Species-Level Paraphyly and Polyphyly: Frequency, Causes, and Consequences, with Insights

from Animal Mitochondrial DNA

Author(s): Daniel J. Funk and Kevin E. Omland
Source: Annual Review of Ecology, Evolution, and Systematics, Vol. 34 (2003), pp. 397-423
Published by: Annual Reviews
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 Annu. Rev. Ecol. Evol. Syst. 2003. 34:397-423
doi: 10.1146/132421
Copyright@ 2003 by AnnualReviews. All rightsreserved
First publishedonline as a Review in Advance on August 14, 2003

AND POLYPHYLY:
PARAPHYLY
SPECIES-LEVEL
Frequency,Causes,and Consequences,with
Insightsfrom Animal MitochondrialDNA

DanielJ.Funk1andKevinE. Omland2
1Departmentof BiologicalSciences,Vanderbilt Nashville,Tennessee
University, 37235;
email:daniel.j.funk@vanderbilt.edu
2Departmentof BiologicalSciences,University
of Maryland,BaltimoreCounty,
Baltimore,
Maryland 21250;email:omland@umbc.edu

Key Words gene trees,introgression,lineagesorting,speciesconcepts,

paraphyleticspecies
U Abstract Manyuses of gene trees implicitlyassumethat nominalspecies are
monophyleticin theirallelesat the studylocus. However,in well-sampledgene trees,
certainallelesin one speciesmayappearmoreclosely relatedto allelesfromdifferent
speciesthanto otherconspecificalleles.Suchdeviationsfromspecies-levelmonophyly
havea varietyof causesandmayleadto erroneousevolutionaryinterpretations if unde-
tected.Thepresentpaperdescribesthe causesandconsequencesof theseparaphyletic
andpolyphyleticpatterns.It alsoprovidesa detailedliteraturesurveyof mitochondrial
DNA studieson low-levelanimalphylogenyandphylogeography, resultsfromwhich
revealthe frequencyof nonmonophylyand patternsof interpretation and sampling.
This surveydetectedspecies-levelparaphylyor polyphylyin 23% of 2319 assayed
species, demonstrating this phenomenonto be statisticallysupported,taxonomically
widespread,and far more commonthangenerallyrecognized.Ourfindingscall for
increasedattentionto samplingandtheinterpretation of paraphyleticandpolyphyletic
gene treesin studiesof closely relatedtaxaby systematistsandpopulationgeneticists
alikeandthusfor a new traditionof "congenericphylogeography."

INTRODUCTION

Intraspecificvariationis at the core of modern evolutionarybiology, its preva-

lence and importancehaving been increasingly documented at the phenotypic
and genotypic levels over the course of the twentiethcentury.Whereasmany bi-
ological disciplines implicitly adopted a more typological approach-studying
the physiology or molecular biology of individuals,then extrapolatingto entire
species andbeyond-evolutionary biology has long emphasizedthe importanceof
appropriatelysamplingany traitor process so as to identify,and thus have the op-
portunityto interpret,importantelements of variation.
1543-592X/03/1215-0397$14.00 397

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398 FUNK0 OMLAND

Interestingly,however, while an early source of molecular data (allozymes)

greatly motivatedinterestin variation,the more recent introductionof DNA se-
quences initially reducedthe emphasis on certainaspects of variationin studies
of evolutionaryhistory.This de-emphasispresumablyoccurredfor the same rea-
son thatvariationis largely ignoredin otherfields, namely,constraintson money
and effort thatrestrictedthe numberof individualsthat could practicallybe stud-
ied. The samplingtraditionsof two groupsof biologists who embracedthese new
data reflect different responses to these constraints(Barraclough& Nee 2001,
Funk 1999). To caricaturethese two traditions:Systematistsbegan to use DNA
sequencesto studythe phylogeneticrelationshipsamongtaxaby samplinga single
individualper species, whereaspopulationbiologists began to evaluatephylogeo-
graphic patternsin DNA sequence variationamong many individualswithin a
single species (Avise 2000, Avise et al. 1987).
In such cases of extremely restrictedintraspecificor interspecificsampling,
the accuracyof various evolutionaryinferences depends on the assumptionthat
individualstudy species are monophyleticwith respect to the alleles at the study
locus. Thatis, theyassumethatall the DNA sequencealleles thatmightbe collected
from individualsof a given species are more closely related to each other than
to any alleles that exist in any other species. In turn, this assumptionrequires
that nominal study species representgenetically and reproductivelyindependent
lineages whose boundarieshave been accuratelyidentified by taxonomists and
whose reconstructedgene trees areaccurateapproximationsof organismalhistory,
i.e., species trees. However,only by samplingmultiple individualsfrom each of
multiple species can both intraspecificand interspecific variationbe assessed,
allowing the hypothesisof species-level monophylyto be tested.
The alternativesto species-levelmonophylyarespecies-levelparaphylyorpoly-
phyly (Figure la) in which gene treesrevealan allele from one species to be more
closely relatedto particularalleles in a differentspecies thananyconspecificallele
(but see Wheeler& Nixon 1990). In this review,we use the termpolyphyly in re-
ferringto both paraphyly-in which all the haplotypesof one or more species are
phylogeneticallynestedwithinthe haplotypesof a second,paraphyletic,species-
and narrow-sensepolyphyly-in which varioushaplotypesfrom the polyphyletic
species are phylogenetically interspersedwith those of other species such that
they are not phylogeneticallycontiguouswith each otheron the gene tree.We use
polyphyly as our more generaltermratherthannonmonophylyto avoid awkward
prose; we use it ratherthan paraphylybecause polyphyly is the older term and
we hope thattemporarilyexpandingits meaningto include paraphylywill be less
discordantwith past systematicsliteraturethanthe reverse.We commonlyuse the
termpolyphyleticspecies as convenientshorthandin referringto currentlyrecog-
nized species taxa whose alleles exhibit a polyphyleticpatternin the broadsense
outlinedabove.This patternis significantbothbecauseof whatit may revealabout
the biology of the polyphyletic species and because of the consequencesit may
have for evolutionaryinferenceif undetected.

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 SPECIES-LEVEL
PARAPHYLY 399

a Definingterms Imperfecttaxonomy
A = monophylyB = paraphyly
C = polyphyly Geographic
Polymorphism variation
A AC DCBB BBC 1111 1212
A BA BC ABAB C

as a generalterm
Here,we use"polyphyly" C) ]d
&polyphyletic
forbothparaphyletic patterns
Lumpingof species
Cryptic
b taxa
"infraspecific" aa b b
ABAB C 11 11
Al Al q A 4242 AA B CAA
m 0 a m

Inadequate
phylogenetic
information
synapomorphies
homoplasies Cei fl

Interspecifichybridization
I

Hybrid
speciation Paralogy
(9)
Introgressive (unidirectional)
hybridization N` #singlegeneduplication
A = maternal
species followedby
12 223 B = hybridspecies
D = paternal
species event
*singlespeciation
AABC AC
ABACDD ABAB

Incompletelineagesorting
selection
Balancing
Incomplete
sorting ancient barrier isolates
Peripheral
recentisolatingbarier isolating
speciation
AA B B C A A B B C 1 1 2 3 3a3a
AAA ABB

fi I k

Figure 1 Species-levelpolyphylyand its causes.This figureillustratespatternsof

gene-treetopologythatareconsistentwith variouscausesof species-levelpolyphyly.
Capitallettersrepresentnominalspecies;numbersrepresentgeographicregions;the
subscriptsin (e) identifyrecognizedsubspecies;the lowercaselettersin (f) represent
subtlephenotypicdifferencessubsequentlyfoundto distinguishcrypticspecies first
identifiedfroma gene tree.See textfor details.

