Delimiting Species Using DNA and Morphological Variation and … · 2013. 1. 27. ·...

Syst Biol 51(1)69ndash91 2002

Delimiting Species Using DNA and Morphological Variation andDiscordant Species Limits in Spiny Lizards (Sceloporus)

JOHN J WIENS12 AND TONYA A PENKROT23

1Section of Amphibians and Reptiles Carnegie Museum of Natural History Pittsburgh Pennsylvania 15213-4080USA E-mail wiensjcarnegiemuseumsorg

2Department of Biological Sciences University of Pittsburgh Pittsburgh Pennsylvania 15260 USA

AbstractmdashHaplotype phylogenies based on DNA sequence data are increasingly being used to testtraditional species-level taxonomies based on morphology However few studies have critically com-pared species limits based on morphological and DNA data and the methods used to delimit speciesusing either type of data are only rarely explained In this paper we review three approaches forspecies delimitation (tree-based with DNA data and tree-based and character-based with morphologi-caldata)and propose explicit protocols for eachWe then compare species limits inferred from these ap-proaches using morphological and mtDNA data for the Yarrowrsquos spiny lizard (Sceloporus jarrovii) atraditionally polytypic species from the southwestern United States and Mexico All three approachessupport division of S jarrovii into ve species but only two species are the same among the threeapproaches We nd the greatest support for the ve species that are delimited based on mtDNAdata and we argue that mtDNA data may have important (and previously unappreciated) advan-tages for species delimitation Because different data and approaches can disagree so extensivelyour results demonstrate that the methodology of species delimitation is a critical issue in systemat-ics [Mitochondrial DNA molecular systematics morphological systematics nested-clade analysisphylogeography population genetics species limits taxonomy]

The two major goals of systematicsare delimiting species and reconstructingtheir phylogenetic relationships Althoughspecies are fundamental units in studiesof evolution ecology phylogeny and con-servation biology surprisingly little atten-tion has been paid to the methods anddata used to recognize and delimit them(Wiens 1999) This trend is particularly ap-parent when the meager literature on themethodology of species delimitation is con-trasted with the extensive body of workon the theory and methods of phyloge-netic analysis Few specic criteria or meth-ods for species delimitation have been pro-posed (eg Avise and Ball 1990 Davis andNixon 1992 Baum and Donoghue 1995Mallet 1995 Brower 1999 Wiens 1999Wiens and Servedio 2000 Puorto et al 2001Templeton 2001)and these criteria are rarelystated explicitly by empirical workers Mor-phological data traditionally have been usedto delimit species and continue to be widelyused today but many recent studies haveused DNA sequence data to test traditionalmorphology-based taxonomies particularlyanalyses of mitochondrial DNA (mtDNA)

3Present address Johns Hopkins University Schoolof Medicine Department of Cell Biology and Anatomy725 N Wolfe St Baltimore Maryland 21205 E-mailtpenkrotjhmiedu

variation in wide-ranging polytypic animalspecies (eg Sperling and Harrison 1994Shaffer and McKnight 1996 Sullivan et al1997 Zamudio et al 1997 Zamudio andGreene 1997 Steppan 1998 Parkinson etal 2000 Rodracuteotilde guez-Robles and de Jesus-Escobar 2000 Serb et al 2001) These studieshave revealed both discordance and concor-dance with previous taxonomies Howeverfew studies have critically compared specieslimits inferred from morphological andDNA sequence variation (eg Puorto et al2001)

Disagreement between species boundariesinferred from different data types raisesseveral important questions How commonare these disagreements and under whatconditions are they most likely to occurAre disagreements between species limitsfrom DNA and morphological data gener-ally caused by deviations between gene andspecies trees (eg Pamilo and Nei 1988Maddison 1997) Or is discordance morefrequently caused by convergence in mor-phological characters Will DNA sequencephylogenies give a very different picture ofspecies diversity and patterns of speciationfrom that obtained from traditional mor-phological characters What approach willgive us the most accurate picture of the truespecies boundaries Addressing these ques-tions requires explicit methods for species

69

70 SYSTEMATIC BIOLOGY VOL 51

delimitation and critical comparisons ofmorphological and DNA variation in empir-ical case studies

The use of mtDNA sequence data inspecies delimitation has been particularlycontroversial and someauthors have arguedthat species should not be delimited basedon these data alone (eg Moritz et al 1992Moritz1994Sites andCrandall 1997Puortoet al 2001) MtDNA data may be problem-atic in that all mitochondrial genes are in-herited as a single linkage group as a resultany mismatch between gene and populationhistories caused by ancestral polymorphismor gene ow between species will simultane-ously affect all mitochondrial genes (Moore1995) Furthermore because mtDNA is ma-ternally inherited mtDNA haplotype phy-logenies will reect only patterns of femalegene ow and dispersal which may bequite different from those patterns in males(Avise 1994) Despite these potential prob-lems we argue that mtDNA has an impor-tant advantage in species delimitation rela-tive to nuclear-based markers that has notbeen widely appreciated As pointed out byMoore (1995) in his defense of mtDNA datain phylogeny reconstruction the smaller ef-fective population size (Ne) of the mitochon-drial genome will cause mtDNA haplotypesof a given species to coalesce (ie becomeldquomonophyleticrdquo) four times more quicklythan will nuclear markers (given some as-sumptions) We suggest that this propertyis important in species delimitation becausenewly formed species should become dis-tinct in their mtDNA haplotype phylogenieslong before they become distinct in nuclear-based markers Thus analysis of mtDNAdata should allow resolution of species limitsin many groups that are difcult to resolvewith nuclear-based markers such as mor-phology We present an empirical study ofspiny lizards (Sceloporus) that may illustratethis phenomenon

The Yarrowrsquos spiny lizard (Sceloporusjarrovii) has traditionally comprised sevensubspecies distributed in the mountains anddeserts of northern Mexico and the south-western United States Wiens et al (1999)presented a phylogeny for 30 populationsof S jarrovii (including the type localitiesof all seven subspecies) that was based onparsimony and maximum likelihood anal-yses of 18 kb of mtDNA sequence datafrom the 12S and ND4 genes (and adjac-ent tRNA genes) They found S jarrovii

to be paraphyletic within the monophyletictorquatus species group (sensu Wiens andReeder 1997) and divided S jarrovii intove species These species correspond tove allopatric clades of haplotypes noneof which are sister taxa Wiens et al (1999)mentioned some diagnostic morphologicalcharacters for these species but did notpresent a critical analysis of morphologi-cal variation in the group Because we wishto evaluate the partitioning of S jarroviimore carefully we herein revert to the tradi-tional taxonomy and refer to these taxa col-lectively as S jarrovii

In this paper we discuss three approachesfor species delimitation using DNA andmorphological variation We propose andreview specic protocols for delimitingspecies using these general approaches andthen apply these methods in an empiricalcase study of the traditionally recognizedSceloporus jarrovii We nd that these meth-ods and data give surprisingly discordantresults which may reect a problematic pat-tern of morphologicalvariationin this groupWe suggest that mtDNA data may generallybe a very powerful tool in species delimita-tion especially in groups that are difcult toresolve with morphological data

APPROACHES TO SPECIES DELIMITATION

Concepts and OverviewBefore we attempt to recognize species

we need a clear concept of what species arede Queiroz (1998) suggested that despitethe long history of dispute over species con-cepts most species concepts agree funda-mentally that species are lineages (Simpson1961 Wiley 1978 Cracraft 1983 de Queirozand Donoghue 1988 Frost and Kluge 1994Baum and Shaw 1995) and for sexual or-ganisms they are lineages that are unitedthrough the process of gene ow (Mayr 1942Dobzhansky 1950 Templeton 1989) Whatprevious authors have generally disagreedabout are the best criteria for recognizingthese lineages (de Queiroz 1998)

In this study we compare the results ofthree approaches (or criteria) to speciesdelimitation (1) tree-based delimitation(sensu Baum and Donoghue 1995) usingDNA haplotype phylogenies (2) tree-baseddelimitation using morphological data and(3) character-based delimitation (sensuBaum and Donoghue 1995) using mor-phological data We discuss these three

2002 WIENS AND PENKROTmdashDELIMITING SPECIES WITH DNA AND MORPHOLOGY 71

approaches below However species are realentities that exist in nature regardless ofwhether they are supported by any some orall of these approaches and we acknowledgethat in some cases all approaches are likelyto fail or give ambiguous results (Frost andKluge 1994)

We do not apply character-based delimita-tion to the DNA sequence data because wend the arguments of Brower (1999) againstthis approach to be compelling Character-based delimitation is concerned only with thedistribution of alleles and ignores their phy-logenetic history and implicitly requires thatthe potentially diagnostic characters evolverelatively slowly Brower (1999) showed aplausible hypothetical example in which twospecies each have many distinct haplotypesfor a rapidly evolving marker with no xeddifferences between thembut a phylogeneticanalysis of these haplotypes easily separatesthem into two distinct lineages For mor-phological characters (to which character-based delimitation is usually applied) thereare generally few ldquoallelesrdquo (ie characterstates) and their history is relatively difcultto estimate

In the following discussion we use theterm ldquoexclusiverdquo instead of monophyletic todiscuss haplotype phylogenies because theterm monophyly may not be applicable be-low the species level (following de Queirozand Donoghue [1990] and subsequentauthors)

Tree-Based Species Delimitationwith DNA Data

OverviewmdashWe propose an explicit pro-tocol for delimiting species based onDNA haplotype phylogenies which canbe integrated with nested-clade analysis(NCA Templeton et al 1995) Despite thewidespread use of DNA data in evolution-ary conservation and taxonomic studiesof population-level differentiation (Avise2000) the methodology by which haplotypephylogenies are used to delimit speciesis only rarely explained (eg Baum andShaw 1995 Brower 1999 Templeton 2001)Our approach is essentially a dichotomouskey for making species-level decisionswhich we outline both verbally (below) andgraphically (Fig 1) For our approach weassume a phylogeny of nonrecombiningDNA haplotypes of known locality andtaxonomic designation We also assume

that the failure of haplotypes from a givenlocality to cluster together is potential evi-dence for gene ow with other populations(Slatkin and Maddison 1989) as is the gen-eral discordance between haplotype cladesand the geographic area from which thehaplotypes are found (eg some haplotypesfrom Australia cluster with some from NewGuinea rather than the haplotypes fromeach region being mutually exclusive) Al-ternative explanations for this discordanceinclude incorrect estimation of the gene treeor incomplete lineage-sorting of ancestralpolymorphisms Incorrect estimation of thegene tree may be unlikely if there is strongstatistical support for discordant clades (egas assessed by bootstrapping Felsenstein1985) The effects of incomplete lineage sort-ing may be difcult to distinguish from geneow Because of the smaller Ne of the mito-chondrial genome the use of mtDNA datamay greatly reduce the possibility of discor-dance caused by ancestral polymorphisms(Moore 1995) and we generally assume inthis paper that discordance is caused by geneow Given that retained ancestral polymor-phisms are most likely in populations thathave split very recently (Neigel and Avise1986) mistaking retained polymorphismfor gene ow should only rarely lead tomistakes in species delimitation using ourapproach given our emphasis on the olderlineages within a putative species

