The subspecies concept in butterflies: has its application in taxonomy and conservation biology...

REVIEW ARTICLE

The subspecies concept in butterflies: has itsapplication in taxonomy and conservation biologyoutlived its usefulness?

MICHAEL F. BRABY1,2*, RODNEY EASTWOOD3 and NEIL MURRAY4

1Museum and Art Gallery of the Northern Territory, GPO Box 4646, Darwin, NT 0801, Australia2School of Biological Sciences, The Australian National University, Canberra, ACT 0200, Australia3Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA, 02138, USA4Department of Genetics, La Trobe University, VIC 3086, Australia

Received 11 November 2011; revised 27 February 2012; accepted for publication 27 February 2012bij_1909 699..716

Subspecies lie at the interface between systematics and population genetics, and represent a unit of biologicalorganization in zoology that is widely used in the disciplines of taxonomy and conservation biology. In this review,we explore the utility of subspecies in relation to their application in systematics and biodiversity conservation, andbriefly summarize species concepts and criteria for their diagnosis, particularly from an invertebrate perspective.The subspecies concept was originally conceived as a formal means of documenting geographical variation withinspecies based on morphological characters; however, the utility of subspecies is hampered by inconsistencies bywhich they are defined conceptually, a lack of objective criteria or properties that serve to delimit their boundaries,and their frequent failure to reflect distinct evolutionary units according to population genetic structure. Moreover,the concept has been applied to populations largely comprising different components of genetic diversity reflectingcontrasting evolutionary processes. We recommend that, under the general lineage (unified) species concept, thedefinition of subspecies be restricted to extant animal groups that comprise evolving populations representingpartially isolated lineages of a species that are allopatric, phenotypically distinct, and have at least one fixeddiagnosable character state, and that these character differences are (or are assumed to be) correlated withevolutionary independence according to population genetic structure. Phenotypic character types include colourpattern, morphology, and behaviour or ecology. Under these criteria, allopatric subspecies are a type of evolution-arily significant unit within species in that they show both neutral divergence through the effects of genetic driftand adaptive divergence under natural selection, and provide an historical context for identifying biodiversity unitsfor conservation. Conservation of the adaptedness and adaptability of gene pools, however, may require additionalapproaches. Recent studies of Australian butterflies exemplify these points. © 2012 The Linnean Society ofLondon, Biological Journal of the Linnean Society, 2012, 106, 699–716.

ADDITIONAL KEYWORDS: biological species concept – conservation genetics – geographical variation –hybrid population – introgression – phylogenetic species concept.

INTRODUCTION

The species level of biological organization is thefundamental unit of analysis in ecology, evolution,and conservation biology (Sites & Crandall, 1997; de

Queiroz, 1998, 2007; Sites & Marshall, 2003; Isaac,Mallet & Mace, 2004; Balakrishnan, 2005; Petit &Excoffier, 2009). Subspecies represent a lower unit ofbiological organization and also are relevant in biodi-versity conservation (Ryder, 1986; Avise, 1989; Zink,2004; Haig et al., 2006), with many taxa listed underthe International Union for the Conservation ofNature (IUCN) Red List criteria (IUCN Standardsand Petitions Subcommittee, 2010), appendices in the

*Corresponding author. Current address: BiodiversityConservation Division, Department of Natural Resources,Environment, the Arts and Sport, PO Box 496, PalmerstonNT 0831, Australia. E-mail: [email protected]

Biological Journal of the Linnean Society, 2012, 106, 699–716. With 4 figures

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© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 699–716 699

Convention on International Trade in EndangeredSpecies of Wild Flora and Fauna (CITES, 2012), andvarious national legislation [e.g. Australian Environ-ment Protection and Biodiversity Conservation Act1999 (EPBC Act), US Endangered Species Act 1973(ES Act)]. Under the most recent edition of theInternational Code of Zoological Nomenclature(ICZN) (ICZN, 1999), subspecies are recognized in thespecies-group category as a formal trinominal, that is,the scientific name of a subspecies is a combination ofthree names (Article 5.2). By contrast, other infraspe-cific units, such as ‘varieties’, ‘races’ and ‘forms’, arenot formally recognized under the ICZN. However,although the ICZN regulates the structure, validationand usage of a particular trinomen, it does notdefine subspecies or say what they should be. TheCode is neutral on all matters of taxonomic theoryand practice, and the methods adopted to circum-scribe subspecies rest very much on the shoulders ofthe taxonomist. Subspecies are deemed to be avail-able names of the species group if they meet certaincriteria.

The subspecies concept was originally conceived inthe late 19th Century as a formal means of document-ing geographical variation or units of variation withinanimal species that had been identified as geographi-cal races based on morphological differences (Mayr,1942, 1963, 1982b; Wilson & Brown, 1953; Mallet,2001, 2004). According to Mayr (1942, 1963), a subspe-cies is an aggregation of phenotypically similarpopulations of a species inhabiting a geographicalsubdivision within the overall range and differing fromother such subdivisions of the species. Taxonomistshave not always adhered to this definition and, conse-quently, subspecies have been ascribed to a range ofpatterns, morphological and otherwise (Mallet, 2001).

In general, most subspecies have been proposedto designate morphologically distinct populationsaccording to two contrasting criteria. First, they havebeen applied to geographically isolated (allopatric)populations that are assumed to be genetically dis-tinct (Wilson & Brown, 1953; Mayr, 1982b; O’Brien &Mayr, 1991; Mallet, 1995; Moritz, 2002). Second, theyhave been used to delineate subsets within contigu-ously distributed (parapatric) populations that areable to interbreed at zones of contact. For allopatricsubspecies, it has been assumed that they representan incipient stage of differentiation, that is, they arein the process of independent speciation with minimalgene flow so that they comprise distinct evolutionarygroups according to neutral molecular markerssuch as mitochondrial DNA (mtDNA), microsatellites,allozymes or amplified fragment length polymor-phisms (AFLP) (Zink, 2004; Gompert et al., 2006;Joyce et al., 2009). For parapatric subspecies, anabrupt change in a character (step) that otherwise

shows clinal variation over much of the geographicalrange and the presence of intermediate forms(intergradation) have been used to define the geo-graphical boundaries or zones of contact, although inpractice delineation of these subspecies often repre-sent arbitrary divisions of a primary cline (Wilson &Brown, 1953; Gillham, 1956; Mayr, 1982b; James,2010). What has not been widely appreciated is that,from a theoretical viewpoint, these two concepts ofsubspecies largely reflect different components ofgenetic diversity (and hence different evolutionaryprocesses operating) within species (Fig. 1). Allopatricsubspecies are expected to show both adaptive andneutral divergence in which phylogeographical struc-ture has been driven primarily by the effects ofgenetic drift, whereas parapatric subspecies reflectunits of local adaptive divergence in which theobserved phenotypic differences are driven primarilythrough the forces of natural selection, whereasneutral genetic markers may introgress extensively.

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Figure 1. Conceptual model of species boundaries in rela-tion to the two major components of genetic diversity(adapted from Moritz, 2002). The two dimensions of geneticdiversity emphasize different evolutionary processes oper-ating within species. Adaptive divergence arises primarilyunder natural selection and is assessed by phenotypicvariation, whereas neutral divergence arises primarilythrough genetic drift as a result of fixed mutations inpopulations that have been isolated historically, eitherthrough vicariance or long-distance dispersal, and is ana-lyzed by molecular phylogeographical structure. Subspecies(zone between solid and dashed lines) have been delineatedaccording to criteria that reflect both dimensions of geneticdiversity, with phenotypic variation predominating inparapatric populations and phylogeographical structure inallopatric populations.

700 M. F. BRABY ET AL.

© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 699–716

A further problem with the subspecies concept,from a practical point of view, is its applicationin legislative policy and biodiversity conservation.Subspecies are frequently the focus of protectivelegislation and conservation programmes, but incon-sistencies in subspecies taxonomy as a result of dis-agreements in species concepts and a lack ofstandardized criteria for their diagnosis, togetherwith biases in taxonomic coverage of groups/regions,have made it difficult for government agencies andnongovernment organizations to evaluate their valid-ity in the listing process for conservation (Stanford,2001; Haig et al., 2006; Gippoliti & Amori, 2007;James, 2010). Given that taxonomy is a major scien-tific discipline that underpins the preservation ofbiological diversity, there is an urgent need for anevidence-based rigorous definition of subspecies toallow prioritization of resources for the protection andeffective conservation management of that diversity.

There are thus two issues in zoology regardingsubspecies that have not been resolved adequately:

(1) should taxonomists continue to recognize subspe-cies in accordance of rules of the ICZN and, if so, howshould they be defined conceptually and in practice(i.e. what objective criteria or properties serve todelimit their boundaries?) and (2) should subspeciescontinue to be used as infraspecific units in conser-vation biology under frameworks such as the IUCN?

