged dorsal fin a nd

use this fin in a courtship display behavior directedoward females, the monophyly of the group argueshat these traits evolved in the ancestor of this clade.

Females of P. latipinna use intraspecific variation indorsal fin morphology and courtship display rates todistinguish am ong ma les from different populations(Pt acek an d Travis, 1997). These sam e behaviora l an dmorphological traits also distinguish males of sailfinspecies from shortfin species (Ptacek, 1998) and mayhave been important in the speciation of this group.

ACKNOWLEDGMENTS

We t ha nk the following people for providing specimens: M. Britt o,C. Moreira, and R. Cunha, P. a raguaiensis; Charles Baer, H. formosa;

M ik e R ya n a n d G il R os en t h a l , X. nigrensis; Bob McKeand, P.nigrofasciata; Hector Espinosa Perez, P. sphenops; David Reznick, P.perugiae; J uan J acobo Schmitter-Soto, P. velifera a n d P. petenensis;

and Joel Trexler, P. gilli, P. latipinn a, a n d P. orri. Jon Durkin, BlairHed ges, Tomas Hr b ek , B r ad Sh affer, an d Jo h n Tay lor p r ov id edhoughtful comments on the manuscript. Fish were collected from

Venezuela under Permit No. 0497; from Guyana with permission ofh e Coordinat or of Environment al Affairs, Office of th e Pr esident, a nd

Dr. Kar en Pilgrim, Wildlife Division, Ministry of Agriculture; and

from Trinidad with permission of the Ministry of Agriculture, Landand Marine Resources. This work was funded by a grant from the

Natural Sciences and Engineering Research Council of Canada toF.B., a Fa culty Research Committee Grant , Idaho Sta te Un iversity to

M.B.P., and National Science Foundation Grant DEB 92-20849 to J.

Travis of Florida St at e Universit y.

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