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400 FUNKN OMLAND

In this review,we providethe firstanalysis of the observedfrequencyand tax-

onomic distributionof species-level polyphyly.We describeand contrastthe vari-
ous mechanismsthatyield polyphyleticpatternsandreporton the frequencywith
which particularcauses are invokedin the literature.We discuss the implications
of polyphyly, describe patternsof sampling from the literature,and recommend
samplingstrategiesfor futureresearch.Oursurveyanddiscussionemphasizestud-
ies of mitochondrialDNA sequence variationin animals,reflectingthe authors'
expertise,the widespreaduse of these data,and practicallimitationson the scope
of this study.MitochondrialDNA offers a particularlyvaluable source of mark-
ers for the study of closely relatedtaxa and the causes of polyphyly owing to its
lack of recombination(but see MaynardSmith & Smith 2002), maternalmode of
inheritance(but see below), simple genetic structure,rapidrate of mutation,and
reducedNe (Avise et al. 1987, Harrison1989, Moore 1995, Moritz et al. 1987).
The general principlesdiscussed here, however,apply to the study of gene trees
across diverseloci and taxa.
The broadestgoals of this review areto provideinvestigatorswith a framework
for thinkingthroughthe unexpectedpatternsrevealedby their gene trees and to
encouragesamplingpracticesthat maximize the detectionof importantelements
of intra-andinterspecificvariation.Some workersdismiss all polyphylyas reflect-
ing bad taxonomy.And indeed, imperfecttaxonomyand inadequatephylogenetic
informationare two of the causes we will discuss below. However,we also em-
phasize introgressionand incomplete lineage sortingfollowing recent speciation
as majorcauses of species-level polyphyly thatreflectfundamentalaspects of or-
ganismalbiology with importantevolutionaryimplications.This review does not
addressthe observationof polyphyly at higher taxonomic levels or its practical
implicationsfor nomenclaturalissues.

THE PREVALENCEOF POLYPHYLY

The Literature Survey

of species-levelpolyphylyasanempiricalobservation,
Toevaluatetheimportance
we conductedan intensivesurveyof studiesthatevaluatemitochondrialDNA vari-
ationin animalsin a phylogeneticcontext.Thissurveyincludedonlythosestudies
witha theoreticalpossibility observingpolyphyly.Sincemanyof thecausesof
of
polyphyly aremost likelyto affectcloselyrelatedtaxa,we furtherandarbitrarily
limitedourdatacollectionto studiesof congeners.In orderto avoidinflatingour
estimatesof polyphyly,we excludedexplicitstudiesof hybridzones,wherepoly-
phyly would be expected.Includedstudieswere all othersthattreatedat least two
congenericspecies, at least two individualsfrom one of these species, and an out-
by multipleindividuals(andthuspotentially
group.Foreachspeciesrepresented
polyphyletic)we recorded:numberof individuals,localities, congeneric species,
and congenericindividualssampled;whetheror not polyphyly was observed;and
(wherepresentedby the authors,when polyphylywas observed)bootstrapsupport

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 SPECIES-LEVEL
PARAPHYLY 401

and possible explanationsfor the observed polyphyletic pattern.Polyphyly was

evaluatedusing the species-level taxonomy adoptedby our studies' authorsand
their publishedmtDNA-only phylogenies. Where multiple trees were published,
the phylogeny derivedfrom the most datawas used. Whereunresolvedhaplotype
relationshipswere consistent with either monophyly or polyphyly, the species
was removedfrom the analysis. When multiple studies treatedthe same species,
the species was recordedas polyphyletic if polyphyly was detected in any study.
The large majorityof included studies treatedmtDNA sequence data,but appro-
priate mtDNA restrictionanalysis studies were also evaluated.Surveyed studies
were those publishedbetween 1990 and 2002 in 14 leading journals:Annals of
the EntomologicalSocietyofAmerica,Biological Journalof the LinnaeanSociety,
Copeia, Evolution,Genetics, Heredity,Journal of EvolutionaryBiology, Journal
of Mammalogy,Journal of Molecular Evolution, Molecular Biology and Evo-
lution, Molecular Ecology, Molecular Phylogenetics and Evolution, Systematic
Biology, and The Auk. Citations for all papers treatedin our survey are avail-
able via the SupplementaryMaterialslink in the online version of this chapterat
http://www.annualreviews.org/.

The Distribution of PolyphyleticSpecies

Our13-yearsurveytreated584 studies,526 genera,and2319 potentiallypoly-
phyletic species (Table 1). Overall,535 species provedto be polyphyletic,repre-
senting 23% of those surveyed. Forty-fourpercent (44%) of genera included at
least one polyphyletic species, with more than half of these study generainclud-
ing at least two polyphyletic species. A numberof studies showed rampantpoly-
phyly involvingmany(up to 12) congeners,phylogeneticallyfar-flunghaplotypes,

TABLE1 Resultsof theliterature

survey
Numberof:
Percentspp.
Taxa Studies Genera Spp. polyphyletica

Mammals 139 102 469 17.0

Birds 74 87 331 16.7
Reptiles 56 45 147 22.4
Amphibians 35 26 137 21.3
Fishes 100 99 371 24.3
Arthropods 143 126 702 26.5
OtherInvertebrates 37 41 162 38.6
TOTAL 584 526 2319 23.1

apercentageof surveyedspecies observedto exhibita paraphyleticor polyphyleticpatternof haplotype

relationships.

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402 FUNK0 OMLAND

extremepolyphyly,or multiplespeciesemergingfromwidespread polyphyletic

forms(e.g., Crandall& Fitzpatrick1996,Demboski& Cook2001, Funk1999,
Porteret al. 2002, Sota & Vogler2001, van Oppenet al. 2001). The incidence
of polyphylywas also taxonomically widespread,observedfor at least 15%of
species in each evaluatedanimal class andphylum(Table1; this is also trueof
cnidarians, mollusks,insects,crustaceans,arachnids andechinoderms whenthese
invertebrate taxaareconsideredindividually). Interestingly,there seemed to be
a negativecorrelation betweenintensityof studyandproportion of polyphyletic
speciesacrosstaxa,withbirdsandmammalsexhibitinglessthanhalftheincidence
of polyphylyobservedin nonarthropod a patternthanmightpartly
invertebrates,
reflectinadequatetaxonomy(seebelow).Insum,theseresultsclearlyindicatethat
species-levelmonophylycannotbe assumedandthatspecies-levelpolyphylyis a
muchmoreimportant phenomenon thanis generallyrecognized.Tothedegreethat
anybiasexistsagainstpublishing untidyresults,thissurveymayyetunderestimate
polyphyly'sactualprevalence.Sucha biasmightexplainan apparent recentde-
clinein thereported incidenceof polyphyly(1990-1999= 28.2%,2000-2002 =
19.7%)thatisn'treadilyexplainedby changesin samplingorphylogenetic infor-
mationcontent.Alternatively, thispatterncouldreflecta tendencyforearlystudies
on a groupto focuson problematic taxa.

OF POLYPHYLETIC
CAUSESAND INTERPRETATIONS
PATTERNS

Anobservation of polyphylyshouldprompta consideration of itsparticularcauses.