In this review, we begin by defining the subspeciesproblem and ask ‘why are there recognizably differentpopulations within a species?’ Given that intraspecificdifferences exist, how do we then define populations ofa species that are distinct in certain characters butnot different enough to be recognized as full species?Recognition of diversity below the species level hasseveral advantages, including a deeper understandingof intraspecific variation, insights into the adaptabil-ity of organisms as opposed to plasticity responsesto the environment, and knowledge of historical evo-lutionary processes and speciation (Fig. 2). Theseconsiderations are then applied to the question ofeffective conservation management (e.g. of discrete

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e - - reproductively isolated - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - ecologically distinct - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - mate recognition systems - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - reciprocally monophyletic - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - limited or no gene flow - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - diagnosable - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - phenotypically distinct - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Figure 2. The general lineage (unified) species concept (adapted from de Queiroz, 1998, 2007) showing a single ancestrallineage (species A′) diverging to form two daughter lineages (species B and C) over evolutionary time. The grey zone betweenthe two solid horizontal lines represents the time during which the daughter lineages acquire different properties (shownas dashed lines), which serve as operational criteria for recognizing species boundaries under different concepts. There isunanimous agreement amongst taxonomists about the number of species represented by the black bars above and below thesolid lines, although there is frequent disagreement in the grey zone between these lines. Subspecies would be recognizedin the grey zone, perhaps early in the phase of lineage divergence with the acquisition of only a few properties.

THE SUBSPECIES CONCEPT 701


populations or distinct population segments) over aspecies’ geographical range. However, do such popu-lations deserve formal taxonomic status and recogni-tion in conservation programmes?

The taxonomic and conservation problems associ-ated with subspecies have been extensively reviewedfor mammals (Stanford, 2001; Gippoliti & Amori,2007), birds (Mayr, 1982b; Zink, 2004; James, 2010),and plants (Hamilton & Reichard, 1992; McDade,1995); however, relatively little attention has beenpaid to the utility of subspecies in invertebrates.Indeed, most attention to subspecies has been forvertebrates and vascular plants, whereas compara-tively few invertebrates and fungi have subspecificnames (Haig et al., 2006). The butterflies (Insecta:Lepidoptera), however, are an exception of an extantinvertebrate group in which trinominal nomenclatureis rife (Gillham, 1956; Mallet, 2001; Haig et al., 2006;Vane-Wright & Tennent, 2011). Here, we review recentstudies on Australian butterflies that have beenaccorded subspecific names.

SUBSPECIES IN AUSTRALIANBUTTERFLIES

Australia followed the European trend set by promi-nent entomologists such as Lord Rothschild andHans Fruhstorfer in the late 1800s and Karl Jordanin the early 1900s to apply trinominal nomenclatureconsistently and name every last geographicalvariety of butterfly as a formal subspecies (Mallet,1995, 2001; New, 1999; Edwards, Newland & Regan,2001; Isaac et al., 2004; Descimon & Mallet, 2009).During the 20th Century, many of the earlier Aus-tralian taxonomists devoted considerable effort inproviding subspecific names for populations from dif-ferent parts of a species range that they recognizedas being qualitatively different, the consequence ofwhich is that a plethora of subspecific names amongAustralian butterflies has been erected over the past100 years (Waterhouse & Lyell, 1914; Common &Waterhouse, 1981). Most of these subspecies havebeen distinguished on the basis that they comprisephenotypically distinct allopatric populations (New,1999). However, parapatric subspecies have alsobeen proposed for many polytypic species (Fig. 3). Inthe butterflies, and other ‘charismatic’ groups suchas birds for which there is considerable morphologi-cal, biological, and ecological information, subspeciesare usually recognized by possessing one or moreminor but usually diagnosable phenotypic or mor-phological differences in size, wing pattern, andgeneral coloration from the nominate subspecies(Gillham, 1956). For example, subspecies may differin quantitative ratio (continuous) character states

such as body size, the extent or width of marginalbands or the size of spots, or differ in qualitativenominal (discrete) character states such as thecolour of patches or presence/absence of wing patternelements (Fig. 3). It has generally been assumedthat these small character (phenotypic) differencesamong subspecies reflect population differentiationthat has a heritable (genetic) basis as a result ofselection and genetic assimilation and not to envi-ronmental (nongenetic) factors interacting with thegenotype.

Historically, if a population of an Australian but-terfly was discovered from a new locality and itappeared rather different, it was named as a subspe-cies, often uncritically with insufficient samples, andwithout any real consideration of the pattern of phe-notypic variation, detailed knowledge of the geo-graphical range, extent of isolation and dispersal, andpopulation size (New, 1999). Indeed, Braby (2000,

Figure 3. Geographical variation and subspecies in theAustralian satyrine butterfly Tisiphone abeona. The sixrecognized subspecies comprise two major groups, three inthe northern areas of the range and three in the southernpart of the range. A limited hybrid zone occurs in centralcoastal New South Wales where the two groups meet inwhich populations are highly variable and individualsfrequently possess character states of both groups. Lettersdenote subspecies: A, T. abeona rawnsleyi; B, T. abeonamorrisi; C, T. abeona regalis; D, T. abeona aurelia; E,T. abeona abeona; F, T. abeona albifascia; G, H, hybridzone ‘joanna’. Images of butterflies modified and repro-duced with permission from Braby (2004: 158–159), withimage of T. abeona aurelia courtesy of Australian Museum,Sydney.



2010) did not recognize many of the ‘traditional’ sub-species of Australian butterflies because either thedistributions were continuous such that the pattern ofvariation appeared to be clinal without obvious steps(i.e. taxa were deemed to represent historical sam-pling artefacts), or reliable diagnostic morphologicalcharacters were lacking (i.e. taxa were based on‘average’ but not unique phenotypic differences), orthe features on which the taxa were established wereinconsistent and too variable. In some cases, sub-species were actually junior synonyms that weresubsequently awarded subspecific status with littleobjectivity rather than being placed in synonymy(Waterhouse & Lyell, 1914; Common & Waterhouse,1981). In other cases, infraspecific taxa were actuallydescribed as geographical races or local forms. More-over, the concept of subspecies among butterflies inthe 20th Century shifted from one that emphasizedallopatric subspecies as ‘incipient species’ to the ideathat subspecies differ mostly because they representan adaptive response to different local environmentalconditions (Braby, 2000).

Perhaps the best-known and the most strikingexample of an Australian butterfly exhibiting complexgeographical variation is the polytypic satyrine Tisi-phone abeona from eastern and south-eastern Austra-lia, with six to eight subspecies being recognized(Waterhouse, 1914, 1928; Lucas, 1969; Conroy, 1971;Braby, 2000) (Fig. 3). Evidence from morphologicaland ecological properties, as well as reproductive com-patibility according to captive breeding experimentsand male reproductive structures (genitalia), suggeststhat the subspecies all belong to one species, unlikethe allopatric population of the congeneric T. helenafrom north-eastern Australia, which is considered tobe a distinct species (Braby, 1993). Similar to manybutterflies in Australia that exhibit complex geo-graphical variation, the subspecies concept in thisexample has been based largely on patterns of adap-tive phenotypic variation for parapatric populationsin which there are steep primary and secondary clinesin a number of quantitative characters (Lucas, 1969;Conroy, 1971). Conroy (1971) demonstrated that atleast three of these phenotypic characters (colour ofdorsal forewing patch, presence of cell bar on dorsalforewing, and width of cell bar on ventral forewing)are under genetic control; however, the various sub-species have not been assayed to determine patternsof phylogeographical structure according to molecularmarkers such as mtDNA.

The subspecies of T. abeona fall naturally into twobroad geographical groups, and there is a limitedhybrid zone where these two groups meet in centralcoastal New South Wales, although some wingpattern elements show more extended clines (Conroy,1971). Three subspecies from the northern end of the

range, the ‘northern group’, have cream markings ofvarying extent on the dorsal surface of both wings butno orange band on the forewing, and the hindwingeyespot is broadly ringed with orange–red; the creammarkings are only faintly developed in the northern-most subspecies. By contrast, the three subspeciesfrom the southern end of the range, the ‘southerngroup’, have a broad orange median band on thedorsal forewing but no cream markings on the hind-wing, and the hindwing eyespot is narrowly ringedwith red. A hybrid population (known as form‘joanna’), from the Port Macquarie region, New SouthWales, is highly variable and has arisen throughintrogression (the infiltration of genes from one set ofdifferentiated populations into another) between thenorthern coastal subspecies T. abeona morrisi and thesouthern coastal subspecies T. abeona aurelia andbetween the montane subspecies T. abeona regalisand T. abeona aurelia (Conroy, 1971). In addition,laboratory hybrids resembling form ‘joanna’ havebeen produced by crossing T. abeona abeona (‘south-ern group’) with T. abeona morrisi or T. abeona raw-nsleyi (‘northern group’) (Waterhouse, 1928).

In this example, recognizably different populationswith varying phenotypes have thus arisen through acombination of local adaptation, introgression involv-ing hybridization after secondary contact, and pre-sumably genetic drift through geographical isolationand limited gene flow.

UTILITY OF SUBSPECIES IN TAXONOMY

Before addressing species concepts and explicit crite-ria, it is pertinent to summarize the utility of sub-species and to explore some of the arguments infavour of and against the use of such infraspecificunits in taxonomy. The utility of subspecies is increas-ingly becoming a pressing issue because the taxo-nomic status of many subspecies is controversialand inconsistent amongst practitioners (Hennig,1966; Vane-Wright, 2003; Vane-Wright & Tennent,2011).