MethodologymdashOur approach is as followsGiven a haplotype phylogeny for a set ofpopulations currently classied as a species(the focal species of the study) and one ormore closely related species the haplotypetree may show the focal species to be eitherexclusive or not If the haplotypes of the focalspecies are exclusive the presence of multi-ple species that are hidden by previous tax-onomy is suggested by well-supported basallineages (by ldquobasalrdquo we mean the oldest splitor splits within the species) that are concor-dant with geography (Fig 1a) The presenceof a single species (Fig 1b) is suggested byevidence for gene ow among the basal lin-eages (ie haplotypes from a given localityappear in both of the basal lineages andorthere is overall discordancebetween the hap-lotype phylogeny and geography) If thehaplotypes of the focal species are not ex-clusive (the focal species is ldquoparaphyleticrdquo orldquopolyphyleticrdquo) with respect to one or morespecies that are each distinct and exclusivethen the focal species may represent multiple


FIGURE 1 Simplied ow chart for species delimitation based on a DNA haplotype phylogeny illustrated witha hypothetical example The hypothetical example involves two species one with two disjunct populations (speciesA) the other with a single contiguous population (species B) Two individuals are sampled from each population(eg A1 D a haplotype from locality 1 of species A) The line above the terminal taxa indicates species limits

species if there is no evidence of gene ow be-tween the basal lineages (Fig 1c) If there isgene ow among the basal lineages (Fig 1d)the focal species may represent a singlenonexclusive species (ldquoferespeciesrdquo [Gray-beal 1995] or ldquoplesiospeciesrdquo [Olmstead1995]) If the focal species is nonexclusivewith respect to a species that is itself not ex-clusive (the haplotypes of the focal speciesinterdigitate with those of another species)and if there is no evidence of gene ow be-tween the basal lineages of each species thenthe focal species and the other species mayeach represent multiple species disguisedby traditional taxonomy (Fig 1e) Alterna-tively the focal species and the other speciesmay be conspecic if there is evidence of ex-tensive gene ow between them (Fig 1f)mdashfor example if some of the haplotypes froma given locality of the focal species clusterwith some of the haplotypes from a given lo-calityof the other species In all of these casesthe support for the hypothesis that multi-ple species are present will be strengthened

if the putatively conspecic populations arenot merely nonexclusive but are also rela-tively distantly related

Assumptions and extensionsmdashTwo aspectsof sampling design are critical to our ap-proach (1) including as many species aspossible that are closely related to the focalspecies to thoroughly test the exclusivity ofthe focal species and (2) including two ormore individuals from as many localities aspossible to evaluate gene ow among pop-ulations (Slatkin and Maddison 1989) In-cluding closely related species is crucial be-cause several previous studies have foundthat the focal species is nonexclusive andconsists of multiple species that are not eachotherrsquos closest relatives (eg Zamudio et al1997 Steppan 1998 Rodracuteotilde guez-Robles andde Jesus-Escobar 2000 Serb et al 2001) Thispattern can be detected only by including theadditional (nonfocal) species in the analysisIf multiple individuals cannot be obtainedfrom each population then delimitation ofspecies will depend on the relationship


(ie exclusivity) of populations of the focalspecies to other species and on the generalconcordance between phylogeny and geog-raphy within the focal species (which can beobjectively tested using NCA) Our approachcan be applied without extensive samplingof individuals from each species but is moreprone to error if sample sizes are small (egfailing to detect putative gene ow betweenpopulations)

Although our method emphasizes thebasal lineages of a focal species as potentiallydistinct species it is theoretically possiblethat each of these basal lineages might con-tain multiple species (which could be exclu-sive or nonexclusive) and we would use thesamereasoningoutlined above to detect suchcases However at these progressively lowerlevels small sample sizes may limit our abil-ity to condently rule out gene ow withother lineages (eg if only two individualsare sampled from a locality they are likely toappear to be exclusive even if there is ongo-ing gene ow with other populations) Ourapproach does not determine when splittingis no longer justied by the available databut this can be evaluated statistically by us-ing NCA

We have assumed that all the putativespecies are allopatric or parapatric with re-spect to each other This seems to be truefor most of the case studies we have citedbut sympatric species may be present thathave been unrecognized by previous taxon-omy and the focal taxon might even con-sist of a mixture of sympatric and allopatricspecies If a focal species contains morpho-logically cryptic sympatricspecies thatoccurtogether at several localities we expect thatthe haplotypes from each species will phy-logenetically segregate into basal clades con-taining haplotypes from each locality How-ever distinguishing between gene ow andthe presence of sympatric species may bedifcult when divergent haplotypes of thesame putative species occur at the samelocality

We have implicitly assumed that any geneow between haplotype clades is evidencethat these clades are conspecic In factspecies may be distinct and still engage inlimited gene ow but the exact level of geneow that determines whether taxa are con-specic is unclear (and will probably alwaysremain so) Templeton (1994) suggested thatspecies could be delimited despite the pres-

ence of hybridization by using random per-mutation methods to test for a signicant as-sociation between the phylogenetic structureof the haplotype tree and prior taxonomiccategories This application of NCA seemspromising even if it is not a universal so-lution In general we consider the criticalfactors in determining the species status ofhybridizing taxa to be the geographic extentof gene ow relative to the geographic rangearea of the species and relative to the areaof sympatry between speciesmdashhybridizingtaxa might still be distinct species if geneow is limited in extent andor is restrictedto a small portion of their ranges andor to asmall part of their area of sympatry

We have also assumed that the haplotypephylogenies are inferred from a single geneor linked set of genes (eg mtDNA) Whenmultiple genes are available one expects thatgene histories will generally be concordantbetween species (and with regard to speciesexclusivity) and discordant within speciesThese expected patterns of concordance anddiscordance may be an additional line of ev-idence for species delimitation when usinghaplotype phylogenies (Avise and Ball 1990Baum and Donoghue 1995 Baum and Shaw1995)

Comparison with other approachesmdashOur ap-proach differs from those of several otherauthors (eg Avise and Ball 1990 Baumand Shaw 1995) in that we do not requirespecies to be exclusive (eg Paetkau 1999Templeton 2001) Furthermore we do notconsider nonexclusive species to be funda-mentally different from exclusive species(contra Graybeal 1995 Olmstead 1995) fol-lowing Frost and Kluge (1994) Because ofincomplete lineage sorting of ancestral poly-morphisms all species may be nonexclu-sive at some point in their history (Neigeland Avise 1986) and species may be dis-tinct and even morphologically diagnosablefrom each other but still have nonexclusivegene genealogies This scenario may be com-mon when a species with a large geographicrange and a large population size gives riseto a diagnosably distinct species with a muchsmaller range (ie a peripheral isolate) suchthat the latter species quickly becomes ex-clusive whereas the former species does not(eg Funk et al 1995 Talbot and Shields1996 Hedin 1997 Paetkau 1999)

Our approach is similar to the cladistichaplotype aggregation method of Brower


(1999) but differs fundamentally in that weexplicitly considerestimatedpatterns ofgeneow in inferring species limits Accordingto Brower (1999202) ldquoeach population is aphylogenetic species if the haplotypes of allof its members are joined in a contiguoussection of an unrooted networkrdquo Howeverwithout utilizing estimated patterns of geneow it is unclear how exactly one divides upthese networks (trees) into species For exam-ple applying Browerrsquos method to the tree inour Fig 1a it appears that populations A1A2 and A3 could each be recognized as dis-tinct species or all four populations (A1ndashA4)might collectively represent a single speciesSimilarly when populations of a putativespecies fail to ldquoaggregaterdquo the method seemsto give little basis for distinguishing the pres-ence of a single species rather than multiplespecies Given the idea that species differ fun-damentally from higher taxa because of geneow it is unclear how any method can hopeto delimit species successfully without tak-ing into account this process

Templeton (2001) recently advocated ap-plication of his NCA method (Templetonet al 1995) to the problem of species de-limitation NCA uses permutation tests todetermine whether there is a nonrandomassociation of haplotype clades with theirgeographic locations given the latitude andlongitude of each locality and the positionof each haplotype within the tree NCAthen applies a dichotomous inference key todistinguish among different causes for thesesignicant associations (eg range fragmen-tation [as in allopatric speciation] isolation-by-distance contiguous range expansionlong-distance colonization) This method re-quires sampling from throughout the rangeof all included species and may thereforebe inapplicable to many species or groupsof species Furthermore NCA does not dis-tinguish among different causes for the lackof signicant association between haplotypeclades and their geographic location (eginsufcient sampling gene ow ancestralpolymorphism) However NCA has at leasttwo important advantages relative to our ap-proach (1) it takes into account all of theavailable information on the geographic andphylogenetic position of haplotypes and sta-tistically tests for their association and (2)it can be applied to many different levelswithin a clade to determine whether a spe-ciation (fragmentation) event can be inferred

with signicant statistical support given thedata available

Combining our approach with NCAmdashWesuggest the strengths of our method andNCA are complementary and we proposeusing them togetherOur method canbe usedto delimit species at the highest levels of di-vergence even when some of the includedspecies are represented by only one or two in-dividuals (as in our studyof Sceloporus) NCAcan then be used to determine whether fur-ther splitting is statistically justied withinwell-sampled clades and to more rigorouslytest the limits of taxa that are closely relatedand extensively sampled We demonstratethis integrated approach using our data fromspiny lizards (Sceloporus)

Tree-Based Delimitation withMorphological Data

We propose an explicit protocol for de-limiting species using population-level treesbased on morphology Tree-based delim-itation with morphology although advo-cated by some authors (eg Baum andDonoghue 1995) has rarely been used byempirical systematists (eg Hollingsworth1998)and the precise methodologyfor itsusehas never been thoroughly explained Thisapproach is facilitated by recent methodsthat allow continuous quantitative charac-ters and polymorphic characters to be in-cluded in phylogenetic analyses with lit-tle loss of information (eg Thiele 1993Wiens 1999 2001) We use populationsrather than individuals as terminal units (fol-lowing Hollingsworth 1998) because mor-phological characters are generally thoughtto be genetically unlinked and using indi-viduals will inappropriately treat all poly-morphisms shared between populations ashomoplasies rather than potential synapo-morphies (Wiens 2000) We consider sets ofpopulations that are strongly supported asexclusive and are geographically coherentto be potentially distinct species (eg for agiven taxon all populations from Australiaform one well-supported clade and the pop-ulations from New Guinea form anotherwell-supported clade) We emphasize cladesthat are strongly supportedmdashfor exampleas assessed by bootstrappingmdashbecause it istheoretically possible that a clade of pop-ulations could be united by slight differ-ences in qualitative trait frequencies or quan-titative trait means but still be engaging in


gene ow with other populations Howevercontinuing gene ow seems unlikely if theclade of populations is well differentiatedand strongly supported Methods for sta-tistically determining the congruence be-tween morphology-based clades and theirgeographic locations (equivalent to NCA)have not yet been developed