Wheninterpreting molecularvariation,however,it is oftentemptingto offerad
hoc explanations forunusualpatternswithoutfullyconsidering This
alternatives.
tendency is exacerbated when certain explanations haveachieved wide recogni-
tion only recentlyor by workersin certainfieldsor studentsof certaintaxa.In
this sectionwe tryto alertworkersto the full rangeof phenomena thatmaypro-
ducespecies-levelpolyphylyandto explainhow theydo so (Avise1994,Funk
1996,Slowinski&Page1999).Insomecases,observedpolyphylyis anartifactof
misidentified specimens,specieslimits,andstudyloci, or of inadequate informa-
tion.In others,it reflectsaspectsof allelichistorythatprovideimportant insights
into speciesbiology.Wherepossible,we recommendmeansof distinguishing
amongthesealternative explanations.Unfortunately, however,clearone-to-one
correspondence betweenspecificcausesandparticular patternsoften does not
existso thatdefinitiveconclusionsmayfrequently remainelusive.

InadequatePhylogeneticInformation
Onepotentiallyquitegeneralcauseof observedpolyphylyis weakphylogenetic
signal, which may resultin poor phylogeneticresolutionor inaccurategene trees
as an artifactof phylogeneticreconstruction.Phylogenetic algorithmscan create
topologiesregardlessof the amountandqualityof the data.Thus,if a gene is evolv-
ing too slowly relativeto the rateof speciationin one's studytaxa or if too small a

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 SPECIES-LEVEL
PARAPHYLY 403

fragmentof thatgene is analyzed,obtaineddatamay providetoo few synapomor-

phies to robustlyrecoverthe underlyinggene tree,a challengethatbecomes greater
if positively misleadinghomoplasiesconfoundthe few variablesites (Figure lb).
Although rapidlyevolving mitochondrialsequences are less prone to inadequate
informationthanmost loci, even mtDNA may exhibitinsufficientvariationfor the
accuratereconstructionof very recentphylogeneticradiations.On the otherhand,
sequences from a gene thatevolves very rapidlyrelativeto speciationrates might
be saturatedand produce an inaccurategene tree owing to high levels of homo-
plasy. Thus, even if all study species are in fact monophyletic, a reconstructed
gene tree may erroneouslyexhibit polyphyletic groupingsthat do not accurately
representthe historyof the analyzedalleles or species.
The studiesin our surveyadoptedvariousapproachesto assess the likely histor-
ical accuracyof polyphyleticgene trees. Some studies tested whethera topology
constrainedto be monophyleticrepresenteda significantlyworse fit to the datathan
the observedpolyphyletictopology using, for example,the methodof Kishino &
Hasegawa(1989). Morecommonly,Bremersupport(Bremer1988) andespecially
bootstrapsupport(Felsenstein 1985) were offered as estimates of the degree to
which the data supportedhaplotypegroupings.Here, we use reportedbootstrap
values to assess the generality of statistical supportfor observed polyphyletic
patterns.Specifically,for each polyphyletic species (A), we recordedthe largest
bootstrapvalue that grouped any haplotypes of A with one or more haplotypes
from any otherspecies to the phylogeneticexclusion of some otherA haplotypes.
This provideda conservativeestimate of the supportfor polyphyly because only
one of potentiallymultiple supportingnodes was considered.
We found that 85% of polyphyletic species were from studies that employed
bootstrapproportions,providing a large sample for this analysis. Among these
studies,thepercentagesof polyphyleticspecies supportedby variousbootstrappro-
portionswere as follows: <50 = 17%of species, 50-69 = 15%,70-94 = 22%,
and >95 = 46%. Thus, in two-thirdsof observedcases polyphyly was supported
by >70% of bootstrapreplicates.These resultsprovidecompelling evidence that
the prevalenceof polyphyly documentedby our surveyreflects a common aspect
of true mitochondrialgene trees and is not simply a common artifactcaused by
inadequatedata.
The remainingcauses of polyphyly resultnot from imperfectphylogeneticre-
constructions,but despite well-supportedgene trees with topologies that likely
depict the true origins and relationshipsamong sampled alleles. Such gene trees
may nonetheless disagree with recognized species boundaries-and produce
polyphyly-for a numberof reasons. To simplify and separateour discussions
of these reasons, we hereafterassume that the phylogeneticpatternsinvoked are
stronglysupported,unless statedotherwise.

ImperfectTaxonomy-Inaccurate SpeciesLimits
One importantreason for the observationof polyphyly is a failure of the taxo-
nomic circumscriptionof a nominalspecies to correspondto patternsof gene flow.

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404 FUNKE OMLAND

Thatis, polyphyly sometimes results from "badtaxonomy"when named species

fail to identify the genetic limits of separateevolutionaryentities. This failure
can occur eitherby underestimatingor by overestimatingthe field of genetic ex-
change among individualsand populations.In both situations,polyphyly can be
validlyeliminatedsimplyby changingcurrenttaxonomy.Moretrivially,polyphyly
can resultfrom the misidentificationof samples,providinga strongargumentfor
maintainingvoucherspecimens.

SPECIES INTRASPECIFIC
OVERSPLIT-MISIDENTIFYING VARIATION AS SPECIES-LEVEL
VARIATIONTaxonomyunderestimatesthe breadthof species limitswhen anatom-
ical (or behavioral,ecological, etc.) variantsof a single species have erroneously
been describedas separatenominal species. This may occur, for example, when
distinctivevariantscoexist within individualpopulationsof a single polymorphic
species. Underthis scenario,no phylogeneticsubstructureas a functionof variant
type is expected(e.g., Demasteset al. 2002, Nice & Shapiro2001, Small & Gosling
2000) (Figure lc). This is because local gene flow amongvariantsshouldproduce
a gene tree in which sympatrichaplotypesfrom each variantare cladisticallyin-
termingledwith those of the other(s), renderingeach polyphyletic.Furthermore,
levels of genetic variationamongthese "oversplit"nominalspecies areexpectedto
be typical of within-speciesvariationin the taxa understudy.Incompletelineage
sorting(see below), however,can producethe same patterns.
Species polyphyly may similarlybe observedif two nominal species actually
representgeographicvariants(races, subspecies)of a single species thatcontinue
to exchangegenes. In this case, the observedphenotypicdivergencemay be either
environmentallyinducedor genetically based and maintainedby strongselection
despite gene flow. If haplotypes of these geographic variantsdo not phyloge-
netically segregateinto separateclades, conspecificity is supported(Figure ld).
Unlike the polymorphismexample above, however,some degree of phylogenetic
substructuringby varianttype might be observed if gene flow is geographically
restricted,yielding isolationby distance.In such cases, distinguishingbetweenin-
traspecificvariationandinterspecificintrogression(see below) as a cause of these
patternsmay be difficult.