Wilson & Brown (1953) and Gillham (1956) recom-mended that the subspecies concept be abandonedbecause subspecies are often poorly diagnosed, theirdelimitation is difficult to determine (especially forparapatric populations), and taxonomic decisionsmade for a particular set of populations are oftenarbitrary, subjective, and based on too few characters.They noted that, too frequently, little attention is paidto the quantitative analysis of clines in geographicalvariation and the establishment of charactersthat are genetically independent, as well as whetherthere is a general tendency for sets of characters toshow discordant variation and recur in more than one



geographical area. In short, it was concluded that ‘thesubspecies was not a concept of evolutionary biologybut simply a handle of convenience for the clericalwork of the museum curator . . . the primary use ofsubspecies is as a sorting device in collections’ (Mayr,1982b, pp. 594–595). Wilson & Brown (1953) con-cluded that subspecies be replaced with a system ofreference based on vernacular locality or geographicalarea names. Further deficiencies in the application ofsubspecies have become apparent during the past twodecades because of confusion with both concept andcriteria on which they are recognized. Moreover, anincreasing number of studies have shown incongru-ence between subspecies taxonomy and populationgenetic diversity as revealed by neutral genetic data(Avise, 1989; Avise & Ball, 1990; Burbrink, Lawson &Slowinski, 2000; Zink, 2004; Eastwood, 2006; Joyceet al., 2009). Several studies of vertebrates haveshown that subspecies often lack population geneticstructure; for example, major differences in allelefrequencies and/or phylogeographical patterns,indicative of evolutionary differentiation but wherephylogeographical diversity is present it is often notreflected in the existing taxonomic classification(Avise, 1989; Burbrink et al., 2000; Zink, 2004).

With respect to Australian butterflies, most subspe-cies have been described without quantitative analy-sis and comparison with other populations, and therehave been few attempts to examine critically whetherpatterns of geographical variation comprise primaryor secondary clines (Lucas, 1969; Pearse & Murray,1981; McQuillan & Ek, 1997; Braby, 2008). Moreover,there have been even fewer studies aiming to establishwhether geographical subspecies are differentiatedgenetically and comprise highly structured discreteallopatric subunits through the effects of drift, orcomprise parapatric subunits that are part of a pan-mictic population with gene flow among them but areresponding differentially to the relative effects ofselection acting over the range of the species (i.e.adaptive phenotypic variation). Schmidt & Hughes(2006) found in the lycaenid Ogyris amaryllis thatgenetic variation and differentiation among the four tosix putative subspecies was better explained by larvalfood plant affiliation than by geography. On theother hand, within Acrodipsas cuprea, which exhibitspolymorphic male colour pattern that is partitionedgeographically into three broad allopatric areas, East-wood & Hughes (2003) found a strong correlationbetween morphotype and haplotype variation fora 582-nucleotide fragment of cytochrome oxidasesubunit I (COI), with little mixing of haplotypesbetween morphotypes, indicating that, if trinominalnames were to be given to these unnamed populations,each would comprise a distinct unit. Conversely, East-wood et al. (2006) identified within Jalmenus evagoras

pronounced genetic structure that was not evident inthe present morphologically-based taxonomy butwhich was associated with minor but consistent geo-graphically correlated differences in adult phenotype.Eastwood et al. (2008) subsequently evaluated thetaxonomic status of J. eubulus, which previously hadbeen treated as a parapatric subspecies of J. evagoras,using a multidiscipline approach in which data frommultiple properties (adult colour pattern, morphology,ecology, phylogenetics, and genomics) were analyzed.Although the mean pairwise difference between thetwo taxa was only 0.85% based on a 615-nucleotidefragment of COI, the analysis indicated fixed differ-ences in the mitochondrial genomes and an absenceof matrilineal gene flow that was associated withpronounced phenotypic and ecological differencesbetween two species (Eastwood et al., 2008). This workalso clarified much confusion in the biology and con-servation status of J. eubulus (cf. Dunn, Kitching &Dexter, 1994; Sands & New, 2002).

In a detailed phylogeographical study of theTheclinesthes albocinctus complex (Eastwood, 2006),it was demonstrated that the species T. hesperiarestricted to south-western Western Australia ispoorly differentiated (Fig. 4). Populations of T. hespe-ria and T. albocinctus were not significantly differentmorphologically, behaviourally, ecologically or geneti-cally. Although considerable genetic diversity occurswithin T. hesperia and the average pairwise differencebetween T. albocinctus and T. hesperia is 1.01%, thetwo species were not recovered as reciprocally mono-phyletic lineages, with the ancestral haplotypesrestricted to south-western Western Australia so thatT. albocinctus was nested within T. hesperia. More-over, analysis of molecular variation indicated thatgenetic variation among populations of these twospecies is better explained by adult phenotype, geog-raphy (biogeographical refugia), and larval food plantthan by present taxonomic classification (Eastwood,2006). Eastwood (2006) hypothesized that the ances-tor of this species complex evolved as a peripheralisolate from T. miskini in the south-western corner ofWestern Australia via a larval food plant shift (fromFabaceae to Euphorbiaceae), and then it colonizedmuch of arid Australia during the Pleistocene. Sub-sequent periods of isolation resulted in some geneticdrift and localized morphological divergence withlittle contemporary gene flow, culminating in fourbroadly discrete allopatric forms or morphotypes,which could be designated as subspecies.

Despite limitations and misuse of the subspeciesconcept, particularly in the early 20th Century,several evolutionary biologists (Mayr, 1982b; Crusz,1986; Avise, 1989; O’Brien & Mayr, 1991; Mallet,1995; Moritz, 2002; Descimon & Mallet, 2009) andconservation biologists (Haig et al., 2006) are in



favour of subspecies, especially for populations inisland regions (Mayr, 1982b). Moritz (2002) concludedthat rather than abandoning units below the level ofspecies altogether, we persist with delineating allo-patric subspecies provided the concept is defined on

sound criteria consistent with our understanding ofevolutionary and ecological processes and taxonomicand conservation objectives in mind. Furthermore, itmay be prudent to retain trinominals for taxa withdiagnosable characters having allopatric ranges in

Figure 4. Phylogeography of the Theclinesthes albocinctus complex showing geographical distribution of the speciesT. albocinctus and T. hesperia, geographical variation in adult phenotype (morphotypes of the male summer form),distinguishing features of morphotypes, haplotype network based on 640-nucleotide fragment of the mitochondrialcytochrome oxidase subunit I gene, and populations sampled and their sample sizes (N) (sensu Eastwood, 2006).Theclinesthes albocinctus comprises at least two geographical forms in inland Australia and coastal South Australia,whereas T. hesperia consists of two subspecies in coastal south-western Western Australia, giving a total of fourmorphotypes (A–D) for the complex (Sibatani & Grund, 1978). Diagnostic character states for each morphotype arehighlighted in italics. The character types comprise wing colour pattern (dorsal ground colour), morphology (size of blacksubtornal spots on ventral hindwing), and a mate recognition character (presence/absence of androconia). The 15haplotypes are separated by single base mutations, with grey haplotypes comprising inferred changes in the nested cladeanalysis. Images of butterflies modified and reproduced with permission from Braby (2004: 287), with the image ofT. hesperia littoralis provided courtesy of Australian National Insect Collection, Canberra.



case future research finds them to be specificallydistinct (Frost, Kluge & Hillis, 1992).

SPECIES CONCEPTS

To address the first part of our subspecies problem,should subspecies continue to be recognized and whatcriteria should be used to delineate them, we mustfirst briefly summarize species concepts within thefield of systematics because the criteria used to definesubspecies hinge very much on the species conceptadopted. Defining species is problematic becausethere is no concept that, when applied to nature, isfree from ambiguities (Hey, 2001): all concepts havepractical and philosophical limitations (Sperling,1993, 2003; Mallet, 1995, 2004; Sites & Marshall,2003; Coyne & Orr, 2004; Balakrishnan, 2005; Abbott,Ritchie & Hollingsworth, 2008).

Two concepts (i.e. the biological and the phyloge-netic species concepts) have been particularly influen-tial (Hey, 2001) and merit comment. Most animalshave been delineated on biological criteria, or morpho-logical criteria as a proxy (e.g. reproductive structuresand wing pattern elements in Lepidoptera, Collins,1996), according to the biological species concept ofMayr (1982a), which has had a profound influencesince its inception almost 70 years ago (Mayr, 1942).Under the biological species concept, speciation isachieved most commonly via reproductive (geographi-cal) isolation; hence, one of the main criteria used todelimit variation within species is allopatry. Repro-ductive isolation is achieved by assortative mating(prezygotic isolation) and/or disruptive or divergentselection against hybrids (postzygotic isolation); it isusually measured in terms of the extent of reproduc-tive incompatibility as a result of reduced gene flowand genetic divergence among geographically-isolatedpopulations (Coyne & Orr, 2004).