As with tree-based delimitation usingDNA data we present our method for mor-phological data verbally and graphically as adichotomous key (Fig 2) If the populationsof the focal species appear as exclusive thefocal species may represent a single speciesif it contains no strongly supported basal lin-eages that are concordant with geography(Fig 2b) whereas it may represent multiplespecies if it contains basal clades of popu-lations that are strongly supported and ge-ographically coherent (Fig 2a) If the focalspecies is not exclusive the basal lineagesof the focal species may represent distinctspecies if they are strongly supported as

FIGURE 2 Simplied ow chart for species delimitation using a population-level phylogeny based on morpho-logical data illustrated with a hypothetical example The hypothetical example involves two species one with twodisjunct populations (species A) the other with a single contiguous population (species B) The line above theterminal taxa indicates species limits Numbers at nodes indicate hypothetical bootstrap proportions

exclusive and concordant with geography(Fig 2c) and especially if they are not on ad-jacent branches such that the focal species ispolyphyletic The focal species may be a sin-gle nonexclusive species if the basal cladesof populations are weakly supported arenot concordant with geography and appearon adjacent branches of the phylogeny suchthat the focal species is paraphyletic and notpolyphyletic (Fig 2d) The basal lineages ofa nonexclusive species could appear to bepolyphyletic and still be conspecic (freelyinterbreeding) but this seems less likely themore distantly related these populations areon the tree The focal species may be con-specic with another species if their popula-tions interdigitate on the tree if relationshipsamong them are weakly supported and ifthe clades of populations are not concordantwith geography (Fig 2e)

Some authors have stated that phyloge-netic analysis should not be attempted be-low the species level (eg Davis and Nixon


1992) and few researchers have attempted toreconstruct phylogenies for populations us-ing morphology Because gene ow amongpopulations and recombination among char-acters should break up hierarchical patternswithin species inferred from morphologicaldata there is unlikely to be a clear phyloge-netic signal within species using these dataOur method rests on the idea that this lack ofintraspecic signal will lead to weakly sup-ported population-level trees that are discor-dant with geography and moreover that wecan detect this lack of signal and use it to in-fer species limits in cases where these limitsare unknown Finally we note that the tree-based approach we outline for morphologi-cal data should be equally applicable to al-lozyme and microsatellite data

Tree-based species delimitation can also beattempted with combined DNA and mor-phological data We expect strong phyloge-netic signal within species from the DNAdata and weak intraspecic signal from mor-phological data Thus combining these datamay simply yield the DNA haplotype treeor some slight modication thereof and thisis basically the pattern we observe in ourresults from spiny lizards (Sceloporus) Wedo not see any clear advantage to delimit-ing species using trees from such combinedanalyses

Character-Based Delimitation withMorphological Data

Character-based species delimitation in-volves nding diagnostic character statesthat represent seemingly xed differences be-tween the putative species (eg frequencyof the diagnostic state in species A D 100vs frequency in species B D 0) or dif-ferences that are at least nonoverlapping(eg range in number of vertebrae in speciesA D 10ndash12 vs range in species B D 14ndash15) This approach has been formalized aspopulation aggregation analysis (Davis andNixon 1992) The approach is advantageousin that there is a clear relationship betweenxed differences and gene owmdashif the di-agnostic traits are genetically based and aretruly xed in each species there is unlikelyto be any gene ow between the speciesHowever given nite sample sizes deter-mining with certainty whether traits aretruly xed is virtually impossible (Wiensand Servedio 2000) Even allowing some

low level of polymorphism in the diagnosticcharacters (eg a frequency of 10) requiresvery large sample sizes to achieve statisticalcondence (using the method of Wiens andServedio 2000) and the best frequency cut-off to use remains uncertain This approachshould be applicable to other types of char-acter data as well (eg allozymes)

Many systematists utilize statistical anal-yses of quantitative morphological charac-ters to test species boundaries often eval-uating the extent to which individuals ofa putative species cluster together usingprincipal components or canonical variatesanalysis This approach may be useful ininferring species limits but lacks the clearrelationship to estimated patterns of geneow that the character-based approach of-fers or the phylogenetic component of thetree-based approach

Puorto et al (2001) recently developedan approach to species delimitation that in-volves testing for signicant association be-tween quantitative variation in morphologyand haplotype trees from DNA data usingMantel tests Their method tests whethermorphological data signicantly conrmgroupings based on DNA by testing for sig-nicant association between matrices of Eu-clidean distances based on morphologicaldata and matricesof patristic distancesbasedon the haplotype tree

Concordance Between Approaches andAdvantages of mtDNA Data

We make the following predictions aboutpatterns of concordance between the threeapproaches In general we predict that thedistinctness of a species based on all threeapproaches will be related to how longthe species has been isolated from geneow with other species Thus given enoughtime distinct species should (1) have ex-clusive DNA haplotype phylogenies relativeto other species (Neigel and Avise 1986)(2) have one or more diagnostic morpholog-ical characters (either xed or at high fre-quency) and (3) form strongly supportedclades of populations based on morphol-ogy We consider the strongest evidencefor distinct species to be concordance be-tween these different approaches (eg Aviseand Ball 1990 Sites and Crandall 1997)Conversely for species that have divergedvery recently we predict that species will


have nonexclusive haplotype phylogenies(ie the individuals or populations will beparaphyletic or polyphyletic with respectto one or more other species Neigel andAvise 1986) will lack diagnostic charactersand will have nonexclusive population-levelphylogenies from morphology

For species that are intermediate in theamount of time they have been repro-ductively isolated we predict that specieswill generally become exclusive in theirmtDNA haplotype phylogenies long beforebecoming exclusive in morphology-basedphylogenies and before acquiring diagnosticmorphological characters (see also Frostet al 1998) We expect this pattern largelybecause mitochondrial genes have an Ne thatis one-fourth that of a given nuclear geneand because Ne strongly inuences the rateat which the haplotypes of a species becomeexclusive (Neigel and Avise 1986 Moore1995) Thus all else being equal a specieswill appear distinct (exclusive) based on amitochondrial gene in a quarter of the timeit will take for its nuclear gene haplotypes tobecome exclusive (Moore 1995) Similarlyit will take four times as long for a givennuclear marker (such as a morphological orallozyme character) to become xed withina species as it will take for the mtDNA hap-lotypes of that species to become exclusive(Kimura 1983) Furthermore for conditionswhere a single mitochondrial gene has a95 probability of correctly resolving aspecies as exclusive 16 or 40 nuclear genes(depending on the null hypothesis used)must be sampled to have the same proba-bility of success (based on Moore 1995) Insummary mitochondrial markers should beable to correctly resolve species boundariesfor many species that are too recently di-verged to be resolved using nuclear markersalone and should do so with much greaterefciency and probability of success

These advantages of mtDNA data de-pend on certain assumptionsHoelzer (1997)responding to Moore (1995) suggested thatmtDNA gene trees would coalesce lessrapidly (relative to nuclear-gene trees) whenthere is polygyny female philopatry andmale-biased dispersal Moore (1997) pointedout that the level of polygyny would haveto be extreme to offset the advantages ofmitochondrial markers and showed that fe-male philopatry and male dispersal wouldonly lengthen the coalescence times for mi-

tochondrial markers if speciation occurredwithout geographic subdivision Apart fromtheir effect on population size male disper-sal and female philopatry can bias estimatesof gene ow that are based on mitochondrialmarkers (Avise 1994 Moritz 1994) whichin some cases might inuence estimates ofspecies boundaries (see studies on Ensatinasalamanders for a possible example Moritzet al 1992 Jackman and Wake 1994 Wake1997 Wake and Schneider 1998) Some au-thors have also suggested that delimitingspecies based on mtDNA data may be prob-lematic because the smaller Ne of the mito-chondrial genome may lead to coalescenceof haplotype lineages in populations that areonly temporarily isolated (eg Moritz 1994Sites and Crandall 1997 Paetkau 1999) Theproblems of male-biased dispersal femalephilopatry and coalescence of temporarilyisolated populations all involve potentialoverresolution of intraspecic mtDNA trees(ie mtDNA clades appear as distinct lin-eages even though they are not) and are mostlikely to affect the more recent branches ofthe haplotype tree Our emphasis on recog-nizing basal haplotype lineages (ie the old-est splits) within a taxon as distinct speciesshould greatly reduce these problems

MATERIALS AND METHODS

DNA Haplotype Phylogeny andNested-Clade Analysis

The mtDNA haplotype phylogeny forS jarrovii and related species was based ona maximum likelihood analysis of the com-bined mitochondrial 12S and ND4 gene re-gions (for details see Wiens et al 1999) The12Sdata consistof883bp and133parsimony-informative characters The ND4 data con-sist of 889bp and 248 parsimony-informativecharacters The data matrix is given inAppendix 1 (available at the website ofthe journal [wwwsystbiolorg]) The maxi-mum likelihood tree is very similar to treesbased on weighted and unweighted parsi-mony Maximum likelihood analyses usedthe GTR C I C 0 model (selected us-ing the model-testing procedure outlinedby Huelsenbeck and Crandall 1997) andthe weighted parsimony analyses used a 51transitiontransversion weighing (based onthe ratio estimated by maximum likelihood)

We applied NCA (Templeton et al 1995)totwo well-sampled haplotype clades revealed