VARIATIONAS INTRASPEC-
SPECIESOVERLUMPED-MISIDENTIFYINGSPECIES-LEVEL
IFICVARIATIONJust as intraspecificvariantsmay be mistakenfor species, traits
diagnostic of species are sometimes assumed to representintraspecificvariation
or are simply difficultto detect at all. This may resultin the taxonomic"lumping"
of multiple species undera single name and the observationof polyphyly when
these species arenot sistertaxa.In such cases, currenttaxonomyoverestimatesthe
breadthof species limits. This is sometimes observed,for example, with respect
to subspecies, geographicforms, morphotypes,and othernominallyinfraspecific
taxa that have been recognized on the basis of divergence in particulartraits.
When one or more infraspecifictaxa within a nominal species prove to be mi-
tochondriallymonophyletic, a substantialhistory of genetic isolation of these
taxa from other"conspecific"populationsis indicated.In the case where distinct

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 PARAPHYLY 405
SPECIES-LEVEL

cladesof thiskindseparatefully sympatrictaxa,reproductive isolationbetween

themis furtherindicated,as is speciesstatusundermostspeciesconcepts.In the
casewheredistinctcladesarealsogeographically separated (Fukatsuet al. 2001,
Kotlik& Berrebi2002, Riddleet al. 2000), evidenceon reproductive compati-
bilityis ambiguous,andthe decisionof whetheror notto recognizethesegenet-
ically differentiatedentitiesas separatespeciesdependson the speciesconcept
applied. In eithercase, the telltalepolyphyleticpatternis causedby the nesting
of one or moreadditionalnominalspeciesamongthe haplotypesof the over-
lumpedspecies(A) (Figurele). Whensuchnestingrenderscertaininfraspecific
taxaof A monophyletic, elevatingthesetaxato speciesrankis one strategyfor
taxonomically removingthespecies-levelpolyphyly(Omlandet al. 1999,Voelker
1999).
Sometimes,cluesto lumpingmaybe scarceowingto thehighlysimilarmor-
phologiesof unrecognizedspecies.If otherdescribedspeciesare moreclosely
relatedto such"crypticspecies"thanthecrypticspeciesareto eachother,a mito-
chondrial genetreemighthintatcryptictaxabyrevealingpolyphylyin theformof
twophylogenetically separated clades(Figure1f)(Omlandet al. 2000;Williams
et al. 2001; D.J. Funk1998,unpublished data).Suchcrypticspeciesmightre-
flecttheretentionof ancestralmorphology (Jarman & Elliott2000).However,the
samepolyphyleticpatternwouldbe expectedif crypticspeciesresultedfromthe
convergent evolutionof similarmorphologies (Kimet al. 2000,Reeset al. 2001,
Richmond&Reeder2002,Suet al. 1996).Thismightbe expectedif divergent lin-
eages were responding to similar selection as in
pressures, threespine stickleback
fishesthathaverepeatedlyevolvedcomplexbenthic-andpelagic-adapted mor-
&
phologies(Bell 1987,Schluter Nagel 1995). Such convergence creates special
problemswhentraitsunderselectionarealsothoseusedby taxonomists to define
species.
Inthescenarios justreviewed,polyphylyresultswhenthedescribed phenotypic
boundaries of nominalspeciesdo notadequately or accuratelyreflectthehistory
of populationdifferentiation andspeciation.Thatis, polyphylyresultsevenif a
speciestreecanbe safelyassumedtobeidenticaltothegenetreeusedtoinferit.By
contrast,theremainingcausesof polyphylygenerallyreflectsituationswherethe
historyof allelesrevealedby a genetreeis incongruent withtheactualorganismal
historyembodiedby the speciestree(butsee Doyle 1997,Maddison1997).This
"genetree/species treeproblem" (Aviseet al. 1983;Broweret al. 1996;Doyle1992;
Goodmanet al. 1979;Maddison1996, 1997;Nichols2001;Pamilo& Nei 1988;
Slowinski& Page1999;Wu 1991)represents a majorlimitationon evolutionary
inferencesfromsingle-locus(e.g.,mitochondrial) genetreesthathasnotyet been
fullyincorporated into certainareas of systematicbiology.

InterspecificHybridization
One potentialcause of gene tree/speciestreediscordanceand accompanyingpoly-
phyly is the occasional mating between otherwise distinct species and resulting
transferof parentalalleles to hybridoffspring.Two aspects are worthnoting.

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406 FUNK0 OMLAND

Alleles from one species may penetratethe gene pool of an-

INTROGRESSION
other throughinterspecificmating and the subsequentbackcrossingof hybrids
into parentalpopulations,a process known as introgressivehybridization,intro-
gression,or interspecificgene flow. Introgressionyields polyphylyby introducing
phylogeneticallydivergentallelic lineages across species boundaries(e.g., Boyce
et al. 1994; Patton& Smith 1994; Shaw 1999, 2002). The phylogeneticeffects of
mitochondrialintrogressionareparticularlygreatbecause a lack of recombination
entailsthatall mitochondrialbase positionsintrogressas a completelylinkedblock
(Smith 1992). Thus, any analyzedfragmentof introgressedmtDNA will entirely
reflect the heterospecificorigin of its mitochondrialgenome. Furthermore,mito-
chondrialalleles might be expected to introgressfarther,on average,thannuclear
loci if their persistence in a foreign gene pool is less constrainedby linkage to
selected loci thanare the alleles of nucleargenes (Barton& Jones 1983, Harrison
et al. 1987, Marchant1988, Tegelstr6m1987; reviewedin Harrison1990, Arnold
1993). For these reasons, mitochondrialgene trees could be particularlysuscep-
tible to the effects of introgression.An interestingexception is offered by female
heterogametictaxa following Haldane'srule, such as birds (Tegelstrim & Gelter
1990) andbutterflies(Sperling1993). In such cases, female hybridsshow reduced
viability that might restrictthe introgressionof maternallyinheritedmtDNA be-
tween species, offering a potential explanationfor low mtDNA introgressionin
severalavianhybridzones (e.g., Allen 2002, Brumfieldet al. 2001, Sattler& Braun
2000). Moregenerally,the exposureof haploidmtDNAloci to selectionin all indi-
viduals may also impedeits introgression(Brumfieldet al. 2001). The differential
introgressionof mitochondrialversus nuclearalleles and its effects on polyphyly
is an importanttopic thatdeservesfurtherattention.
Recognizing mitochondrialintrogressionrequiresevaluatinga mitochondrial
gene tree againsta nuclearbackgroundthatidentifiesthe participatingtaxa. This
backgroundcan be providedby gene trees from nuclear loci or simply by con-
sistenttaxon-specificphenotypicdifferencesthatpresumablyhave a nuclearbasis
(Smith 1992). The clearest signatureof introgressionis the sympatricsharingof
geographicallylocalized mtDNA sequence haplotypesbetween otherwise genet-
ically and morphologicallydivergentspecies (Figure 1g). Such a patternis hard
to interpretas anythingbut ongoing (or very recent)and geographicallylocalized
interspecificgene flow. Importantly,introgressionmay not be detected in such
situationsunless populationsare indeed sympatricallysampled because species
that sharehaplotypesin regions of geographicoverlapmay otherwiseexhibit re-
ciprocal monophyly in gene trees based on allopatricsamples (e.g., Masta et al.
2002, Redenbach& Taylor2002).
Unfortunately,confidentlyattributingpolyphylyto introgressionbecomes pro-
gressivelymoredifficultthe fartherin the pastthatgene flow last occurred.Species
thathave ratherrecentlyceased exchanginggenes may no longer sharehaplotypes
(becauseof post-introgressionmutation)yet still possess very closely relatedhap-
lotypes that are nested togetherwithin the gene tree. However,as the time since
last gene flow increases, those introgressedallelic lineages that do persist are
more likely to be phylogeneticallybasal (as a result of the sorting out of allelic