The phylogenetic species concept of Cracraft (1983,1989, 1997), and its various forms (diagnostic concept,monophyletic concept), was proposed as an alternativeto the biological species concept with the rise of phy-logenetic systematics and the recognition of severalpractical and conceptual limitations with the biologi-cal species concept, most notably the assumption ofreproductive isolation, lack of consideration of histori-cal processes and relationships, and the tendency tolump taxa that might otherwise be evolutionarilydistinct (Nixon & Wheeler, 1990; Baum, 1992; Frostet al., 1992; Vogler & DeSalle, 1994; Baum &Donohue, 1995; Davis, 1996; Groves, 2004). The phy-logenetic species concept, however, is also not withoutits problems, one of which is that infraspecific unitssuch as significant populations or evolutionarily sig-nificant units (ESUs), distinct population segments,subspecies, and races are not distinguished (Avise,

1989; Mallet, 1995; Fraser & Bernatchez, 2001;Moritz, 2002; Isaac et al., 2004; Haig et al., 2006). Thisleads to substantial over-splitting of taxa resulting intaxonomic inflation, eroding the usefulness of speciesas a robust measure of the diversity of life (Mallet,2001, 2008; Isaac et al., 2004; Brooks & Helgen, 2010;James, 2010). That is, application of the phylogeneticspecies concept results in much higher estimates ofspecies diversity and higher levels of endemism as aresult of a smaller geographical range sizes of speciescompared to the biological species concept. A recentexample of the application of the phylogenetic speciesconcept in butterflies is the division of the singlewidely distributed monobasic pierid Pseudopontiaparadoxa from West Africa into five species, of whichtwo (Pseudopontia gola, Pseudopontia mabira) aredifferentiated according to molecular characters(AFLPs and nucleotide sequences) only but show neg-ligible and inconsistent or no morphological differ-ences compared to their sister species Pseudopontiaparadoxa s.s. (Mitter et al., 2011). Under the biologicalspecies concept P. gola and P. mabira, both of whichare allopatric, with the former having a particularlysmall, peripheral geographical range, would beregarded as ESUs of P. paradoxa.

The unified species concept (Fig. 2) proposed by deQueiroz (1998, 2007) is an alternative approach tothe biological and phylogenetic concepts in that itattempts to separate the concept from the propertiesor criteria that are used to delimit the boundariesof species (Sites & Marshall, 2003; Hey, 2006; Wiens,2007). The unified species concept is a generallineage-based concept that recognizes nominal speciesas diverging lineages through evolutionary time. Thatis, if a species is viewed as a lineage, or at least ahypothesis of a lineage, the underlying concept is thatit should either already have a history as a separatelineage or at least be forecast to have a separatefuture. According to de Queiroz (2007), species aredefined as ‘separately evolving metapopulation lin-eages’ (i.e. an inclusive population made up of con-nected subpopulations). This concept of a species thusunites the underlying theory or primary concept of allother species concepts (biological, phylogenetic, diag-nostic, ecological, evolutionary, genealogical, cohesion,morphological, genotypic cluster, etc.) that an ances-tral lineage (species) diverges to form two or moredaughter lineages or species. Moreover, the attributesby which alternative species concepts are defined areregarded as secondary properties (criteria) that defineor delimit the boundaries of species. Properties ofthese lineages (and hence species) include phenotypicdistinctiveness, diagnosable in terms of fixed morpho-logical character states that are non-overlapping orunique (a morphological gap in the sense of Darwin;Mallet, 2008), limited or no gene flow, component



genes reaching a state of reciprocal monophyly (i.e. alldescendant haplotypes of each group are derived froma single common ancestor), specific mate recognitionsystems, distinct ecologies, and reproductive isolation.As the daughter lineages diverge from their ancestrallineage, the number of different properties is expectedto accumulate (Fig. 2). The concept covers lineagesdiverging in both geographical space (allopatric) andecological space (broadly sympatric).

The properties serve as alternative operationalcriteria or lines of evidence relevant to assessing theseparation of lineages and hence for delimitingspecies. de Queiroz (1998, 2007) argued that most ofthe major disagreements amongst biologists ofwhether a particular set of metapopulations consti-tutes one or two species occur because the proper-ties arise at different times during the process ofspeciation, and not necessarily in the same order.Indeed, because any one property provides evidenceof lineage separation, species may be delimitedbased simply on one criterion, an action that inevi-tably will lead to incompatible species delimitationamongst taxonomists. Where both long-term isola-tion and differential adaptation arising under strongselective pressures are high, two different popula-tions would be regarded as two distinct species(evolutionary lineages) by most taxonomists (Moritz,2002) (Fig. 1). de Queiroz (2007) recommends that ahighly corroborated hypothesis of lineage separation(i.e. existence of two species) should be based onmultiple lines of evidence. That is, the more prop-erties (evidence) to distinguish between two lin-eages, the more confidence one has in rejecting thenull hypothesis of a single species and accepting thealternative hypothesis that the lineages have indeeddifferentiated (i.e. speciated) into two or morespecies (Mallet, 1995) (Table 1). Yeates et al. (2011)argued that this hypothesis testing process be itera-tive, that is, the species boundaries should be con-tinually refined and tested with new sources of data.

CRITERIA FOR SUBSPECIES

For the purposes of our subspecies problem, we haveadopted the general lineage (unified) species conceptas a framework to guide the selection of criteria. Theunified species concept does not explicitly deal withsubspecies and an obvious challenge for zoologists isto reconcile how subspecies (should they continue tobe recognized) might be defined under this conceptualframework. For simplicity, we have limited our dis-cussion to allopatric lineages diverging in geographi-cal space.

Hennig (1966) pointed out the lack of clear guide-lines for recognizing subspecies, noting discrepanciesin both the extent of divergence and quality of differ-ences among characters used to delineate subspecies.In an extensive review of taxonomic publications,Haig et al. (2006) found that none provided standard-ized criteria for delimiting subspecies boundaries.The selection of characters on which to delineatesubspecies requires careful consideration because it isapparent that some morphological characters are toolabile and unreliable (Burbrink et al., 2000), whereasothers are environmentally plastic and lack evidenceof heritability (Vogler & DeSalle, 1994; Joyce et al.,2009). In addition to environmental factors influenc-ing geographical variation, as opposed to genotypiceffects, selection of characters in butterflies is par-ticularly important because of the confounding effectsof seasonal variation (polyphenism), polymorphism,and mimicry causing intraspecific variation in pheno-type. Vane-Wright & Tennent (2011) argued that theextent of phenotypic plasticity in butterflies (andother biological considerations such as migration,local extinction and founder effects) needs to bestudied using a variety of techniques before attemptsare made to recognize subspecies.

In terms of criteria, Amadon (1949) proposed aquantitative 75% threshold for morphological charac-ters; that is, a subspecies is recognized when 75% ormore of a sample of specimens differs for a givencharacter state from the reference population.O’Brien & Mayr (1991) recommended that the sub-species concept be limited to geographical subunits orallopatric populations of extant species that are‘reproductively isolated’ by a physical barrier underthe framework of the biological species concept. Theysuggested the following criteria be used for recogni-tion of subspecies: (1) allopatry with a unique geo-graphical range (or habitat); (2) phylogeneticallyconcordant phenotypic characters; (3) geneticallydivergent as a result of an absence of gene flow;and (4) a unique natural history relative to othersubdivisions of the species. Under this set of fourproperties, they predicted that most subspecies willbe monophyletic and have the potential to become

Table 1. Hypothesis testing for taxonomic status of allo-patric populations. The null hypothesis of a single speciesis the default hypothesis and is rejected only if evidencefrom other multiple data sources (colour pattern, morphol-ogy, behaviour, ecology, genetics etc) supports the alterna-tive hypothesis of lineage divergence and monophyly; theprocess should be iterative so that the species boundariesare progressively refined and tested when new evidencebecomes available

Hypothesis Taxonomic status

H0 One species (with two or more subspecies)H1 Two or more species



new species over evolutionary time, with increasinglikelihood of further genetic differentiation and eco-logical adaptation. However, they noted that theproperty of monophyly may be violated becauseof ancestral hybridization and reproductive compat-ibility. Haig et al. (2006) emphasized two minimumcriteria for recognizing subspecies as practical man-agement units (i.e. the discreteness and the biologicalsignificance or distinctiveness of the population) in anattempt to reconcile inconsistencies in taxonomictreatments and their listing/delisting under protec-tive legislation.

Descimon & Mallet (2009) argued that subspeciesought to be based on one or more criteria (morphology,ecology or genetics) and applied to allopatric popula-tions that are ‘moderately differentiated’, that is,below that expected among closely-related sympatricspecies. Tobias et al. (2010) developed this ideafurther and proposed a quantitative method usingexplicit criteria for distinguishing between speciesand subspecies among allopatric populations of birdsunder the biological species concept. They firstassessed the level of divergence between closelyrelated, non-interbreeding sympatric species in fourindependent phenotypic character types determinedby multiple genes (i.e. morphology, vocalization,colour pattern, and behaviour or ecology) and thenused this calibration as a yardstick to assess thetaxonomic status of allopatric forms. Allopatric taxathat fell below a particular threshold were treated assubspecies. Importantly, they employed a standard-ized sampling technique, with traits sampled acrossthe geographical range; capped the number of scoresfor each category type; and scored only two morpho-logical characters, thereby minimizing the effect oftrivial differences and non-independence of charac-ters. Molecular characters, although desirable, wereexcluded because it was argued that genetic samples/data are currently unavailable for the majority oftaxa, especially from the tropics where much of thebiological diversity resides (Tobias et al., 2010). Thus,in ornithology, a subspecies is now typically definedas a breeding population that occupies a distinctsegment of the geographical range of its species andthat is measurably distinct in phenotype or genotype,or both (James, 2010).