FIGURE 3 Map of northern Mexico and adjacent United States showing geographic location of populations ofSceloporus jarrovii (sensu lato) used in this study See Appendix 3 for explanation of localities Three populationsincluded in the molecular analysis are not included in the morphological analysis because of inadequate samplingof specimens I D S j oberon Coahuila N of El Diamante II D S j minor Nuevo Leon 71 km north of Doctor ArroyoC D S j immucronatus Queretaro near Pinal de Amoles Figure is modied from Wiens et al (1999) Lines indicateinferred range limits of each taxon based on a broader sampling of museum specimens The ldquoNrdquo and ldquoSrdquo indicatethe northern and southern sets of populations of the two subspecies with highly disjunct ranges (S j immucronatusand S j minor) Distribution maps for other included members of the torquatus group are provided by Sites et al(1992) and are summarized as follows S cyanogenys southern Texas eastern Nuevo Leon and western TamaulipasS dugesii southern Nayarit to Guanajuato S insignis narrowly distributed in southwestern montane Mexico inJalisco and Michoacan S lineolateralis just east of the range of S j jarrovii in the state of Durango S mucronatusmontane southern Mexico from southern Hidalgo to western Oaxaca and Guerrero S ornatus north and west of Sj oberon and south of S j cyanostictus in southern Coahuila S poinsettii northern Chihuahuan desert from ArizonaNew Mexico and Texas south to Durango S torquatus widely distributed in central Mexico from Nuevo Leon toPuebla


in the mtDNA trees (B and C in Fig 4) inorder to test for the presence of additionalspecies and to further test the distinctnessof these two lineages from each other Weused the nesting algorithms described byTempleton et al (1987) and Templeton andSing (1993) with modications for DNA se-quence data described by Crandall (1996) todivide the maximum likelihood tree withineach of these lineages into nested clades ofhaplotypes The nesting designs for clades Band C are shown graphically in Appendix 2The number of mutational steps separat-ing clades was estimated using equallyweighted parsimony Permutation tests wereperformed using GeoDis version 20 (Posadaet al 2000)and the causesof signicant asso-ciation of haplotype clades with geographywere evaluated using the inference key givenby Templeton et al (1995)The NCA was con-sidered to potentially support the presenceof distinct species if the inference chain for agiven clade ended in ldquorange fragmentationrdquoor ldquoallopatric fragmentationrdquo (Templeton2001) The status of clades as ldquointeriorrdquo ver-sus ldquotiprdquo was determined using outgrouprooting In the analysis that included bothclades B and C we picked clade B to be inte-rior because it is much more geographicallywidespread than clade C and is thereforemore likely to be ancestral based on coales-cent theory (Crandall and Templeton 1993)

Morphology-Based Phylogeny

Sampling of populationsmdashWe used 21 pop-ulations of S jarrovii as terminal units in ourphylogenetic analysis (see Fig 3 for map andAppendix 3 for localities and specimens ex-amined) Wiens et al (1999) examined 30populations for their molecular phylogeneticanalyses Some of these populations wereseparated by only a few kilometers and werecombined for our analysis to increase samplesizes and a few populations that lacked ade-quate material (ie one or more adult males)were not included In addition to specimensobtained through eldwork by Wiens ANieto T Reeder and R Reyes-Avila mostspecimens of S jarrovii from all major USherpetological collections were examinedduring the course of the study Only pre-sumed adults were included Institutionalabbreviations follow Leviton et al (1985)

Eight other species of the torquatus speciesgroup that were included in the molecularanalysis of Wiens et al (1999) were also in-

cluded A representative of the sister taxonof the torquatus group (S grammicus of thegrammicus group) was included to root thetree (Wiens and Reeder 1997) Apart fromS jarrovii all species were treated as a singleterminal taxon in the phylogenetic analysesbecause these species were generally repre-sented by a single individual in the molec-ular analyses and most are morphologicallydistinct (eg Smith 1939)

Character samplingmdashMorphological char-acters consisted of those used previouslyin diagnosing subspecies of S jarrovii (egSmith 1939) those used in a prior phy-logenetic analysis of Sceloporus (Wiens andReeder 1997) and new characters found dur-ing the course of the study Many of themorphological characters used by Wiens andReeder (1997) showed little or no variationamong populations of S jarrovii or othermembers of the torquatus group and were notincluded

Characters were derived largely fromsquamation coloration and morphometricfeatures Because of limited sample sizes formany populations osteological preparationsand characterswere not used and these char-acters show relatively little variation in thetorquatus group (Wiens and Reeder 1997)Characters were not excluded because of in-traspecic variability or overlap in quanti-tative trait values between populations be-cause both polymorphic (Wiens 1995 1998aWiens and Servedio 1997) and continuoustraits (Thiele 1993 Wiens 2001) have beenshown to contain useful phylogenetic infor-mation A few morphometric and meristiccharacters were excluded because of prob-able correlations with included charactersThe characters used are listed in Appendix 4Terminology for squamation and colorationfeatures follows Smith (1939) and Wiens andReeder (1997)

Character coding and weightingmdashMost ofthe qualitative characters showed extensivevariation within populations Qualitativepolymorphic characters were coded usingthe frequency-based step-matrix approach(Wiens 1995 1999 Berlocher and Swofford1997) Studies using simulations (Wiens andServedio 1997 1998) congruence betweendata sets (Wiens 1998a) and statistical cri-teria (Wiens 1995) suggest that frequency-based methods may be the most accuratemethods for coding polymorphic data (seereview and discussion of controversy byWiens 1999 2000)


FIGURE 4 Molecular phylogeny for populations of Sceloporus jarrovii (sensu lato) and related species of thetorquatus species group based on maximum likelihood analysis (GTR C I C 0 model) for the combined ND4 and12S gene regions (Wiens et al 1999) The lengths of branches are proportional to the maximum likelihood branchlength estimates (the expected number of changes per site the scale bar is roughly equal to 10 substitutions) Num-bers at branches indicate bootstrap values gt50 based on the same maximum likelihood model (100 pseudorepli-cates with TBR-branch swapping) Figure is modied from Wiens et al (1999) Letters indicate the ve putativespecies inferred from tree-based delimitation using DNA data and the ve species we recognize in this studyA D S cyanostictus (S j cyanostictus) B D S minor (S j erythrocyaneus S j immucronatus and southern S j minor)C D S oberon (northern S j minor and S j oberon) D D S sugillatus (S j sugillatus) E D S jarrovii (S j jarrovii andpossibly S lineolateralis) Lowercase letters associated with population numbers indicate individual specimens froma given locality corresponding to those in Table 1 of Wiens et al (1999) with the following exceptions (designationin this paper D locality or specimen designation in Wiens et al 1999) I D 27 II D 18 III D 7 5a D 6a 5b D 6b 5 D 5c6a D 8 6b D 9 7 D 11 8 D 10 9 D 21 10a D 22 10b D 23 11 D 24 12a D 19 12b D 20 13a D 16 13b D 17 14 D 1515 D 14 16a D 25 16b D 26 17 D 12 18 D 13 19 D 28 20 D 29 21 D 30


For a given character each terminal taxonwas given a unique character state in thedata matrix The cost of a transition betweeneach pair of character states was then enteredinto a step matrix and the cost was basedon the Manhattan distance between the fre-quencies of each pair of taxa One charac-ter (dorsal color in life) was coded using thescaled method (Campbell and Frost 1993)implemented using a step matrix (Mabee andHumphries 1993) The scaled method wasused because estimating the frequencies ofthe different conditions was difcult for thischaracter Meristic and morphometric char-acters were coded using step-matrix gap-weighting (Wiens 2001) a modication ofthe gap-weighting method of Thiele (1993)Conversion of trait frequencies and ldquoscoresrdquoto step matrices was performed with a pro-gram written in C by JJW The raw data arepresented in Appendix 5

Three morphometric characters were in-cluded Populations differ considerably inoverall body size and maximum male snout-to-vent length (SVL) was used as a characterMaximum SVL was used rather than meanSVL because of the difculty in determin-ing sexual maturity with certainty and onlymale size was considered to reduce biasescaused by the combination of sexual-size di-morphism and unequal sex ratios in the sam-ple of a given population The other mor-phometric characters were the length of thehindlimb relative to the SVL and the length ofthe head relative to SVL These measures ofshape were obtained by using the residualsof hind limb length and head length re-gressed against SVL and were scored formales only to avoid possible sex-biased dif-ferences in shape within species

We scaled all quantitative characters tohave the same maximum cost as a xedbinary character (between-character scalingWiens 2001) This is a common ldquodefaultrdquo ap-proach and may be appropriate for our studybecause many of the meristic charactershad very large ranges of trait values Largeranges of trait values may be problematic forbetween-state scaling (see Wiens 2001)

Phylogenetic analysismdashPhylogenetic anal-yses of the data matrix (Appendix 6) wereperformed using PAUPcurren (Swofford 1998)Shortest trees were sought using the heuris-tic search option with 100 random-additionsequence replicates Relative support forindividual branches was assessed usingnonparametric bootstrapping (Felsenstein

1985) with 100 bootstrap pseudoreplicatesEach bootstrap pseudoreplicate used aheuristic search with ve random-additionsequence replicates to nd the shortest treefor that matrix Branches with bootstrap val-ues gt70 were considered to be stronglysupported following Hillis and Bull (1993but see their caveats)

Combined Analysis of Morphologicaland Molecular Data

We performed a combined analysis of themtDNA and morphological data for the 21populations analyzed using both data setsAlthough we did not use the combined anal-ysis in species delimitation it may providethe best estimate of species or populationphylogeny We performed two combined-data analyses In the rst the DNA sequencedata were weighted equally (transitions andtransversions were given equal weight)as were the morphological and molecularcharacters such that the maximum cost ofa morphological character-state change wasequivalent to a nucleotide substitution (seematrix in Appendix 7) In the second analysis(Appendix 8) transversions were weightedve times as much as transitions (see Wienset al 1999) and the DNA sequence datawere weighted such that the maximum costof a morphological change was equivalentto that of a transversion (assuming that mor-phological changes areequivalent to therarerclass of molecular changes because manymolecular changes may contribute to a sin-gle morphological change) Before combin-ing the data sets we examined trees fromthe separately analyzed datasets to identifyregions of strongly supported incongruence(following Wiens 1998b)

Character-Based Species DelimitationThe character-based approach was im-

plemented by comparing the frequenciesof qualitative characters and the rangesof trait values for quantitative characters(Appendix 5) across all populations to seekpotentially diagnostic characters Characterswere considered to diagnose one species orset ofpopulations from others if thosecharac-ters were invariant for alternative characterstates or showed no overlap in trait values

We used the methodof Wiens andServedio(2000) to evaluate statistical condence inthe distinctness of sets of populations thatwere delimited using the character-based


approach Specically we used Equation 3of that paper which tests whether samplesizes (for a given set of populations) are largeenough to argue that at least one of the diag-nostic characters is not polymorphic above aselected frequency cutoff For this test weused 10 as the frequency cutoff (ie themaximum frequency of the alternative char-acter state allowed in the diagnostic charac-ter) Thus failing this test means that thereis a gt5 probability that all of the diagnosticcharacters for a given taxon are actually poly-morphic with the unobserved characterstateoccurring at a frequency of 10 or greaterWe treated overlap in trait values betweenpopulations as equivalent to polymorphismin qualitative traits

RESULTS

DNA Haplotype Phylogeny andNested-Clade Analyses

Phylogenetic analysis of the mtDNA se-quence data using parsimony and likelihoodshows that the haplotypes of S jarrovii are