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 SPECIES-LEVEL
PARAPHYLY 407

polymorphisms datingto the time of introgression) andless likelyto showany

geographicassociationwiththe populationfromwhichtheyintrogressed (to the
degree that populations
change distributions over time).
Otherfactorscanfurthercomplicatetherecognition of introgression
as a cause
of polyphyly.If mitochondrial gene flow is bidirectional,very common, oroccurs
amongmultiplespecies, its affectonmitochondrial tree be
topologymay profound,
makingit difficultorimpossibleto confidentlyinferpatternsof geneticexchange
orevento determine whichmitochondrial claderepresents the"native"lineageof
particular species.Mitochondrial gene trees be
may especiallymisleadingin cases
whereintrogressed haplotypelineages become fixed,leavingno hintthattheyare
of heterospecific The
origin. smallerNe of mtDNA compared withnuclearlocimay
facilitatethisprocess,suchthatevenlow levelsof introgression maybe sufficient
to establisha neutralmitochondrial genotypein a foreignpopulation (Takahata &
Slatkin1984).Patton& Smith(1994),for example,attributed complicated poly-
phyleticpatterns inpocketgophersto sporadicepisodesof hybridization combined
withsmall,patchygopherpopulations thatfacilitatedthefixationof introgressed
alleles.Recurrenthybridization has been similarlyinvokedto explainrampant
in a
polyphyly variety of taxa (Freeland & Boag 1999,Funk1999,Shaw2002,
Sota& Vogler2001).

HYBRIDSPECIATIONPolyphyly may also result from the spontaneousforma-

tion of a new speciesthroughinterspecific hybridization,a mechanismthathas
beendemonstrated in variousanimaltaxa(e.g., Moritzet al. 1992;reviewedby
Dowling& Secor1997).In suchinstances,theinitialrelationship amongparental
anda new hybridspecies'mitochondrial alleleswill dependon the numberand
symmetryof hybridspeciationevents.Mosthybridspeciesappearto originate
via asymmetrical A hybridspeciesformedby a singlesuchevent
hybridization.
will itself be mitochondrially monophyletic,while specificallyrenderingthe
mitochondria-contributing maternalspeciesparaphyletic(Figurelh). A hybrid
species formed throughrepeatedasymmetric hybridizations(allinvolving,e.g., a
femaleof speciesA anda maleof speciesB) willbemonophyletic if theparticipat-
ing females have identical
mitochondrialhaplotypes,polyphyletic otherwise(e.g.,
Mantovani et al. 2001).A hybridspeciesformedthroughsymmetric hybridization
eventswouldbe polyphyletic, aswouldbothparentalspecies.Becausehybridspe-
ciationis oftenassociatedwithpolyploidyor asexualreproduction, knowledgeof
suchtraitsmaybolstera suspicionthathybridspeciationis thecauseof observed
polyphyly(e.g., Johnson& Bragg1999).However,in severalcases of putative
hybridspeciation(Hedricket al. 2002,Wayne& Jenks,1991;alsosee Salzburger
et al. 2002)alternative explanationshaveprovendifficultto ruleout.

IncompleteLineageSorting
The incomplete sorting of ancestrallypolymorphicallelic lineages representsa
very general source of polyphyly,potentiallyafflictingany single-locus gene tree
in any taxon. Within any species, the various alleles at a particularlocus have

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408 FUNK0 OMLAND

theirownhistory,withsomeallelessharingmorerecent,andothersmoreancient,
coalescentevents(Pamilo&Nei 1988).Thus,therandomdivisionof allelecopies
at speciationwill generallyresultin each daughterspeciespossessingcertain
allelesthataremostcloselyrelatedto thosein theotherdaughter species.Forthis
new are
reason, species initiallyexpected to exhibitpolyphyletic genetrees(Figure
li). Over time, allelic lineages in each daughterspecies will be randomlylost by
drift,and new alleles will be formed by mutation until eventuallyonlyone of the
(ancestrallypolymorphic) allelic lineagespresent in the parentspeciessurvives
in each daughterspeciesand all intraspecific variationreflectspost-speciation
mutation.At this point,sortinghas gone to completion,andallelesin the two
daughterspeciesarereciprocally monophyletic. Thisprogression frompolyphyly
(narrow-sense) to paraphylyto monophylyis expectedto takeon the orderof
4Ne generations for mitochondrial loci andultimatelyresultsin a gene treethat
accurately reflectsthespeciestree(Avise1989,Avise&Ball1990,Harrison1991,
Neigel& Avise1986,Pamilo& Nei 1988,Tajima1983,Takahata & Nei 1985).
Becausethemitochondrial is and
genome haploid maternally inherited,theNe
of mitochondrial loci is generallyone-quarter thatof nuclearloci (butsee Hoelzer
1997),andstochasticlineagesortingis expectedto progressmorerapidlyformi-
tochondrial alleles.Thus,incompletesortingis less of a concernformitochondrial
thanfor nuclearloci, otherthingsbeingequal,providingone advantageto us-
ing mitochondrial genetreesas estimatesof speciestreesforcloselyrelatedtaxa
(Hudson & Turelli 2003).Indeed,theorypredictsthatif one canbe 95%certain
thataninternodein a singlemitochondrial genetreehasnotbeenaffectedby in-
completesorting,16 independent nuclear genetreeswouldbe requiredto justify
an equallevelof confidence(Moore1995).Nonetheless,incompletesortingalso
affectsmitochondrial genetreesandcanhaveespeciallymajoreffectsin thecase
of rapidlyradiating taxa,in whichsucceedingspeciationeventsoccurbeforesort-
ing is completed.Thisscenariohasbeeninvokedto explainthesharingof alleles
amongmultiplespeciesin therampant polyphylyexhibitedby cichlidfishesand
othertaxa(Moran& Kornfield1993,1995;also see Crandall & Fitzpatrick 1996,
Goodacre& Wade2001,Klein& Payne1998).
Unfortunately, it is impossibletodemonstrate conclusively thatincompletesort-
ingexplainsany particular case of polyphyly. One problem is thatbecausespecies
remainincompletely sortedfora narrowwindowof evolutionary time,a dearthof
accumulated synapomorphies may often make it difficult
to distinguish incomplete
sortingfrominadequate phylogenetic information asacauseof observedpolyphyly
(Slowinski& Page1999).Anotherproblemis thedifficultyof distinguishing the
effectsof incompletesortingandintrogression, anissueof considerable interest.A
phylogenetically basalpositionof polyphylyrendering haplotypes hintsatretained
ancestralpolymorphism, whilerecentlyintrogressed allelesmayassumea highly
derivedpositionin thegenetree.Also,incompletesortingis notpredictedto pro-
mote the geographicproximityof interspecificallysharedalleles thatmay be seen
underlocal introgression(Hare& Avise 1998, Mastaet al. 2002). However,these
criteria are often inadequateto distinguish ancient mitochondrialintrogression

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 SPECIES-LEVEL
PARAPHYLY 409

from incompletesorting(Schneider-Broussard et al. 1998). Comparisonswith nu-

clear markersand geographycan provide additionalinsights (e.g., Redenbach&
Taylor2002, Tegelstr6m1987, Wecksteinet al. 2001), as may nested clade analy-
sis (Templeton1998, Templetonet al. 1995; but see Knowles & Maddison2002).
Moore (1995) suggestedcomparingmaximumintraspecificsequencedivergences
between a polyphyletic species and related species in an empirical method for
evaluating the likelihood of incomplete sorting (see Baker et al. 2003, Holder
et al. 2001, Knowles 2000, Mason et al. 1995, Palumbiet al. 2001, Rees et al.
2001). Other,more statistical,methodshave also been described(e.g., Nielsen &
Wakeley2001, Sang & Zhong 2000, Wakeley 1996). However,a generallydiag-
nostic and widely agreed-uponapproachfor documentingincompletesortingand
distinguishingit from introgressionhas not yet emerged(Holderet al. 2001).