It is clear from the general lineage (unified) speciesconcept that for any given pair of sister lineagesinterpretations of species’ (or subspecies’) boundarieswill vary because the distinctions between taxa, basedon various properties, hold only for the considera-tion of a single time-transect (Fig. 2). Indeed, it hasbeen noted that delimiting species boundaries anddiscriminating between species or subspecies statusfor some geographically isolated populations issomewhat arbitrary because the populations are

evolving through time (Darwin, 1859; Wilson &Brown, 1953; Mayr, 1982a). In other words, speciesmay be viewed as an arbitrary cut-off along a branchin the tree of life, and allopatric subspecies are simplya point along the continuum of population differen-tiation comprising nested smaller interbreedinggroups within species.

Hence, for a lineage undergoing allopatric specia-tion under a general lineage-based concept, subspe-cies would be recognized in the ‘grey zone’ (Fig. 2),perhaps early in the phase of lineage divergence withthe acquisition of only a few properties; for example,the lineage is phenotypically distinct, has one or afew fixed (heritable) diagnosable character differ-ences, and is genetically divergent. Under somespecies concepts, such as the phylogenetic speciesconcept or even phenetic concepts incorporatingmolecular divergences based solely on DNA barcodethresholds (Hebert et al., 2003, 2004), these proper-ties would qualify taxa as full species. However, ifthere is additional evidence from other propertiessuch as a lack of specific mate recognition systems(e.g. identical male genitalia, scent-organs, ultra-violet reflectance pattern or pheromones), similarontogeny and development (e.g. identical immaturestages), limited reproductive isolation with occasionalgene flow (e.g. shared haplotypes or lack of reciprocalmonophyly according to mtDNA), and similar ecologi-cal niche (e.g. same larval food plant and habitatspecialization), then the null hypothesis is acceptedand subspecific status is recommended for theseotherwise morphologically distinct allopatric popula-tions (Table 1).

EVOLUTIONARY SIGNIFICANT UNITSAND CONSERVATION

To address the second part of our subspecies problem,should subspecies continue to be used in conservationbiology, we must explore contemporary approachesadopted in population genetics. Uncertainties in sub-species concepts and the criteria by which subspeciesare defined become compounded in a conservationcontext because most management agencies lack suf-ficient taxonomic expertise to evaluate the validity ofsubspecies in relation to the listing process and theneed to optimize resources for protection and conser-vation management (Stanford, 2001; Haig et al., 2006;Gippoliti & Amori, 2007). In the State of Victoria, forexample, three narrow range endemic subspecies oflycaenid butterflies (Acrodipsas brisbanensis cyrilus,Paralucia pyrodiscus lucida, and Ogyris genovevaaraxes) have been listed under the Flora and FaunaGuarantee Act 1988 (FFG Act), and two of these areranked as threatened (Endangered or Vulnerable)



(Sands & New, 2002; New, Field & Sands, 2007).However, all of these subspecies are poorly diagnosed(Braby, 2000) and the species to which they belong arenot threatened at the national level, with each beingwidely distributed in eastern and south-eastern Aus-tralia. At the municipal and state levels, populationsof A. brisbanensis ‘ssp. cyrilus’ and P. pyrodiscus ‘ssp.lucida’ are truly threatened near the city of Mel-bourne and elsewhere in Victoria, although it is notknown if these populations are genetically distinct.

It has been suggested that subspecies ought torepresent populations possessing genetic structureindicative of evolutionarily potential to allow priori-tization of conservation efforts (Ryder, 1986; Avise,1989; Zink, 2004; Degner et al., 2007; Joyce et al.,2009). This notion that subspecies ought to representdistinct genetic units is, in part, synonymous with theconcept of the ESU (Ryder, 1986; Moritz, 1994, 2002;Vogler & DeSalle, 1994; Waples, 1995; Fraser & Ber-natchez, 2001), which was developed to provide anexplicit basis for prioritizing minimal units for con-servation by ensuring that the evolutionary history(and potential) within species is maximized, pro-tected, and maintained. Although there is lack ofagreement of criteria on which ESUs are recognized(Crusz, 1986; Vogler & DeSalle, 1994; Crandall et al.,2000; Goldstein et al., 2000; Kizirian & Donnelly,2004), in part because of disagreement over speciesconcepts, most definitions of the ESU concept avoidthe issue of the amount or extent of DNA sequencedivergence by considering the phylogenetic pattern orevolutionary processes for a set of populations thathave been historically isolated in time and space.Several criteria have been proposed to define ESUs,including a set of populations that is reciprocallymonophyletic for mtDNA alleles and showing signifi-cant divergence of allele frequencies at nuclear loci(Moritz, 1994), as well as a lineage demonstratinghighly restricted gene flow from other such lineageswithin the higher organizational level of the species(Fraser & Bernatchez, 2001); however, a consensushas not been reached on how to delineate suchinfraspecific units for conservation (Crandall et al.,2000; Moritz, 2002), although lack of ecologicalreplaceability (Crandall et al., 2000) is a clearprimary criterion for conserving local adaptedness.The working definition adopted by the US Fish andWildlife Service and the National Marine FisheriesService is that a population must satisfy two criteriato be considered an ESU: it must be substantiallyreproductively isolated from other conspecific popula-tion units (i.e. discrete), and it must represent animportant component in the evolutionary legacy ofthe species (Waples, 1995).

The criterion of reciprocal monophyly for ESUs(Moritz, 1994) may not hold at the species level, let

alone within species. Funk & Omland’s (2003) exten-sive review of the literature revealed significant levelsof polyphyly and paraphyly within species across awide spectrum of animals. Aside from problemsof incomplete geographical sampling, inadequatephylogenetic information (i.e. weak phylogeneticsignal), and imperfect taxonomy (e.g. misidentifiedspecimens, species boundaries misinterpreted), non-monophyletic patterns in gene trees of nominalspecies may arise through interspecific hybridization(introgression, hybrid speciation), incomplete lineagesorting (including peripatric or peripheral isolatesspeciation), and recent speciation events (Paetkau,1999; Crandall et al., 2000; Funk & Omland, 2003;Mallet, 2005; Petit & Excoffier, 2009). Furthermore,different molecular markers may have different genetrees as a result of varying rates of substitution; forexample, the faster evolving uni-parental haploidmitochondrial genes may reach reciprocal monophylybefore bi-parental diploid nuclear genes underneutral processes. In butterflies, however, mtDNAmay be more informative because of Haldane’s Ruleand the heterogametic sex being the female.

Reciprocal monophyly is probably a property morecharacteristic of sister species where the divergencesare particularly deep (Carstens & Knowles, 2007)than infraspecific categories such as ESUs and sub-species, and should not be a necessary criterion foridentifying units below the level of species (James,2010). Simulation studies indicate that from the timethat two daughter populations separate, it takesalleles a number of generations that is approximatelyfour times the effective population size (4Ne fornuclear genes) to reach a stage of reciprocal mono-phyly, proceeding successively through stages of poly-phyly and paraphyly (Neigel & Avise, 1986; Avise,2000). In cases of peripheral isolates speciation inwhich a small allopatric population has differenti-ated, the larger populations of the non-isolatedspecies will remain polymorphic in alleles for manygenerations and hence will be paraphyletic for thosealleles (Paetkau, 1999). In other words, paraphylyshould not be considered a criterion for rejecting unitsbelow the species level because relationships amongpopulations are reticulate. In such cases, species/subspecies boundaries can only be delimited by ref-erence to other properties, such as morphology,behaviour, and ecology (e.g. butterflies; Sperling,1993; Brower, 2006).

Historically, subspecies were proposed to documentgeographical patterns of morphological variation withlittle or no consideration of population genetics,whereas ESUs aim to capture the evolutionaryhistory within species for conservation purposes, irre-spective of that geographical variation. The ESUconcept does not apply to continuously distributed



populations, except in some cases of parapatry.Because ESUs are based on sound knowledge of thedistribution and historical relationships of extantpopulations, it is tempting to equate subspecies withESUs because both possess a spatial or biogeographi-cal criterion. But what of the converse: should allESUs be recognized as subspecies? The answer to thisquestion will probably be case-specific and dependent,for example, with respect to whether a pattern ofmtDNA divergence is correlated with divergence ofother properties such as phenotype; under Moritz’s(1994) concept, ESUs have diverged primarilythrough genetic drift with minimal or cryptic pheno-typic differentiation (Fig. 1). We contend that thecurrent trend to recognize and describe numerouscryptic species or subspecies based solely on historicreproductive isolation (mtDNA) but that do not differin any other attributes is misguided and that, unlessthe divergences are particularly deep, these popula-tions are probably better regarded as ESUs.

It is undeniable that some subspecies have beenerected or promoted so that taxon-based conservationlegislation will allow the listing of locally distinctpopulations as legally threatened, with a notoriousexample in Australia being the descriptions of variousreptiles by R. Hoser (Wüster et al., 2001). Some leg-islation allows the recognition of important popula-tions as ‘distinct’ (e.g. Australian EPBC Act and USES Act). Although the criteria for this designation aredebatably worded, we suggest that it is a more appro-priate path to the conservation of adaptability thanthe formal erection of subspecific taxa.