FIGURE 5 Graphical summary of results of NCA for haplotype clade B (Fig 4) which shows restricted geneow caused by isolation-by-distance Nesting levels are indicated from top to bottom starting with the individualhaplotypes (0-step clades) at the top The numbering of clades accounts for empty clades (clades containing inferredancestral haplotypes but no observed haplotypes) which are not shown and the complete nesting design is givenin Appendix 2 For clades that show a signicant (P lt005) probability of association between geography andclade distribution based on an exact contingency test (Templeton et al 1995) the clade distance (Dc) and thenested clade (Dn) distances are given Clades that contain two or more clades and two or more localities but donot show a signicant association are asterisked Distances signicantly smaller than expected are indicated with asuperscripted S and distances signicantly larger than expected are indicated with a superscripted L (given a nullexpectation of randomgeographical distribution of haplotypes) Distances for a few clades (5-1 6-2) that do not showoverall signicant association are included for interpretation of results from higher-level clades Results contrastinginterior versus tip clades are indicated by I-T and the numbers of interior clades are boldfaced and italicized Theinference chain (IC) from the key provided by Templeton et al (1995) is used to interpret the statistical results

not exclusive and that they instead interdig-itate among the other species of the torquatusgroup (Fig 4) These haplotypes are parti-tioned among ve lineages none of whichare sister taxa Each of these ve lineagesis allopatric and concordant with geography(Fig 3) Application of our protocol to tree-based delimitation using DNA data suggeststhat the focal species is nonexclusiveand con-sists of ve basal lineages with no gene owamong them (Fig 1d) and supports the di-vision of S jarrovii into ve species as sug-gested by Wiens et al (1999)

Application of NCA to the entire ingroupwould be impractical because extensive geo-graphic sampling was undertaken only forS jarrovii and not the other eight speciesof the torquatus group that were included(and which interdigitate among populationsof S jarrovii) We applied NCA within thetwo lineages of S jarrovii that were sam-pled most extensively (B and C in Fig 4)Our results (Figs 5 6a) suggest that thereis a signicant association between haplo-type clades and their geographical locations


FIGURE 6 Graphical summary of results of NCA for (a) haplotype clade C (Fig 4) which shows restricted geneow caused by isolation-by-distance and (b) a clade including clades B and C which shows range fragmentation(speciation) See Figure 5 for explanation and Appendix 2 for nesting design

within each of these clades at the highestclade levels However within each of thesetwo clades restricted gene ow appears to bethe result of isolation-by-distance within therange of a single lineage rather than rangefragmentationby speciation When these twolineages are analyzed together (combinedanalysis of the molecular and morphologicalsuggests they might be sister taxa Fig 8a)NCA indicates that the geographical asso-ciation of haplotypes within each lineage iscaused by range fragmentation supportingthe hypothesis that these are two distinctspecies (Fig 6b)

Combined application of our approachwith NCA supports division of S jarroviiinto ve lineages and suggests that fur-ther division of these ve lineages into ad-ditional species is currently unwarrantedOne of these ve lineages contains haplo-types from both S j jarrovii and an indi-vidual of S lineolateralismdasha taxon that someconsider to be morphologically indistin-guishable from S j jarrovii (Sites et al 1992)Two likely possibilities are thatS lineolateralisis conspecic with S j jarrovii or S j jarroviiis a nonexclusive species relative to a distinctS lineolateralis Although our sampling wastoo limited to resolve the separation of S jjarrovii and S lineolateralis it is clear that S jjarrovii is not conspecic with other popula-tions of S jarrovii

Sceloporus j cyanostictus is known onlyfromtwopopulations thatarewell-separatedgeographically Two individuals were se-quenced from each of these populations andthe haplotypes of each are exclusive and rela-

tively divergent Although these two popula-tions might represent distinct species furthersampling from these localities and geo-graphically intermediate localities is neededto distinguish speciation from isolation-by-distance Sceloporus j sugillatus was sampledfrom the type localityand isknown only fromthere and a nearby locality Because of theextreme geographic proximity of these pop-ulations they most likely are members of asingle species

Sceloporus j oberon and the northern pop-ulations of S j minor form a clade (clade C)that is weakly supported as the sister taxonof S ornatus The populations of S j oberonand northern S j minor are not mutually ex-clusive and the haplotype phylogeny sug-gests geographically widespread gene owamong these populations (eg population10 appears in two disparate locations on thehaplotype tree Fig 4) It is not possible todelimit any basal exclusive lineages withinthis clade and NCA does not support fur-ther division of this lineage into additionalspecies

The most extensively sampled clade ofS jarrovii haplotypes (B) contains S jerythrocyaneus S j immucronatus and thesouthern populations of S j minor Noneof these three taxa is exclusive in the hap-lotype tree and the disparate placementsof the haplotypes from near Doctor ArroyoNuevo Leon (population II Fig 3) suggestgeographically widespread gene ow in thislineage (Fig 4) Although there is a well-supported basal split within this clade be-tween the two haplotypes from Concepcion


FIGURE 7 Population-level phylogeny of Sceloporusjarrovii and related species of the torquatus group basedon parsimony analysis of morphological data Branchlengths are drawn proportional to estimated characterchange and numbers at branches indicate bootstrap val-ues gt50 Letters indicate the ve species suggested bytree-based delimitation A D S j cyanostictus B D S joberon C D S j erythrocyaneus S j immucronatus and Sj ldquominorrdquo D D S j jarrovii E D S j sugillatus

del Oro (population 18) and all other popula-tions NCA does not support recognition ofadditional species within this lineage

Tree-Based Morphology

A total of 44 characters were scored (43parsimony-informative Appendix 4) con-sisting of 24 scale characters (11 qualitativeand polymorphic 13 meristic) 17 colorationcharacters (15 qualitative and polymorphic1 qualitative and xed and 1 meristic) and3 morphometric characters (Appendix 5)

Phylogenetic analysis of the morphologi-cal data matrix (Appendix 6) yielded a sin-gle shortest tree with a length of 171404a consistency index of 0268 (excluding un-informative characters) and a retention in-dex of 0400 Like the mtDNA phylogenythe morphology-based phylogeny showsS jarrovii to be nonexclusive with popu-lations of S jarrovii interdigitating amongother species of the torquatus group Inthe morphological phylogeny the popula-tions of S jarrovii form eight basal clades

(Fig 7) none of which are sister taxa Inother words eight lineages would have tobe elevated to distinct species to avoid pa-raphyly of S jarrovii However only twoof these basal clades are well-supported(S j jarrovii and S j cyanostictus) and manyof the weakly supported clades areobviously

FIGURE 8 Population-level phylogeny of Sceloporusjarrovii and related species of the torquatus group basedon parsimony analysis of combined molecular and mor-phological data Branch lengths are drawn proportionalto estimated character change and numbers at branchesindicate bootstrap values gt50 (a) Unweighted anal-ysis with all DNA substitutions weighted equally toeach other and to maximum cost of each morphologicalcharacter change (b) Weighted analysis with transver-sions weighted ve times as much as transitions andweighted equal to the maximum cost of each morpho-logical character change Clades AndashE are essentially thesame as in Figure 4


discordant with the geographic proximity ofpopulations For example the populationsof southern S j minor fail to cluster to-gether (ie population 14 clusters withS cyanogenys instead of with populations 13and 15ndash18) as do the populations of north-ern S j minor (population 9 clusters withS torquatus and S j cyanostictus insteadof with populations 10ndash12) The two pop-ulations of S j oberon also form a stronglysupported clade that is concordant with ge-ography (suggesting that S j oberon also rep-resents a distinct species) although this cladeseems to emerge from among the northernpopulations of S j minor

Using our criteria the morphological re-sults suggest that S j minor S j immu-cronatus and S j erythrocyaneus form a sin-gle nonexclusive species (clade C Fig 7)from which arises the seemingly distinctspecies S torquatus S cyanogenys S j oberonand S j cyanostictus The supporting evi-dence is the nonexclusive nature of thesepopulations on adjacent branches of the phy-logeny the weakly supported relationshipsamong them and the discordance of manyof these clades with geography (Fig 2d)Our criteria also support S j jarrovii S jsugillatus and S j cyanostictus as distinctspecies because each isphylogenetically sep-aratedfromother S jarroviipopulationseachis strongly supported as exclusive and eachis concordant with geography (although theinclusion of S j sugillatus is based on a sin-gle population) In summary application ofour protocol for tree-based delimitation us-ing morphology suggests that S jarrovii rep-resents four exclusive species and one nonex-clusive species

Combined Data AnalysisPhylogenetic analysis of the combined

data using the two weighting schemes (ma-trices in Appendices 7 and 8) yields phylo-genies that are similar to the tree based onthe molecular data alone (Fig 8) as predictedabove Nevertheless there is a strongly sup-ported conict between the morphologicaland DNA data that is apparent from exam-ining bootstrap values on the separately an-alyzed trees involving the exclusivity of S joberon (supported by the morphologicaldatarejected by the mtDNA data)The combined-data trees although not fully congruentbetween weighting schemes support theexclusivity of the same ve clades based on

the mtDNA dataalone However the equallyweighted analysis shows weak support forplacing the two clades that contain S j minorpopulations as sister taxa a result not seenin separate analyses of either the molecularor morphological data The combined-datatrees were not used in species delimitation

Character-Based MorphologyThe character-based approach using mor-

phological data supports recognition ofve sets of populations as distinct species(1) S j sugillatus (2) S j cyanostictus (3) S jimmucronatus C S j erythrocyaneus (4) pop-ulation 18 of S j minor (Concepcion delOro) and (5) all the remaining populations ofS jarrovii including S j jarrovii S j minorand S j oberon However three of theseve (2ndash4) are consistently diagnosed by onlya single character (male dorsal coloration)which can be difcult to score and the lastspecies is simply an amalgam of the popula-tions that lack the diagnostic characters seenin the other species Sceloporus j sugillatushave unique dark transverse stripes on theanks of adult males and a wider blackcollar than other populations of S jarrovii(6ndash85 scales in S j sugillatus vs 2ndash55 inother S jarrovii populations) Furthermorethe large number of scales around the fore-limb in S j sugillatus (14ndash18) shows onlyminimal overlap with other populations ofS jarrovii Sceloporus j cyanostictus has aunique dorsal coloration in adult males thatranges from green to blue-green Sceloporusj erythrocyaneus and S j immucronatusare diagnosed by blue dorsal coloration Thepopulation of S j minor from Concepciondel Oro (population 18) has a distinct yellowdorsal coloration but this was scored fromonly a few individuals and may not be diag-nostic for the population as a whole

Applying the test of Wiens and Servedio(2000 their Equation 3) shows that samplesizes are insufcient to argue that any of theseve sets of populations are statistically dis-tinct given a frequency cutoff of 10 and aP-value of 005 This test was not applied tothe composite species (S j jarrovii S j minorS j oberon) because the other species had al-ready failed