SPECIATIONAND SORTING Although the progression of new species from initial

polyphyly throughparaphylyto monophyly follows quite generally on the heels
of speciation, the particularpatternand time course of this progressionmay be
ratherdistinctivein the case of peripatric,peripheralisolates, or "budding"spe-
ciation (Frey 1993; Harrison1991, 1998; Rieseberg & Brouillet 1994), in which
populationsalong the peripheryof a species rangebecome spatiallyisolated and
speciate. To the degree that a "parental"species exhibits geographicsubstructure
and a peripherallyspeciatingpopulationis small and local, this populationmay
be predictedto initially possess a phylogeneticallyrestrictedsubset of parental
alleles and may lose alleles underdriftat a fasterratethanthe largerparentalpop-
ulation. For these reasons, peripheralisolates speciation may commonly yield a
geographicallyrestricteddaughterspecies whose monophyleticset of haplotypes
is embedded within a widely distributedand still paraphyleticparentalspecies
(termeda ferespecies by Graybeal1995; also see Baum & Shaw 1995, Olmstead
1995) (e.g., Avise et al. 1990, Funket al. 1995a, Hedin 1997, Marko1998; but see
Knowles et al. 1999) (Figure 1k). This deep phylogeneticnesting is not expected
under large-scale vicariantor parapatricmodes of speciation, althoughit might
also be observed (in a differentphylogeographiccontext) in the case of rapid,
local sympatricspeciation(Harrison1998). This asymmetricallyparaphyleticre-
lationshipwill persistuntil sortingrendersthe parentalspecies monophyletic.
In the case of buddingspeciation,forcing taxonomyto reflectgene tree mono-
phyly by synonymizing the nested and parent species or by elevating lineages
in the paraphyleticlineage to species status ignores the distinctivenatureof the
nested lineage (de Queiroz & Donoghue 1988; Harrison1991, 1998; Olmstead
1995; Rieseberg& Brouillet 1994; Rodriguez-Robles& De Jesis-Escobar 2000;
Sosef 1997; Wiens & Penkrot2002). Underbuddingspeciation,the cause of pa-
raphyly is incomplete lineage sorting, yet the gene tree accuratelyreflects the
history of populationdivergence. Thus, although gene trees from different loci
are ordinarilyexpected, by chance, to be incongruentunder incomplete sorting,
budding speciation is predictedto produceparallelpatternsof paraphylyacross
nuclearand mitochondrialloci (Hedin 1997, Marko 1998, Petrenet al. 1999; but

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410 FUNK0 OMLAND

see Ballard2000, Tosiet al. 2000). Becauseit reflectspopulationhistory,thisnested

is evolutionarily
pattern allowingthepolarization
informative, of thespeciation
eventandof transitions
betweentraits(hostplantassociations,
plumagepatterns,
ranges, thataccompany
geographic etc.) andmayhavepromotedspeciation(e.g.,
Brown et al. 1994, 1996; Funk et al. 1995b; Omland1997).

SELECTIONAND SORTING The expected time to complete sorting invoked above,

4Ne generations,appliesto strictlyneutralmitochondrialalleles. However,mtDNA
variationmay often be subjectto selection (Ballard& Kreitman1995, Hudson&
Turelli2003, Rand2001), which will affectthe rateat whichreciprocalmonophyly
is attained.While positive selection will accelerateallele fixationand sorting,bal-
ancing selection may preserveancestrallypolymorphicalleles withina population
indefinitely(Figure1j).PolymorphicnuclearMHCalleles, for example,areshared
betweenotherwisegeneticallydivergentspecies in severalanimaltaxa(Kleinet al.
1993, 1998). However,althoughthereis some evidence for balancingselection on
mtDNA in animals(James& Ballard2000) and plants(Stdidler& Delph 2002), it
has not yet been documentedas a cause of species-level mitochondrialpolyphyly.

UnrecognizedParalogy
Orthologousalleles derive from the same locus whereas paralogousalleles de-
rive from differentloci that originatedby a gene duplicationevent. A gene tree
thatincludesparalogousalleles may depictpolyphyleticspecies becauseits topol-
ogy reflects gene duplicationas well as speciation (Figure 11).The cause of this
polyphyly may be misinterpretedif the orthologyof alleles is assumed.Because
mitochondrialloci are single-copy genes ratherthanmembersof multigenefami-
lies, it was long consideredsafe to assumethe orthologyof alleles sequencedwith
mitochondrialprimers.Twophenomenaillustrateexceptionsto this rule thatcause
polyphyly.

NUCLEARPSEUDOGENES It is now well understood that segments of mitochon-

drial DNA are sometimes transposedinto the nucleus where they become func-
tionless pseudogenes(Bensassonet al. 2001, Collura& Stewart1995, Sorenson&
Fleischer 1996, Sunnucks & Hales 1996, Zhang & Hewitt 1996). When such
nuclear copies of mtDNA exist, using mitochondrialprimersfor PCR amplifi-
cation from whole-genomic DNA extractions(a common approach)may yield
sequences of nuclear as well as mitochondrialorigin. Indirectevidence for nu-
clear copies may be providedby unusualpatternsof molecularevolutionthat are
consistent with the reduced functional constraint(e.g., elevated frequencies of
nonsynonymoussubstitutions,indels, frameshifts,and stop codons) or nuclearlo-
cation (slowed ratesof substitution)of pseudogenes.Nuclearcopies may be more
directly detected throughthe isolation of mtDNA, cloning, and rtPCR(Collura
et al. 1996). Nuclear copies of mtDNA and their effects on polyphyly have now
been documentedin a varietyof taxa.

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 PARAPHYLY
SPECIES-LEVEL 411

PATERNAL
INHERITANCE
In a few cases, paternallyinheritedmitochondriallin-
eageshavebeenshownto originatefrommaternally inheritedancestors,muchas
new loci areformedby a gene duplicationevent.Thesedivergentmaternaland
paternallineagescancoexistwithinspecies,yieldingspecies-levelpolyphylyin
gene treesthatincludeallelesfromboth.To date,suchinstanceshavegenerally
beentaxonomically restrictedto variousbivalvemollusks(e.g.,Rawson& Hilbish
1995), so thisphenomenon notknownto presenta generalcauseof polyphyly.
is
Recentresultsfromhumans(Bromham et al. 2003),however,illustratethatother
taxamayalsobe affected.

LiteraturePatterns
Attemptingto elucidatethe actualcausesof polyphylyin the studiesfromour
surveyis beyondthescopeof thepresentreview.However,someobservations on
authors'tendenciesin reportingpotentialcausesareworthnoting.First,24%of
paperswithpolyphyleticgenetreesofferedno discussionof thispattern.Second,
of thosethatevaluatedpolyphyly,50%specificallysuggestedfaultytaxonomy
as one plausibleexplanation, introgressivehybridizationwas invokedin 32%of
and
papers, incompletelineagesorting was citedin 30%. Inadequate phylogenetic
information andunrecognized paralogyreceivedmentionin only a few papers
each.Third,closerinspectionof a subset(~-onehalf)of the polyphyleticpapers
foundthatin 56%of theseonlyone or anotherof threemajorcauses(taxonomy,
introgression, sorting)receivedanymentionat all;twocauseswerementionedin
25%, and all three in only 16%of thestudies.
Although it is encouragingthatmost authorsfindpolyphylya worthysub-
of
ject comment,thesepatternssuggestthata fullypluralisticappreciation of its
causeshasyet to takeroot.Thegeneraldisregard of nuclearcopiesof mtDNAas
a possibleexplanationis especiallyconcerning(butsee Wecksteinet al. 2001).
The equivalentinvocationof introgression andincompletesortingandthe con-
siderablygreaterfrequencyof taxonomicexplanations mayrevealthe biasesof
biologistsorilluminate therelativeimportanceof differentcauses.Werecommend
thatfuturestudiesseekthemostaccurateandinformative interpretations
by sys-
tematicallyconsidering thefullrangeof alternativeexplanationsin accumulating
datasets.