The objectives of conservation are to maintain theadaptedness and adaptability of organisms (Frankel,1970; Frankel & Soulé, 1981; Frankham, 2010). Thesecond part of our question relates to whether ornot subspecies are a useful or sufficient category toachieve these objectives of adaptability. We suggestthat, as defined below, the subspecies category servesthe purpose of identifying groups of populations thatare differentiated from other groups in such a waythat actually and potentially important genetic bio-logical diversity is recognized. Importantly, however,some populations may not satisfy all the criteria wesuggest, although they may represent gene poolsexhibiting local adaptedness and be of value forpotential adaptation to environmental change. Thereis therefore still a conservation need to identify andmanage genetically divergent populations. The ESUidea was developed partly to take account of thisneed, and not just because subspecific taxonomy wasinconsistent (Ryder, 1986). We consider that a broadapproach to ESU definition (Crandall et al., 2000) isstill required to meet this need. That is, we see agreater role for conservation genetics (i.e. one thatextends beyond simple definitions of ESUs), espe-

cially with respect to identifying and characterizinglocalized adaptation, as well as adaptive potential, inallopatric populations that are likely to become morecommon as landscape fragmentation and climatevariability intensifies. For example, in the Australianendemic butterfly Tisiphone abeona (Fig. 3), we do notsuggest that the ‘hybrid’ populations be formally rec-ognized as subspecies, or indeed designated as ESUs,but they are the outcome of selective processes thathave produced particular combinations of charactersin a particular place, and are therefore the mostlocally adapted gene pool available. Therefore, anargument can be made to allocate resources to con-serve these populations because they may be impor-tant reservoirs of adaptive genetic potential. In manycases, particularly for species that are widely distrib-uted across multiple land tenures, it may be morepractical to focus conservation efforts on managingESUs or distinct population segments as subset areaswithin the overall geographical range without theneed to list and protect the species or subspecies as awhole (Haig et al., 2006).

Hybrid populations and hybrid zones, either narrowor broad, pose special challenges to taxonomists andconservation biologists (Hewitt, 1988). Hybrid zonescomprise an area in which adjacent populations oftwo parapatric species (or subspecies) interbreed andexchange genes to produce viable hybrids, usuallyshowing recombinant forms or a set of clines in mor-phological characters that distinguish two or moreparental taxa. They may be primary in which intro-gression persists for some time period after initiallineage divergence, or secondary in which populationsof two taxa exchange genes as a result of the break-down of reproductive isolating mechanisms. Sands(2009) recently suggested that hybrid populationswithin species exhibiting complex geographical varia-tion, such as that observed in T. abeona, or indeedbetween two parapatric species in which the individu-als show intergradation or steep secondary clines in anumber of genetically independent morphologicalcharacters, be accorded trinominal names. Thissuggestion would be difficult to apply because thedistinction between primary and secondary intergra-dation is not always clear (Endler, 1977), and zones ofsecondary contact are now known to allow extensiveintrogression for some genes, whereas others showsharp clines governed by local environmental gradi-ents (Hewitt, 1988) that may be displaced from oneanother.

Although hybridization among recently divergedanimal species is more common in nature than pre-viously realised (Mallet, 2005, 2008; Abbott et al.,2008), as estimated to be 16% for European butter-flies for example (Descimon & Mallet, 2009), hybridspeciation (the spontaneous formation of new species



through interspecific hybridization) is much rarer(Mallet, 2007; Petit & Excoffier, 2009). However,unless hybrid speciation can be established beyonddoubt (Kunte et al., 2011), the utility of formallynaming hybrid populations must be seriously ques-tioned because of the extent of gene flow and inherentcharacter variability, and hence a lack of diagnosticcharacters. Nevertheless, hybrid populations andhybrid zones are of evolutionary significance and ofconservation importance because they may representthe best locally adapted gene pools, and thereforeintrinsically worth preserving regardless of taxonomicstatus (Hewitt, 1988; Mallet, 1995, 2008). In the caseof T. abeona, the hybrid population ‘joanna’ has bothconservation and evolutionary significance: the criti-cal breeding habitat is seriously threatened (Common& Waterhouse, 1981; New, 1984; Sands & New,2002), and field evidence indicates a heterozygoteadvantage at one major locus that determines aninter-subspecific difference in wing pattern (Conroy,1971). It provides a notable example of the evolution-ary processes operating within species (New, 1984),although it is generally not recognized as a formalsubspecies (Common & Waterhouse, 1981; Braby,2000).

DISCUSSION

It is clear from our review that the utility of subspe-cies in taxonomy and conservation biology is ham-pered by inconsistencies by which they are definedconceptually, a lack of standardized criteria or prop-erties that serve to delimit their boundaries, andtheir frequent failure to reflect distinct evolutionaryunits according to population genetic structure. Yet,the subspecies concept as a unit of biological organi-zation is recognized by the ICZN and entrenched inprotective legislation such as the EPBC Act, FFG Act,and ES Act, and many subspecific taxa listed underframeworks such as the IUCN receive substantialfunds for conservation to the extent that the conceptis unlikely to be abandoned in the immediate future,Australian butterflies being no exception (New, 1999).We contend that, unless subspecies capture elementsof recent evolutionary history, the concept has limitedutility in taxonomy and little relevance in modernpractical conservation. Moreover, there is a criticalneed to resolve the subspecies problem to assist man-agement agencies with the task of identifying unitswithin species for legislative protection and for allo-cating resources for conservation of genetic biodiver-sity (Haig et al., 2006).

For the subspecies concept to have broad utility inrecognizing that there are different populationswithin a species, such as butterflies that show pro-nounced patterns of phenotypic (geographical) varia-

tion, as well as reflecting evolutionary processesunder the general lineage (unified) species concept,there needs to be clearer definition and articulation ofcriteria on which to delimit their boundaries. Weembrace some of the properties advocated by earlierworkers and recommend that for extant animalgroups:

Subspecies comprise evolving populations that represent par-tially isolated lineages of a species that are allopatric, pheno-typically distinct, have at least one fixed diagnosablecharacter state, and that these character differences are, orassumed to be, correlated with evolutionary independenceaccording to population genetic structure.

We have provisionally limited our definition to popu-lations (lineages) that are demonstrably allopatricdiverging in geographical space, as opposed to para-patric populations, although we see no reason whythe subspecies concept could not be applied to broadlysympatric lineages diverging in ecological space. Forexample, sympatric lineages in ecological space mightbecome differentiated via partitioning time (e.g.diurnal activity, seasonality, voltinism), larval foodresources (e.g. plant taxa, plant parts), microhabitat(e.g. altitude, environmental gradients), etc., leadingto the evolution of well-defined ‘ecological races’within species (Santos et al., 2011). If these ecologicalraces are phenotypically distinct with at least onefixed diagnosable character state, then subspeciesstatus may be appropriate. In practice, however, mosttaxonomists are likely to regard such races as fullspecies that have diverged under sympatric or allo-chronic speciation.

In terms of phenotypic character differences amonglineages, we recommend the approach undertaken byTobias et al. (2010). For butterflies, at least threeindependent phenotypic character types determinedby multiple genes should be assessed: (1) colourpattern (up to three qualitative characters); (2) mor-phology (two quantitative characters); and (3) behav-iour or ecology (one character), giving a maximumtotal of six characters. Their quantitative methoddemands that each of these characters be scored andthen combined among undisputed sympatric speciesto determine critical thresholds for assessing taxo-nomic status of allopatric populations. However, aconsiderable amount of groundwork for each taxo-nomic group must be carried out before the methodcan be applied consistently and objectively. An alter-native qualitative approach might be to regard allo-patric populations as subspecies when they differ inonly one diagnosable phenotypic character type(either colour pattern, morphology or behaviour/ecology, with a total of one to three characters) but totreat such populations as species if they differ in allthree character types (but with a total of four to six



characters). Such an approach is possibly too simplis-tic but, if there is additional evidence, such as differ-ences in population genetic structure (e.g. reciprocalmonophyly) or characters that are associated withreproductive isolation (e.g. prezygotic – mate incom-patibility, or postzygotic – hybrid sterility, hybridinviability), as has been demonstrated in several sub-species of Australian butterflies (Pearse, 1978; East-wood et al., 2008), then those lineages are probablybetter regarded as distinct species. In other words, weadvocate the recognition of a single species (with twoor more subspecies) as the null hypothesis for a givenset of allopatric populations (lineages) and only acceptthe more complex alternative hypothesis of two ormore species if evidence from other multiple proper-ties provides a better fit supporting lineage diver-gence and monophyly (Fig. 2, Table 1).

Amadon (1949) proposed a frequency rule of 75%for phenotypic characters for a population to be rec-ognized as a subspecies; however, to reject the nullhypothesis of no difference among samples (i.e. < 75%of individuals have the character trait) with 95% levelof significance (a = 0.05), a minimum sample size of18 specimens is required to detect significant differ-ences (i.e. at least 17 individuals of the sample wouldhave to possess the trait to be 95% confident that75% of the population had the trait). An alternativeapproach might be to compare two samples, one fromthe reference population (nominate subspecies) andthe second from the population in question, and ask‘what is the probability that the two samples aredrawn from the same population?’ If the samples aresignificantly different at a given probability level ofcertainty, then the null hypothesis is rejected. In thisway, the frequency of character difference, and hencesample size, do not have to be specified a priori for thetest to have sufficient power.