Contrary to our predictions S j jarroviiand S j oberon have strong bootstrapsupportin the morphology-based phylogeny but lackseemingly xed diagnostic characters There


are two variable features that distinguishS j jarrovii from mostother S jarroviipopula-tions First S j jarrovii usually have three ormore interpostanals (one of the six individu-als from Arizona had only two) versus two orfewer in the other populations (except for thesingle male S j minor from Saldana) Secondthe reduced blue gular blotch distinguishesS j jarrovii from most populations althoughthis was observed at low frequencies in S jsugillatus and S j minor from population 18The clade of S j oberon populations issupported by several polymorphic charac-ters including the distinctive black dorsalcoloration The lack of diagnostic charactersmay be related to hybridization between thistaxon and geographically adjacent popu-lations of S j minor

Summary of Concordance Conictand Taxonomic Conclusions

Given that these three approaches are notfully congruent (Table 1) what are the specieslimits in S jarrovii Sceloporus j cyanostictusand S j sugillatus are distinct species usingall three approaches The morphology- andmtDNA-based phylogenies agree that S jjarrovii is not closely related to other S jarroviipopulations the only question is whetherS lineolateralis is inside or outside the cladeof S j jarrovii populations Furthermore al-though S j jarrovii lacks seemingly xeddiagnostic morphological characters somemorphological characters do strongly differ-entiate these populations even though thesecharacters exhibit some intraspecic varia-tion in S j jarrovii and other populations

There is conict over the relationships ofS j oberon populations with the morpholog-ical tree strongly suggesting exclusivity andthe mtDNA tree strongly suggesting nonex-clusivity with respect to S j minor The ob-

TABLE 1 Comparison of the results of three different approaches to species delimitation applied to mtDNAand morphological variation for populations of Sceloporus jarrovii Listed is each taxon that is considered a distinctspecies by each approach as listed

Tree-based DNA Tree-based morphology Character-based morphology

S j cyanostictus S j cyanostictus S j cyanostictusS j sugillatus S j sugillatus S j sugillatusS j jarrovii (C S lineolateralis) S j jarrovii S j jarrovii C S j minor C S j oberonS j oberon C northern S j minor S j oberon population 18 of S jminor

(Concepcion del Oro)S j erythrocyaneus C S j S j erythrocyaneus C S j erythrocyaneus C S j immucronatus

immucronatus C southern S j immucronatus C allS j minor S j minor

vious explanation for this conict seems tobe lateral transfer of mitochondrial genes be-tween S j oberon and adjacent S j minorthese two taxa share a contact zone wherethere aremorphologically intermediate spec-imens and mixing of mtDNA haplotypes(Wiens et al 1999) Thus we consider S joberon to be conspecic with northern S jminor

The character-based approach to mor-phology provides weak evidence that S jimmucronatus andS j erythrocyaneus togetherrepresent a single species that is distinct fromother S jarroviipopulations based on the bluedorsal coloration In contrast the mtDNAdata place these two subspecies among thesouthern populations of S j minor and sug-gest that the blue immucronatus morph hasevolved independently in Tamaulipas andin a geographically distant region in Quere-taro and Hidalgo The morphology-basedphylogeny can be interpreted as favoring ei-ther the distinctness of these populations (be-cause they cluster together) or their conspeci-city with S j minor (because the clade isweakly supported and interspersed amonggeographically disparate populations of S jminor) Because of its stronger statisticalsupport greater concordance with the geo-graphic proximity of populations and pos-sible support from the morphology-basedphylogeny we favor the interpretation thatS j erythrocyaneus S j immucronatus andsouthern S j minor are conspecic Themorphology-based phylogeny also suggeststhat S j minor represents a single nonex-clusive species (including S j immucronatusand S j erythrocyaneus) In contrast themtDNA tree and NCA show strong supportfor division of S j minor into two clades anorthern one (including S j oberon) and asouthern one (including S j immucronatusand S j erythrocyaneus) This division is


not supported by any diagnostic morpho-logical characters but some of the cladesof the morphology tree are concordantmdashnamely a clade including mostnorthernpop-ulations of S j minor plus S j oberon anda clade containing most southern popula-tions of S j minor We hypothesize that Sj minor does represent two species (as sug-gested by the mtDNA tree) and that these twospecies have not yet become mutually exclu-sive in the morphology-based population-level phylogeny

In summary based on the data and argu-ments presented above we support divisionof S jarrovii into ve species S cyanostictus(for S j cyanostictus) S jarrovii (for S jjarrovii which may also include S lineolater-alis) S minor (for S j erythrocyaneus S j im-mucronatus and southern populations of S jminor) S oberon (for S j oberon and northernpopulations of S j minor) and S sugillatus(for S j sugillatus) These are the samespecies recognized by Wiens et al (1999) Foreach species the oldest available name waschosen andbecause of the absence of sympa-try among these species and the collection ofmaterial from at or close to the type localitiesthere is no question that the type specimenpertains to the appropriate species (typespecimens examined by Wiens for S cyanos-tictus S minor S oberon and S sugillatus)

DISCUSSION

In this paper we outline explicit protocolsfor species delimitation using DNA and mor-phological data and provide the rst criti-cal comparison of species delimitation basedon mtDNA haplotype phylogenies with tree-based and character-based species delim-itation based on morphology We foundthe results to be surprisingly discordant(Table 1) Although all three approaches sup-port recognition of ve species only two ofthese species are the same between the threeThis discordanceis importantbecause it sug-gests that at least two of these approaches aregiving a misleading picture of species bound-aries in this group

Our study differs from many previousanalyses in showing real discordance be-tween species limits suggested by DNA andmorphology and not merely disagreementwith traditional taxonomy Previous stud-ies have shown incongruence between thespecies limits suggested by mtDNA andmorphology-based taxonomy(eg Zamudio

et al 1997 Steppan 1998 Serb et al 2001)Specically those authors found mtDNA ev-idence suggesting that one or more sub-species should be recognized as distinctspecies However on closer inspection thesedistinct subspecies were found to be di-agnosed by one or more morphologicalcharacters (using data from the literature)thus demonstrating concordance betweenthe mtDNA phylogenies and the character-based approach to morphology One previ-ous study (Hollingsworth 1998) analyzedmorphological variation using both tree-based and character-based approaches for agroup (the lizard genus Sauromalus) in whichthere was also an mtDNA haplotype phy-logeny (Petren and Case 1997) althoughthere was no explicit comparison of the threeapproaches Despite some incongruenceover interspecic relationships the three ap-proaches can be interpreted as being highlyconcordant in terms of the species limits theysuggest (our interpretation of species limitsin Sauromalus matches that of Hollingsworth1998) The results of these previous studiescontrast with those of our study of Scelo-porus jarrovii in which there is real conictbetween species limits inferred by the threeapproaches

An obvious explanation for discordancebetween morphological and mtDNA specieslimits is failure of the mtDNA gene tree tomatch the species tree This may explainone case of small-scale incongruence in ourstudymdashnamely the strongly supported con-ict between DNA and morphological dataover the exclusivity of S j oberon How-ever two mtDNA clades that have little sup-port from morphology (northern and south-ern S j minor and their relatives) are eachhighly concordant with geography whereasthe species boundaries implied for thesepopulations by the two morphological ap-proaches are not concordant with each otherand those implied by the character-basedapproach show some discordance with thegeographical proximity of the populationsWe would not expect lateral transfer of genesbetween taxa or stochastic retention of an-cestral polymorphisms to generate large-scale patterns that are concordant with ge-ography Thus mismatch between gene andspecies trees seems to explain relatively lit-tle of the discordance in our study Recentstudies have also shown little evidence tosuggest that this phenomenon is generally


problematic in species delimitation withmtDNA data (eg Zamudio et al 1997Steppan 1998 Parkinson et al 2000 Serbet al 2001)

We propose that much of the discordanceinvolving S jarrovii is caused by a partic-ular pattern of morphological variation inthe group in which between-species differ-entiation is small relative to within-speciesvariation in some taxa Interspecic cladesare generally weakly supported by morphol-ogy in the torquatus group (Fig 7) Boot-strap values for interspecic clades are gen-erally low as was found in a previousmore comprehensive analysis of Sceloporusphylogeny (Wiens and Reeder 1997) Thenumber of parsimony-informative charac-ters within the group is also relatively smalland most of these characters show extensivevariation within populations and within pu-tative species Some of the species also showstriking within-species divergence For ex-ample two mtDNA clades were recognizedas species that lacked diagnostic morpho-logical characters and were not supportedas clades by morphological data These twoputative species (S minor and S oberon)show remarkable within-species divergencein dorsal coloration possibly driven by sex-ual selection or an interactionbetween sexualselection and habitat features (Wiens et al1999) Thus instead of showing strong dif-ferentiation in morphology between speciesand weak differentiation within speciessome species in the torquatus group showstriking differentiation within species andlimited differentiation between species aldquoworst-casescenariordquo for morphology-basedspecies delimitation This hypothesis is alsosupported by qualitative inspection of thebranch lengths in the morphology-basedphylogeny (Fig 7) which suggests thatintraspecic branch lengths are similar tointerspecic branch lengths (although thisobviously depends on which branches areconsidered to be intraspecic versus inter-specic) Whether the specic pattern seenin S jarrovii is common remains unclearbut we suggest there may be many cases inwhich species have split too rapidly to al-low time for many diagnostic morpholog-ical differences to evolve Haplotype phy-logenies from mtDNA may be particularlyuseful in these cases because of the rel-atively fast rate at which species becomedifferentiated

An alternative view of the discordancebetween the results from morphology andmtDNA is that morphological species de-limitation in the group has been biased orcompromised by small sample sizes (forsome populations) or problematic phyloge-netic methods Although small sample sizescan reduce the accuracyof phylogenetic anal-ysis when intraspecic variation is exten-sive (eg Wiens 1998a Wiens and Servedio1997 1998) small sample sizes should in-crease the probability of nding diagnos-tic characters that appear to be xed Yetonly two of the ve species that we con-sider distinct have any ldquoxedrdquo diagnosticcharacters The method that we used for cod-ing polymorphic data (frequency-based cod-ing) has been found to perform well rela-tive to other methods (Wiens 1999)Methodsfor coding quantitative data have not beentested thoroughly Both methods use ne-grained continuous data and should extractthe maximum information possible from themorphological data Furthermore althoughthe morphological phylogeny was generallyweakly supported and somewhat discordantwith the mtDNA tree the topology was farfrom random The morphology-based phy-logeny supported the exclusivity of six ofthe seven subspecies of S jarrovii whereasthe mtDNA tree supported the exclusivityof only two We suggest that our analysisaccurately summarized morphological vari-ation in the group but that morphologi-cal and molecular evolution have not beenconcordant