CONSEQUENCESFOREVOLUTIONARY
INFERENCE

ErroneousEstimates
If undetected,
species-levelpolyphylycompromisesevolutionaryinferencesbased
ongenetreesthatareerroneously assumedto accurately
depictspeciestrees(Funk
1996, 1999) (Figure 2). The extent of these problems will depend on several
aspectsof polyphyly,amongthem:(a) how commonlypolyphyleticspecies occur
in the study taxon, (b) how "polyphyletic"a given species is, i.e., how many

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412 FUNK OMLAND

(1) (2) (1) (3)

(1) (2) (1) (3)
(1) (2) (3) (4) (1) (5) (6) (7)

bebbianae
bebbianae
bebbianae
bebbianae
cribripennis
cribripennis
chamaedaphnes bebbianae
chamaedaphnes cribripennis
bebbiannae
bebbiannae
bebbiannae
chamaedaphnes
chamaedaphnes
chibripennis
N. N. N. N. N. N. N. N. N. N. N. N. N. N. N.

99
63 100 100 77
86 88
77

Max. seg. div.

= 1.7%
N.bebbianae
84100 Max. seg. div.
=9.3%
N.bebbianae 100 100
= 0.3%
N.cribripennis = 10.0%
N.cribripennis
N.chamaedaphnes= 0.4% N.chamaedaphnes= 0.7%

Hostassociation: = Betulaceae = Ericaceae

Figure 2 Erroneousevolutionaryinterpretations dueto polyphyly.Eachtreedepicts

the reconstructedrelationshipsamongmitochondrialhaplotypescollectedfrom dif-
ferentsubsetsof individualsrepresentingthreeNeochlamisusleaf beetlespecies(data
fromFunk1999).Haplotypesfromdifferentindividualbeetlesareindicatedby differ-
ent numbers.Resultsillustratehow drasticallyestimatesof phylogeneticrelationship,
characterevolution,andgeneticdivergencecanvaryas a functionof theparticularindi-
vidualssampledwhenstudyspeciesarehighlypolyphyletic.Bootstrapvaluesindicate
strongsupportof eachdataset for a differenttopology.

distinctallelic lineages are represented,(c) how genetically and phylogenetically

diversethese allelic lineages are, and (d) how evenly alleles are distributedamong
these lineages. The likelihood thatevolutionaryinferenceswill vary dramatically
accordingto the individualssampledwill be greatestwhen polyphyletic species
bearingmany, diverse, and equally frequentallelic lineages are common. In this
context, the consequences of polyphyly may be more or less severe, on average,
accordingto the particularcause. Incompletelineage sorting,for example, is less
likely to involve highly divergentallelic lineages than is an ancient duplication
event or introgressionbetween distantlyrelatedtaxa. Some importantinferential
problemsresultingfrom polyphyly are describedbelow.
First and most basically,the phylogeneticrelationshipsdepictedby a gene tree
may vary accordingto the particularindividualssampled when sequences from
one or a few specimens are used as exemplarsof polyphyletic species (Ballard
2000, Barraclough& Nee 2001, Funk 1999, Melnick et al. 1993, Omlandet al.
1999, Smouse et al. 1991, Zink et al. 1998) (Figure 2). Systematistsgenerally
agreethatmultipleexemplarsshouldbe includedat the level below the taxonomic
rank of interest, and this should also apply to the species level (Ballard 2000,
Barraclough& Nee 2001, Graybeal 1995, Omlandet al. 1999, Wiens 1999; see
also Lanyon 1994). Intensivesamplingis more likely to documentthe underlying
polyphylyandalertthe systematistthatsomethingis amiss. Second, inferredtimes
andratesof evolutionarydivergencemay be considerablyinflatedor deflated(de-
pending on the cause of polyphyly) when alleles are sampledfrom polyphyletic

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 SPECIES-LEVEL
PARAPHYLY 413

species(Ballard2000,Funk1999,Melnicket al. 1993;alsosee Edwards& Beerli

2000) (Figure2). Third,whengenetreesareusedto reconstruct theevolutionary
historyof particular
traits,polyphylypresentsespeciallyegregiousproblemsbe-
causea singlemisplacedtaxoncanhavemajoreffectsoncharacter transformations
throughoutthe tree (Omland 1997; also see Graybeal 1995) (Figure 2). Fourth,
polyphylymaycompromise population geneticandphylogeographic studies.This
may occur,for example,when sampling of relatedspeciesis insufficient
to iden-
tify the heterospecific
(e.g., introgressive) of
origin divergenthaplotypesin the
focalspecies,leadingto mistakenconclusionsaboutdemography andevolution-
aryprocesses thatarebased on allelic and
frequencies relationships (Redenbach &
Taylor2002,Tegelstrim1987; also see Ballard 2000).Fifth, mitochondrialpoly-
phyly complicatesthe identification of species-diagnosticmolecularcharacters
for practicalissues of managementand conservation,such as identifying endan-
gered species (Bakeret al. 1996, Daleboutet al. 1998) and definingevolutionarily
significantunits (e.g., Moritz 1994, Paetkau1999).

IntriguingInsights
Although species-level polyphyly can be quite problematicif undetected,when
recognizedit canprovideinformative cluesthatmotivatefuturework(Funk1996,
1998;Harrison1998;Omland1997).Forexample,appropriately sampledmito-
chondrialsurveyscanbe anefficientmeansof detectinginitialevidenceforgene
flowandinsightson its direction,geographical
andbiologicalcorrelates, andpar-
ticipatingtaxa.Suchobservations candirectworkersto informative investigations
of hybridzones,mechanismsof reproductive isolation,or the reexaminationof
specieslimits.Therevisedtaxonomicandphylogeneticassessmentsof species-
level taxathatare provokedby polyphylymay providemorestableclassifica-
tionsandmoreaccuratehistoricalinferences,forexample,on character evolution
(Omland1997,Omlandet al. 2000). The discoveryof morphologically cryptic
or unusuallyvariablespeciesmaypromptstudieson the evolutionary causesof
convergence/stasisor polymorphism, respectively.Findingsconsistentwith in-
complete sortingmay lead to studiesof demographyand speciationrates.Patterns
consistent with budding speciation may identify taxa that contributeto ongoing
debateson speciationmechanisms(Harrison1991, Turelliet al. 2001). Even nu-
clearcopiesof mtDNAarenow beingexploitedfor novelevolutionary
insights
(Bensassonet al. 2001).