Although desirable, we have not included informa-tion on genetic differences as a criterion for tworeasons. First, most species lack sufficient taxonomicsampling required to assess genetic variation withinand among populations. Second, there is currently alack of meaningful criteria as to what actually con-stitutes ‘genetic distinctness’ among populations.However, we assume that these populations aregenetically distinct. Phenotypic data (whether it isbased on colour pattern, morphology or behaviour/ecology) can imply limited gene flow, although geneticdata (microsatellites, AFLPs, mtDNA, allozymes,karyotypes, etc.) are needed to measure and quantifyit, and such data may be needed to distinguishbetween genotypic and environmental effects. In ourview, phenotypically divergent populations resultingfrom nonheritable environmental factors, rather thanfrom heritable factors, are best regarded as ‘localforms’, not subspecies. The definition by Fraser &

Bernatchez (2001) for ESUs, which are lineages dem-onstrating highly restricted gene flow from other suchlineages within the higher organizational level of thespecies, might be appropriate for subspecies. In otherwords, although we consider allopatry to be a neces-sary criterion, it does not follow that these popula-tions are necessarily reproductively isolated with nogene flow. Subspecies might be viewed as one type ofESU: a partially ‘isolated’ lineage that has not quiteseparated as a result of recent or contemporary geneflow but, all things being equal, is considered likely toachieve separate gene-pool status eventually throughreproductive isolation. Subspecies are populationswithin species that, although they are distinct incertain heritable phenotypic and molecular charac-ters, should not be expected to be reciprocally mono-phyletic according to mtDNA phylogeography. Assuch, subspecies reflect natural (evolutionary) group-ings that serve as operational units for biodiversityconservation. Populations exhibiting pronouncedgenetic structure in neutral markers but not differ-entiated phenotypically according to adaptive traitsare better regarded as ESUs in the broad senserather than as subspecies; as noted above, recognitionof these minimal units for biodiversity conservationensures that the evolutionary history within speciesis conserved without the need to apply trinominalnomenclature.

In general, we do not support the recognition ofparapatric subspecies because of problems in deter-mining the pattern of clinal variation, geographicalboundaries, and the location of steps (if they exist), alack of fixed or unique character states, extent ofintrogression (which may be primary or secondary inorigin), and because the populations frequently reflectdifferent components of genetic diversity subjected todifferent evolutionary processes compared to allopat-ric subspecies. Indeed, Mayr (1963) recommendedthat, when the geographical variation of a speciesis clinal, it is inadvisable to recognize subspecies.However, exceptions can be made for parapatric sub-species that are connected by a narrow hybrid zonethrough secondary contact (Barton & Hewitt, 1985;Hewitt, 1988). Under our definition, polytypic speciessuch as T. abeona (Fig. 3) would be reduced to threesubspecies (a single southern subspecies T. abeonaabeona s.l., and a ‘northern group’ with two subspe-cies T. abeona rawnsleyi and T. a. morrisi s.l.) inwhich two (T. abeona abeona, T. abeona morrisi) haverecently come into secondary contact following aperiod of range contraction and geographical isolationcaused by a putative barrier, the Cassilis Gap in theupper Hunter Valley during one or more of the Pleis-tocene glacial cycles, producing a limited hybrid zoneof intergradation through introgression (Conroy,1971).



Under our subspecies concept and criteria, theexample illustrating the four morphotypes and 15haplotypes identified in the T. albocinctus complex(Fig. 4), which includes the species T. hesperia, wouldbe regarded as belonging to a single widespreadspecies, T. albocinctus s.l., with minimally divergentallopatric populations that are equivalent to four sub-species. These populations are similar in morphology,behaviour, and ecology, although each shows minorbut consistent differences in wing colour pattern (onequalitative character), morphology (one quantitativecharacter), and a behavioural mate recognitionsystem character (the presence/absence of androco-nia), with each morphotype comprising a total of oneor two fixed diagnosable character states. They arealso distinct genetically but not reciprocally mono-phyletic according to mtDNA.

There is an urgent need to further integrate thedisciplines of taxonomy and conservation biology(Vane-Wright, 1996; Crozier, 1997; Sarkar et al., 2006;Margules & Sarkar, 2007; Braby, 2010; Cranston,2010; Yeates et al., 2011), especially conservationgenetics (Kohn et al., 2006). Such integration wouldprovide genomic information from multiple loci todetermine whether geographical patterns of pheno-typic variation within species correspond with pat-terns of genetic variation. For example, routinemolecular analysis can now readily identify deep evo-lutionary divergences (ESUs) that may not be selfevident in the present morphologically based tax-onomy, although there is still a need to evaluate thebiological, behavioural and ecological attributes ofthese lineages to clarify their taxonomic status. More-over, it is these infraspecific units or ESUs, whichinclude subspecies, that ought to guide conservationefforts in order to protect biological diversity and theprocesses that sustain it (Avise, 1989; Moritz, 1994,2002; Crozier, 1997; Zink, 2004). Within those areas,Moritz (2002) recommends protection of contiguousenvironmental gradients and heterogeneous land-scapes to maximize population viability and represen-tation of the adaptive component of genetic diversity.

ACKNOWLEDGEMENTS

We thank Dick Vane-Wright, James Mallet, DanSchmidt, John Trueman, Jane Hughes, MelanieNorgate, Paul Sunnucks, Chris Glasby, RichardWillan, and Neil Collier for their constructive andhelpful comments on earlier drafts of the manuscript,and Chris Baker for statistical advice. Scott Ginnand You Ning Su provided the digital images usedin Figures 3D and 4C, respectively, and CSIROPublishing kindly provided several images forreproduction.

REFERENCES

Abbott RJ, Ritchie MG, Hollingsworth PM. 2008. Intro-duction. Speciation in plants and animals: patterns andprocess. Philosophical Transactions of the Royal Society ofLondon Series B, Biological Sciences 363: 2965–2969.

Amadon D. 1949. The seventy-five percent rule for subspe-cies. The Condor 51: 250–258.

Avise JC. 1989. A role for molecular genetics in the recogni-tion and conservation of endangered species. Trends inEcology and Evolution 4: 279–281.

Avise JC. 2000. Phylogeography. The history and formation ofspecies. Cambridge, MA: Harvard University Press.

Avise JC, Ball RM. 1990. Principles of genealogical concor-dance in species concepts and biological taxonomy. OxfordSurveys in Evolutionary Biology 7: 45–67.

Balakrishnan R. 2005. Species concepts, species boundariesand species identification: a view from the tropics. System-atic Biology 54: 689–693.

Barton NH, Hewitt GM. 1985. Analysis of hybrid zones.Annual Review of Ecology and Systematics 16: 113–148.

Baum DA. 1992. Phylogenetic species concepts. Trends inEcology and Evolution 7: 1–2.

Baum DA, Donohue MJ. 1995. Choosing among alternative‘phylogenetic’ species concepts. Systematic Botany 20: 560–573.

Braby MF. 1993. Early stages, biology and taxonomic statusof Tisiphone helena (Olliff) (Lepidoptera: Nymphalidae:Satyrinae). Journal of the Australian Entomological Society32: 273–282.

Braby MF. 2000. Butterflies of Australia. Their identification,biology and distribution. Melbourne: CSIRO Publishing.

Braby MF. 2004. The complete field guide to butterflies ofAustralia. Melbourne: CSIRO Publishing.

Braby MF. 2008. Taxonomic review of Candalides absimilis(C. Felder, 1862) and C. margarita (Semper, 1879) (Lepi-doptera: Lycaenidae), with descriptions of two new subspe-cies. The Beagle, records of the Museums and Art Galleriesof the Northern Territory 24: 33–54.

Braby MF. 2010. The merging of taxonomy and conservationbiology: a synthesis of Australian butterfly systematics(Lepidoptera: Hesperioidea and Papilionoidea) for the 21stcentury. Zootaxa 2707: 1–76.

Brooks TM, Helgen KM. 2010. A standard for species.Nature 467: 540–541.

Brower AVZ. 2006. Problems with DNA barcodes for speciesdelimitation: ‘ten species’ of Astraptes fulgerator reassessed(Lepidoptera: Hesperiidae). Systematics and Biodiversity 4:127–132.

Burbrink FT, Lawson R, Slowinski JB. 2000. Mitochon-drial DNA phylogeography of the polytypic North Americanrat snake (Elaphe obsoleta): a critique of the subspeciesconcept. Evolution 54: 2107–2118.

Carstens BC, Knowles LL. 2007. Estimating species phy-logeny from gene-tree probabilities despite incompletelineage sorting: an example from Melanoplus grasshoppers.Systematic Biology 56: 400–411.



CITES. 2012. Convention on international trade in endan-gered species of wild flora and fauna. Available at: http://www.cites.org/

Collins MM. 1996. Charles Remington’s contributions of thespecies concept. Journal of the Lepidopterists’ Society 50:268–270.

Common IFB, Waterhouse DF. 1981. Butterflies of Austra-lia. Sydney: Angus and Robertson.