We also found surprising discordance be-tween the tree-based andcharacter-basedap-proaches for delimiting species using mor-phology with only two of ve species agreedon by both approaches (Table 1) As far aswe know this is the rst time such incon-gruence has been shown for morphologicaldata (but see Brower [1999] for an examplewith molecular data) This discordance mayalso be related to the problematic nature ofmorphological variation within the torquatusgroup The character-basedapproachto mor-phological data has been widely used todelimit and describe species for the past200 years or more whereas the tree-basedapproach has been argued for on theoreti-cal grounds (Baum and Donoghue 1995) buthas rarely been applied to morphology Bothapproaches are somewhat problematic Thecharacter-based approach is questionable


without some statistical test (otherwise it ispossible to describe a species based on a sin-gle individual with a single diagnostic char-acter) The only such test currently available(Wiens and Servedio 2000) requires settinga frequency cutoff and requires large sam-ple sizes to achieve statistical signicance Ageneral problem of the character-based ap-proach is that it ignores all but a few of thecharacters that differ between species usingonly those that show no within-species vari-ation On the other hand the tree-based ap-proach can potentially use all characters ifmethods that incorporate polymorphic andquantitative characters are employed In ourstudy the tree-based approach appears togive results that are marginally more con-gruent with the mtDNA results than doesthe character-basedapproach(Table 1) How-ever the theory behind using population-level trees from morphology to infer specieslimits remains poorly explored Despite thelong history of morphology-based taxon-omy the best way to delimit species usingmorphological data remains an open andlargely unexplored question and one thatcritically impacts the issue of congruence be-tween species limits from morphology andDNA data

Appendices for this paper are avail-able at the website of the journal (wwwsystbiolorg)

ACKNOWLEDGMENTS

For valuable comments on the manuscript wethank P Chippindale D Frost R Espinoza B LivezeyC Parkinson T Reeder J Serb M Servedio C SimonJ Sites C Smith T Trepanier and K Zamudio Wiensthanks R Reyes Avila T Reeder and A Nieto Montesde Oca for assistance in the eld and J Fetzner andD Posada for help with NCASupport for eldwork wasprovided by grants from the Netting and OrsquoNeill fundsof the Carnegie Museum of Natural History to J J WWe thank the following individuals and institutions forloan of specimens D Frost and C Cole (AMNH Amer-ican Museum of Natural History) W Duellman andJ Simmons (KU University of Kansas Museum of Natu-ral History) H Voris and A Resetar (FMNH Field Mu-seum of Natural History) R Bezy (LACM Los AngelesCounty Museum) J Rosado (MCZ Museum of Com-parative Zoology Harvard University) H Greene andB Stein (MVZ Museum of Vertebrate Zoology Univer-sity of California Berkeley) A Nieto (MZFC Museo deZoologia Universidad Nacional Autonoma de Mexico)D Cannatella (TNHC Texas Natural History Collec-tion University of Texas Austin) A de Queiroz and RHumphrey (UCM University of Colorado Museum) AKluge and G Schneider (UMMZ University of Michi-gan Museum of Zoology) J Campbell and C Stewart

(UTA University of Texas at Arlington) and especiallyE A Liner (EAL)

REFERENCES

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AVISE J C 2000 Phylogeography The history and for-mation of species Harvard Univ Press CambridgeMassachusetts

AVISE J C AND R M BALL 1990 Principles of ge-nealogical concordance in species concepts and bio-logical taxonomy Oxford Surv Evol Biol 745ndash67

BAUM D A AND M J DONOGHUE 1995 Choosingamong alternative ldquophylogeneticrdquo species conceptsSyst Bot 20560ndash573

BAUM D A AND K L SHAW 1995 Genealogical per-spectives on the species problem Pages 289ndash303 inExperimental and molecular approaches to plantbiosystematics (P C Hoch and A G Stephensoneds) Missouri Botanical Garden St Louis

BERLOCHER S H AND D L SWOFFORD 1997 Search-ing for phylogenetic trees under the frequency parsi-mony criterion An approximation using generalizedparsimony Syst Biol 46211ndash215

BROWER A V Z 1999 Delimitation of phylogeneticspecies with DNA sequences A critique of Davis andNixonrsquos population aggregation analysis Syst Biol48199ndash213

CAMPBELL J A AND D R FROST 1993 Anguid lizardsof the genus Abronia Revisionary notes descriptionof four new species a phylogenetic analysis and keyBull Am Mus Nat Hist 2161ndash121

CRACRAFT J 1983Species concepts andspeciation anal-ysis Curr Ornithol 1159ndash187

CRANDALL K A 1996 Multiple interspecies transmis-sions of human and simian T-cell leukemialymphoma virus type I sequences Mol Biol Evol134115ndash131

CRANDALL K A AND A R TEMPLETON 1993 Empiri-cal tests of some predictions from coalescent theorywith applications to intraspecic phylogeny recon-struction Genetics 13959ndash969

DAVIS J I AND K C NIXON 1992 Populations ge-netic variation and the delimitation of phylogeneticspecies Syst Biol 41421ndash435

DE QUEIROZ K 1998 The general lineage concept ofspecies species criteria and the process of speciationA conceptual unication and terminological recom-mendations Pages 57ndash75 in Endless forms Speciesandspeciation (D J HowardandSHBerlocher eds)Oxford Univ Press Oxford England

DE QUEIROZ K AND M J DONOGHUE 1988 Phyloge-netic systematics and the species problem Cladistics4317ndash338

DE QUEIROZ K AND M J DONOGHUE 1990 Phylo-genetic systematics or Nelsonrsquos version of cladisticsCladistics 661ndash75

DOBZHANSKY T 1950 Mendelian populations and theirevolution Am Nat 84401ndash418

FELSENSTEIN J 1985 Condence limits on phylogeniesAn approach using the bootstrap Evolution 39783ndash791

FROST D R AND A G KLUGE 1994 A considerationof epistemology in systematic biology with specialreference to species Cladistics 10259ndash294

FROST D R H M CRAFTS L A FITZGERALD ANDT A TITUS 1998 Geographic variation species


recognition and molecular evolution of cytochromeoxidase I in the Tropidurus spinulosus complex (Igua-nia Tropiduridae) Copeia 1998839ndash851

FUNK D J D J FUTUYMA G ORTI AND A MEYER1995 A history of host associations and evolutionarydiversication for Ophraella (Coleoptera Chrysomel-idae) New evidence from mitochondrial DNAEvolution 491008ndash1017

GRAYBEAL A 1995 Naming species Syst Biol 44237ndash250

HEDIN M 1997 Speciational history in a diverseclade of habitat specialized spiders (Araneae Nes-ticidae Nesticus) Inferences from geographic-basedsampling Evolution 511929ndash1945

HILLIS D M AND J J BULL 1993 An empirical test ofbootstrapping as a method for assessing condencein phylogenetic analysis Syst Biol 42182ndash192

HOELZER G A 1997 Inferring phylogenies frommtDNA variation Mitochondrial-gene trees versusnuclear-gene trees revisited Evolution 51622ndash626

HOLLINGSWORTH B D 1998 The systematics of chuck-wallas (Sauromalus) with a phylogenetic analysis ofother iguanid lizards Herpetol Monogr 1238ndash191

HUELSENBECK J P AND K A CRANDALL 1997 Phy-logeny estimation and hypothesis testing using max-imum likelihood Annu Rav Ecol Syst 28437ndash466

JACKMAN T R AND D B WAKE 1994 Evolution-ary and historical analysis of protein variation inthe blotched forms of salamanders of the Ensatinacomplex Evolution 48876ndash897

KIMURA M 1983 The neutral theory of molecular evo-lution Cambridge Univ Press Cambridge England

LEVITON A E R H G IBBS E HEAL ANDC E DAWSON 1985 Standards in herpetology andichthyology Part 1 Standard symbolic codes for in-stitutional resource collections in herpetology andichthyology Copeia 1985802ndash832

MABEE P M AND J HUMPHRIES 1993 Coding poly-morphic data Examples from allozymes and on-togeny Syst Biol 42166ndash181

MADDISON W P 1997 Gene trees in species trees SystBiol 46523ndash536

MALLET J 1995 A species denition for the ModernSynthesis Trends Ecol Evol 10294ndash299

MAYR E 1942 Systematics and the origin of speciesColumbia Univ Press New York

MOORE W S 1995 Inferring phylogenies from mtDNAvariation Mitochondrial gene trees versus nuclear-gene trees Evolution 49718ndash726

MOORE W S 1997 Mitochondrial gene trees versusnuclear-gene trees A reply to Hoelzer Evolution49627ndash629

MORITZ C 1994 Application of mtDNA in conserva-tion A critical review Mol Ecol 3401ndash411

MORITZ C C J SCHNEIDER AND D B WAKE 1992Evolutionary relationships within the Ensatinaescholtzii complex conrm the ring species interpreta-tion Syst Biol 41273ndash291

NEIGEL J E AND J C AVISE 1986 Phylogenetic rela-tionships of mitochondrial DNA under various de-mographic models of speciation Pages 515ndash534 inEvolutionary processes and theory (E Nevo andS Karlin eds) Academic Press New York

OLMS TEAD R G 1995 Species concepts and plesiomor-phic species Syst Bot 20623ndash630

PAETKAU D 1999 Using genetics to identify intraspe-cic conservation units A critique of current methodsCons Biol 131507ndash1509

PAMILO P AND M NEI 1988 Relationships betweengene trees and species trees Mol Biol Evol 5568ndash583

PARKINSON C L K R ZAMUDIO AND H WGREENE 2000 Phylogeography of the pitviperclade Agkistrodon Historical ecology species sta-tus and conservation of cantils Mol Ecol 9411ndash420

PETREN K AND T J CASE 1997 A phylogenetic analy-sis of body size evolution and biogeography in chuck-wallas (Sauromalus) and other iguanines Evolution51206ndash219

POSADA D K A CRANDALL AND A R TEMPLETON2000 GeoDis A program for the cladistic nested anal-ysis of the geographical distribution of genetic haplo-types Mol Ecol 9487ndash488

PUORTO G M DA GRACA SALOMAO R D GTHEAKS TON R S THORPE D A WARRELL ANDW WUSTER 2001 Combining mitochondrial DNAsequences and morphological data to infer speciesboundaries Phylogeography of lanceheadedpitvipers in the Brazilian Atlantic forest andthe status of Bothrops pradoi (Squamata SerpentesViperidae) J Evol Biol 14527ndash538

RODRacuteotilde GUEZ-ROBLES J A AND J M DE JESUS-ESCOBAR2000 Molecular systematics of New World gopherbull and pinesnakes (Pituophis Colubridae) atranscontinental species complex Mol PhylogenetEvol 1435ndash50