THE IMPORTANCEOF SAMPLING:PATTERNS

AND PROSPECTS

Detecting polyphyly requires the sampling of multiple individuals of the poly-

phyletic species as well as other species with which it sharesrelated alleles. As
noted earlier,however,the phylogenetic and phylogeographictraditionsinitially

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414 FUNK0 OMLAND

adoptedsamplingstrategiesthatsometimesprecludedpolyphylydetection.These
strategiesreflectthe tendenciesof these traditionsto deemphasizeintraspecificand
interspecificsampling,respectively,in their studies of closely relatedtaxa. These
differentsamplingstrategiesin turnreflectthe differinginterestsof these scientific
disciplines-phylogenetic structureversusmicroevolutionaryprocess-as well as
practicalconstraintson datacollection.
Yet sampling is clearly important(Ballard2000, Funk 1999, Hedin & Wood
2002, Omlandet al. 1999). The more individualssampled, the greaterthe like-
lihood of detecting polyphyly and when the interspecificallysharedalleles that
cause polyphyly are rare, very intensive samplingmay be requiredto document
this pattern(Wiens & Servedio 2000). For a given samplingintensity,polyphyly
detectionshouldgenerallybe increasedby dividingsamplesacrossthe phenotypic,
geographic,andphylogeneticdiversityof individualsand species thatmightplau-
sibly shareallelic lineages. An ideal studywould thus include all species believed
a priorito be closely related(e.g., congeners),maximize the geographicdiversity
of samples and the numberof samples collected from areasof sympatrybetween
study species, and sample broadly from known sources of biological variation
(subspecies,ecotypes, morphologicalvariants,etc.).
Our literaturesurvey providedempiricaldata on the distributionof sampling
patternsbetween 1990 and 2002, beginning with some of the earliest molecular
systematicstudiesusingDNA sequencedata.Becausewe includedonly those stud-
ies theoreticallycapableof detectingpolyphyly(those includingmultiplysampled
species), our estimatesof samplingintensitymay be somewhatupwardlybiased.
This survey shows a regularincrease and possible plateau in the frequency of
such studies (yearly frequencies from 1990 to 2002 = 3, 6, 7, 12, 22, 28, 39,
36, 53, 76, 96, 116, and 89, respectively). Two patternsare worth emphasizing
here (Figure 3). First, although studies vary greatly in sampling intensity, the
majority included no more than a few individuals and sampling localities per
study species. Second, median levels of four samplingparameters(total number
of congeneric species and individualssampled,mean numberof individualsand
localities sampledper study species) thatmightbe expectedto correlatepositively
with polyphyly detection have not notably increased over the 13 years of this
survey (Figure 3). While it is certainlytrue that investigationstreatingmultiple
well-sampledspecies arebecomingmorecommon,our surveysuggests thatmany
studies continueto adhereto an implicit early establishedstandardof acceptable
sampling.
The collection of mtDNA sequences is no longer nearly as onerous as during
the earlyyearsof our survey.However,the high-volumeautomaticsequencingand
declining costs thathave allowed the genomics revolutionhave not yet translated
into generally and considerablyimprovedsampling in mitochondrialstudies of
closely relatedanimal species. The contributionof improvedsamplingto the de-
tectionof polyphylyis indicatedby our survey,which shows thatspecies observed
to be polyphyleticwere represented,on average,by significantlymoreconspecific

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 SPECIES-LEVELPARAPHYLY 415

of
reflect
800 aspect
'02 '02 40 illustrates
1990-1993
700 partly
30
600 eachthe
500 20 different
for1990s
400 10 a
300 10 forSecond, early
individuals values
200
9 the
100 survey.
90 8 in
80 7 theMedian
localities/species
90 70
congeneric 90 of information
in values
3 2 6 of
47M .30 20 10 60
of (inset).
50 5 time.
studies
kinds median
40 4 number all surveythis
30 3 two at
20 number theHigher
10 2 Mean across of
0 1
0 Total 0 presents conducted
180160140120100806040 20 350300250200150100
50 years
intensity
plots 13 separately.
60 the studies
four
'02 50 300
'02
200 of samplingplotted
40 100 of across analysis
was
30 90 Each
80
20 70
year
10 60 intensity
survey. restriction
species
50 distribution
40
10 the following
30
9 20 sampling
8 10 literature
of each
individuals/species
90 t 7
103 10
9 of theillustrates
and
7 6 5 4 3 2 congeneric
6 4 2 8 level
6
of 7 fromplot data,
resource-inte
5 6
4 5 number median
each (less
4
3 3 thepooled
patterns of
Number
2
2 Mean First,
in on
1
0
14012010080 60 40 20 0 50 Samplingbased
250 200 150 100 proportion
intensity.
variation
3 were
studies of Number
higher
sampling
Figure
temporal
period
the

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416 FUNK0 OMLAND

individuals(22.6 versus 10.7), collection localities (6.1 versus3.8), andcongeneric

individuals(49.1 versus 26.9) thanotherspecies (one-way ANOVA,P < 0.001 in
all cases).
Werecommendthatimprovedanddiversifiedsamplingbe embracedas a timely
andimportantgoal thatmightallow a highly productivemergerof the phylogenetic
andphylogeographictraditions.Specifically,we encourageworkersto collect and
simultaneouslyanalyzephylogeographicdatafrommultipleclosely relatedspecies
and their infraspecificvariants(also see Barraclough& Nee 2001, Hey 1994).
These investigationsmight be facilitatedby the increasedexploitationof museum
materialand sequence data from public databasesand by increased investment
in the field work necessary to obtain diverse material.Investigationsin this new
traditionof "congenericphylogeography"will improvethe likelihoodof detecting
and appropriatelyinterpretingcritical patternsof intraspecificand interspecific
allelic variationto the benefit of systematicand populationbiology alike.

CONCLUSIONSAND FUTUREDIRECTIONS

This review describesthe diversecauses and consequencesof species-level poly-

phyly and representsthe firstlarge-scaleempiricalsurveyof polyphyleticspecies
and samplingpatternsin mitochondrialstudies of closely relatedanimaltaxa. We
demonstratepolyphylyto be a common, statisticallysupported,andtaxonomically
generalaspectof mitochondrialgene trees.We findthatsamplingintensityhas not
increased concurrentlywith the increasing ease of collecting mitochondrialse-
quence data. We call for the combinationof phylogenetic and phylogeographic
approachesto samplingin a new traditionof congenericphylogeography.Simi-
lar surveys of nuclear loci might be informative,althougha greaterdiversity of
loci and many fewer studies may as yet make numericalcomparisonsdifficult.
Surveys of, and comparisonswith, the botanicalliteraturemight be particularly
informativeas polyphyly is suspectedto be more common in plantsand has been
embracedas a fundamentalaspectof phylogeneticvariationby the botanicalcom-
munity(Crisp& Chandler1996, Rieseberg& Brouillet 1994). Increasedattention
to samplingandthe interpretationof polyphyly acrossgenes andtaxawill provide
improvedinsightsin systematics,populationgenetics, andevolutionarybiology in
general.

ACKNOWLEDGMENTS
We thank John Avise, William Ballard, Jason Baker, Mike Braun, John Burke,
Matt Hare, Rick Harrison,Marshall Hedin, Leo Joseph, Beatrice Kondo, Jeff
Peters, Ken Petren,Brad Shaffer,KerryShaw, Sonja Scheffer, and Paul Wilson
for helpful discussionsor commentson this manuscript.ElizabethHumphriesand
Paul Prasnikassisted in the compilationof the literaturedatabase.

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The Annual Reviewof Ecology,Evolution,and Systematicsis online at
http://ecolsys.annualreviews.org
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