SERB J M C A PHILLIPS AND J B IVERSON2001 Molecular phylogeny and biogeography ofKinosternon avescens based on complete mitochon-drial control region sequences Mol Phylogenet Evol18149ndash162

SHAFFER H B AND M L MCKNIG HT 1996 Thepolytypic species revisited Genetic differentiationand molecular phylogenetics of the tiger salamanderAmbystoma tigrinum (Amphibia Caudata) complexEvolution 50417ndash433

S IMPSON G G 1961 Principles of animal taxonomyColumbia Univ Press New York

S ITES J W JR J W ARCHIE C J COLE AND O FVILLELA 1992 A review of phylogenetic hypothe-ses for the lizard genus Sceloporus (Phrynosomatidae)Implications for ecological and evolutionary studiesBull Am Mus Nat Hist 2131ndash110

S ITES J W JR AND K A CRANDALL 1997 Testingspecies boundaries in biodiversity studies Cons Biol111289ndash1297

SLATKIN M AND W P MADDISON 1989 A cladisticmeasure of gene ow inferred from the phylogeniesof alleles Genetics 123603ndash613

SMITH H M 1939 The Mexican and Central Americanlizards of the genus Sceloporus Field Mus Nat HistZool Ser 261ndash397

SPERLING F A H AND R G HARRISON 1994 Mito-chondrial DNA variation within and between speciesof the Papilio machaon group of swallowtail butteriesEvolution 48408ndash422

STEPPAN S J 1998 Phylogenetic relationships andspecies limits within Phyllotis (Rodentia Sigmodon-tinae) Concordance between mtDNA sequence andmorphology J Mammal 79573ndash593

SULLIVAN J J A MARKERT AND C W KILPATRICK1997 Phylogeography and molecular systematics ofthe Peromyscus aztecus species group (Rodentia Muri-dae) inferred using parsimony and likelihood SystBiol 46426ndash440


SWOFFORD D L 1998PAUPcurren Phylogenetic analysis us-ing parsimonycurren version 400d63Sinauer AssociatesSunderland Massachusetts

TALBOT S L AND G F SHIELDS 1996Phylogeographyof brown bears (Ursus arctos) of Alaska and paraphylywithin the Ursidae Mol Phylogenet Evol 5477ndash494

TEMPLETO N A R 1989 The meaning of species and spe-ciation A genetic perspective Pages 3ndash27 in Specia-tion and its consequences (D Otte and J Endler eds)Sinauer Associates Sunderland Massachusetts

TEMPLETO N A R 1994 The role of molecular genet-ics in speciation studies Pages 455ndash477 in Molecularecology and evolution Approaches and applications(B Schierwater B Streit G P Wagner and R DeSalleeds) Birkhauser-Verlag Basel Switzerland

TEMPLETO N A R 2001 Using phylogeographic analy-ses of gene trees to test species status and processesMol Ecol 10779ndash791

TEMPLETO N A R E BOERWINKLE AND C F S ING 1987 A cladistic analysis of phenotypic associationswith haplotypes inferred from restriction endonucle-ase mapping I Basic theory and an analysis of al-cohol dehydrogenase activity in Drosophila Genetics117343ndash351

TEMPLETO N A R E ROUTMAN AND C A PHILLIPS 1995 Separating population structure from popula-tion history A cladistic analysis of the geographi-cal distribution of mitochondrial DNA haplotypes inthe tiger salamander Ambystoma tigrinum Genetics140767ndash782

TEMPLETO N A R AND C F S ING 1993 A cladisticanalysis of phenotypic associations with haplotypesinferred from restriction endonuclease mapping IVNested analyses with cladogram uncertainty and re-combination Genetics 134659ndash669

THIELE K 1993 The holy grail of the perfect characterThe cladistic treatment of morphometric data Cladis-tics 9275ndash304

WAKE D B 1997 Incipient species formation in sala-manders of the Ensatina complex Proc Natl AcadSci USA 947761ndash7767

WAKE D B AND C J SCHNEIDER 1998 Taxonomyof the plethodontid salamander genus EnsatinaHerpetologica 54279ndash298

WIENS J J 1995 Polymorphic characters in phyloge-netic systematics Syst Biol 44482ndash500

WIENS J J 1998a Testing phylogenetic methods withtree-congruence Phylogenetic analysis of polymor-phic morphological characters in phrynosomatidlizards Syst Biol 47 427ndash444

WIENS J J 1998b Combining data sets with differentphylogenetic histories Syst Biol 47568ndash581

WIENS J J 1999Polymorphism in systematics and com-parative biology Annu Rev Ecol Syst 30327ndash362

WIENS J J 2000 Coding morphological variation forphylogenetic analysis Polymorphism and interspe-cic variation in higher taxa Pages 115ndash145 in Phy-logenetic analysis of morphological data (J J Wiensed) Smithsonian Institution Press Washington DC

WIENS J J 2001 Character analysis in morphologi-cal phylogenetics Problems and solutions Syst Biol50688ndash699

WIENS J J AND T W REEDER 1997 Phylogeny ofthe spiny lizards (Sceloporus) based on molecular andmorphological evidence Herpetol Monogr 111ndash101

WIENS J J T W REEDER AND A NIETO 1999 Molec-ular phylogenetics and evolution of sexual dichroma-tism among populations of the Yarrowrsquos spiny lizard(Sceloporus jarrovii) Evolution 531884ndash1897

WIENS J J AND M R SERVEDIO 1997 Accuracy ofphylogenetic analysis including and excluding poly-morphic characters Syst Biol 46332ndash345

WIENS J J AND M R SERVEDIO 1998 Phylogeneticanalysis and intraspecic variation Performance ofparsimony likelihood and distance methods SystBiol 47228ndash253

WIENS J J AND M R SERVEDIO 2000 Species delimi-tation in systematics Inferring diagnostic differencesbetween species Proc R Soc London Ser B 267631ndash636

WILEY E O 1978 The evolutionary species concept re-considered Syst Zool 2717ndash26

ZAMUDIO K R AND H W GREENE 1997 Phylogeog-raphy of the bushmaster (Lachesis muta Viperidae)Implications for neotropical biogeography systemat-ics and conservation Biol J Linn Soc 62421ndash442

ZAMUDIO K R K B JONES AND R H WARD 1997Molecular systematics of short-horned lizards Bio-geography and taxonomy of a widespread speciescomplex Syst Biol 46284ndash305

Received 19 January 2001 accepted 9 October 2001Associate Editor Jack Sites


delimitation and critical comparisons ofmorphological and DNA variation in empir-ical case studies

The use of mtDNA sequence data inspecies delimitation has been particularlycontroversial and someauthors have arguedthat species should not be delimited basedon these data alone (eg Moritz et al 1992Moritz1994Sites andCrandall 1997Puortoet al 2001) MtDNA data may be problem-atic in that all mitochondrial genes are in-herited as a single linkage group as a resultany mismatch between gene and populationhistories caused by ancestral polymorphismor gene ow between species will simultane-ously affect all mitochondrial genes (Moore1995) Furthermore because mtDNA is ma-ternally inherited mtDNA haplotype phy-logenies will reect only patterns of femalegene ow and dispersal which may bequite different from those patterns in males(Avise 1994) Despite these potential prob-lems we argue that mtDNA has an impor-tant advantage in species delimitation rela-tive to nuclear-based markers that has notbeen widely appreciated As pointed out byMoore (1995) in his defense of mtDNA datain phylogeny reconstruction the smaller ef-fective population size (Ne) of the mitochon-drial genome will cause mtDNA haplotypesof a given species to coalesce (ie becomeldquomonophyleticrdquo) four times more quicklythan will nuclear markers (given some as-sumptions) We suggest that this propertyis important in species delimitation becausenewly formed species should become dis-tinct in their mtDNA haplotype phylogenieslong before they become distinct in nuclear-based markers Thus analysis of mtDNAdata should allow resolution of species limitsin many groups that are difcult to resolvewith nuclear-based markers such as mor-phology We present an empirical study ofspiny lizards (Sceloporus) that may illustratethis phenomenon

The Yarrowrsquos spiny lizard (Sceloporusjarrovii) has traditionally comprised sevensubspecies distributed in the mountains anddeserts of northern Mexico and the south-western United States Wiens et al (1999)presented a phylogeny for 30 populationsof S jarrovii (including the type localitiesof all seven subspecies) that was based onparsimony and maximum likelihood anal-yses of 18 kb of mtDNA sequence datafrom the 12S and ND4 genes (and adjac-ent tRNA genes) They found S jarrovii

to be paraphyletic within the monophyletictorquatus species group (sensu Wiens andReeder 1997) and divided S jarrovii intove species These species correspond tove allopatric clades of haplotypes noneof which are sister taxa Wiens et al (1999)mentioned some diagnostic morphologicalcharacters for these species but did notpresent a critical analysis of morphologi-cal variation in the group Because we wishto evaluate the partitioning of S jarroviimore carefully we herein revert to the tradi-tional taxonomy and refer to these taxa col-lectively as S jarrovii

In this paper we discuss three approachesfor species delimitation using DNA andmorphological variation We propose andreview specic protocols for delimitingspecies using these general approaches andthen apply these methods in an empiricalcase study of the traditionally recognizedSceloporus jarrovii We nd that these meth-ods and data give surprisingly discordantresults which may reect a problematic pat-tern of morphologicalvariationin this groupWe suggest that mtDNA data may generallybe a very powerful tool in species delimita-tion especially in groups that are difcult toresolve with morphological data

APPROACHES TO SPECIES DELIMITATION

Concepts and OverviewBefore we attempt to recognize species

we need a clear concept of what species arede Queiroz (1998) suggested that despitethe long history of dispute over species con-cepts most species concepts agree funda-mentally that species are lineages (Simpson1961 Wiley 1978 Cracraft 1983 de Queirozand Donoghue 1988 Frost and Kluge 1994Baum and Shaw 1995) and for sexual or-ganisms they are lineages that are unitedthrough the process of gene ow (Mayr 1942Dobzhansky 1950 Templeton 1989) Whatprevious authors have generally disagreedabout are the best criteria for recognizingthese lineages (de Queiroz 1998)

In this study we compare the results ofthree approaches (or criteria) to speciesdelimitation (1) tree-based delimitation(sensu Baum and Donoghue 1995) usingDNA haplotype phylogenies (2) tree-baseddelimitation using morphological data and(3) character-based delimitation (sensuBaum and Donoghue 1995) using mor-phological data We discuss these three

















































































RESULTS















































DISCUSSION













ACKNOWLEDGMENTS



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RESULTS















































DISCUSSION













ACKNOWLEDGMENTS



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DISCUSSION













ACKNOWLEDGMENTS



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ACKNOWLEDGMENTS



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Delimiting Species Using DNA and Morphological Variation and … · 2013. 1. 27. ·...

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