Conroy BA. 1971. Geographic variation and speciation in thesword grass brown butterfly, Tisiphone abeona Donovan.PhD Thesis, The University of Sydney.

Coyne JA, Orr HA. 2004. Speciation. Sunderland, MA:Sinauer Associates.

Cracraft J. 1983. Species concepts and speciation analysis.Current Ornithology 1: 159–187.

Cracraft J. 1989. Speciation and its ontology: the empericalconsequences of alternative species concepts for understand-ing patterns and processes of differentiation. In: Otte D,Endler JA, eds. Speciation and its consequences. Sunder-land, MA: Sinauer Associates, 28–59.

Cracraft J. 1997. Species concepts in systematics and con-servation biology – an ornithological viewpoint. In: ClaridgeMF, Dawah HA, Wilson MR, eds. Species: the units ofbiodiversity. London: Chapman and Hall, 325–339.

Crandall KA, Bininda-Emonds ORP, Mace GM, WayneRK. 2000. Considering evolutionary processes in conserva-tion biology. Trends in Ecology and Evolution 15: 290–295.

Cranston PS. 2010. Insect biodiversity and conservation inAustralasia. Annual Review of Entomology 55: 55–75.

Crozier RH. 1997. Preserving the information content ofspecies: genetic diversity, phylogeny, and conservationvalue. Annual Review of Ecology and Systematics 28: 243–268.

Crusz H. 1986. The dilemma of subspecies. Trends in Ecologyand Evolution 1: 104.

Darwin C. 1859. On the origin of species by means of naturalselection. London: John Murray.

Davis JI. 1996. Phylogenetics, molecular variation, andspecies concepts. BioScience 46: 502–511.

Degner JF, Stout IJ, Roth JD, Parkinson CL. 2007.Population genetics and conservation of the threatenedsoutheastern beach mouse (Peromyscus polionotus niveiven-tris): subspecies and evolutionary units. ConservationGenetics 8: 1441–1452.

Descimon H, Mallet J. 2009. Bad species. In: Settele J,Konvicka M, Shreeve TG, Van Dyck H, eds. Ecology ofbutterflies in Europe. Cambridge: Cambridge UniversityPress, 219–249.

Dunn KL, Kitching RL, Dexter EM. 1994. The conserva-tion status of Australian butterflies. Canberra: Unpublishedreport to Australian National Parks and Wildlife Service.381.

Eastwood RG. 2006. Ant association and speciation in Lycae-nidae (Lepidoptera): consequences of novel adaptations andPleistocene climate changes. PhD Thesis, Griffith Univer-sity.

Eastwood RG, Braby MF, Schmidt DJ, Hughes JM. 2008.Taxonomy, ecology, genetics and conservation status of the

pale imperial hairstreak (Jalmenus eubulus) (Lepidoptera:Lycaenidae): a threatened butterfly from the Brigalow Belt,Australia. Invertebrate Systematics 22: 407–423.

Eastwood RG, Hughes JM. 2003. Phylogeography of therare myrmecophagous butterfly Acrodipsas cuprea (Lepi-doptera: Lycaenidae) from pinned museum specimens.Australian Journal of Zoology 51: 331–340.

Eastwood RG, Pierce NE, Kitching RL, Hughes JM.2006. Do ants enhance diversification in lycaenid butter-flies? Phylogenetic evidence from a model myrmecophile,Jalmenus evagoras. Evolution 60: 315–327.

Edwards ED, Newland J, Regan L. 2001. Lepidoptera:hesperioidea, papilionoidea. Zoological catalogue of Austra-lia, Vol. 31.6. Melbourne: CSIRO Publishing.

Endler JA. 1977. Geographic variation, speciation, andclines. Princeton, NJ: Princeton University Press.

Frankel OH. 1970. Genetic conservation in perspective. In:Frankel OH, Bennett E, eds. Genetic resources in plants –their exploration and conservation. Oxford: Blackwell Scien-tific Publications, 469–489.

Frankel OH, Soulé ME. 1981. Conservation and evolution.Cambridge: Cambridge University Press.

Frankham R. 2010. Challenges and opportunities of geneticapproaches to biological conservation. Biological Conserva-tion 143: 1919–1927.

Fraser DJ, Bernatchez L. 2001. Adaptive evolutionary con-servation: towards a unified concept for defining conserva-tion units. Molecular Ecology 10: 2741–2752.

Frost DR, Kluge AG, Hillis DM. 1992. Species in contem-porary herpetology: comments on phylogenetic inferenceand taxonomy. Herpetological Review 23: 46–54.

Funk DJ, Omland KE. 2003. Species-level paraphyly andpolyphyly: frequency, causes, and consequences, withinsights from animal mitochondrial DNA. Annual Review ofEcology, Evolution and Systematics 34: 397–423.

Gillham NW. 1956. Geographic variation and the subspeciesconcept in butterflies. Systematic Zoology 5: 110–120.

Gippoliti S, Amori G. 2007. The problem of subspecies andbiased taxonomy in conservation lists: the case of mammals.Folia Zoologica 56: 113–117.

Goldstein PZ, DeSalle R, Amato G, Vogler AP. 2000.Conservation genetics at the species boundary. Conserva-tion Biology 14: 120–131.

Gompert Z, Nice CC, Fordyce JA, Forister ML, ShapiroAM. 2006. Identifying units for conservation using molecu-lar systematics: the cautionary tale of the Karner bluebutterfly. Molecular Ecology 15: 1759–1768.

Groves C. 2004. The what, why and how of primate tax-onomy. International Journal of Primatology 25: 1105–1126.

Haig SM, Beever EA, Chambers SM, Draheim HM,Dugger BD, Dunham S, Elliott-Smith E, Fontaine JB,Kesler DC, Knaus BJ, Lopes LF, Loschl P, Mullins TD,Sheffield LM. 2006. Taxonomic considerations in listingsubspecies under the U.S. Endangered Species Act. Conser-vation Biology 20: 1584–1594.

Hamilton CW, Reichard SH. 1992. Current practice in theuse of subspecies, variety, and forma in the classification ofwild plants. Taxon 41: 485–498.



Hebert PDN, Cywinska A, Ball SL, Dewaard JR. 2003.Biological identifications through DNA barcodes. Proceed-ings of the Royal Society of London Series B, BiologicalSciences 270: 313–321.

Hebert PDN, Penton EH, Burns JM, Janzen DH, Hall-wachs W. 2004. Ten species in one: DNA barcoding revealscryptic species in the neotropical skipper butterfly Astraptesfulgerator. Proceedings of the National Acadamy of Sciencesof the United States of America 101: 14812–14817.

Hennig W. 1966. Phylogenetic systematics. Urbana, IL:University of Illinois Press.

Hewitt GM. 1988. Hybrid zones – natural laboratories forevolutionary studies. Trends in Ecology and Evolution 3:158–167.

Hey J. 2001. The mind of the species problem. Trends inEcology and Evolution 16: 326–329.

Hey J. 2006. On the failure of modern species concepts.Trends in Ecology and Evolution 21: 447–450.

ICZN. 1999. International code of zoological nomenclature.London: International Trust for Zoological Nomenclature.

Isaac NJB, Mallet J, Mace GM. 2004. Taxonomic inflation:its influence on macroecolgy and conservation. Trends inEcology and Evolution 19: 464–469.

IUCN Standards and Petitions Subcommittee. 2010.Guidelines for using the IUCN red list categories and crite-ria, Version 8.1. Gland: IUCN Species Survival Commission.

James FC. 2010. Avian subspecies. Ornithological Mono-graphs 67: 1–5.

Joyce DA, Dennis RLH, Bryant SR, Shreeve TG, ReadyJS, Pullin AS. 2009. Do taxonomic divisions reflect geneticdifferentiation? A comparison of morphological and geneticdata in Coenonympha tullia (Müller), Satyrinae. BiologicalJournal of the Linnean Society 97: 314–327.

Kizirian D, Donnelly MA. 2004. The criterion of reciprocalmonophyly and classification of nested diversity at thespecies level. Molecular Phylogenetics and Evolution 32:1072–1076.

Kohn MH, Murphy WJ, Ostrander EA, Wayne RK. 2006.Genomics and conservation genetics. Trends in Ecology andEvolution 21: 630–637.

Kunte K, Shea C, Aardema ML, Scriber JM, Juenger TE,Gilbert LE, Kronforst MR. 2011. Sex chromosome mosa-icism and hybrid speciation among tiger swallowtail butter-flies. PLoS Genetics 7: e1002274.

Lucas AM. 1969. Clinal variation in pattern and colour incoastal populations of the butterfly Tisiphone abeona(Donovan) (Lepidoptera: Satyrinae). Australian Journal ofZoology 17: 37–48.

Mallet J. 1995. A species definition for the modern synthesis.Trends in Ecology and Evolution 10: 294–299.

Mallet J. 2001. Subspecies, semispecies, superspecies. In:Levin S, ed. Encyclopedia of biodiversity, Vol. 5. London:Academic Press, 523–526.

Mallet J. 2004. Poulton, Wallace and Jordan: how discoveriesin Papilio butterflies led to a new species concept 100 yearsago. Systematics and Biodiversity 1: 441–452.

Mallet J. 2005. Hybridization as an invasion of the genome.Trends in Ecology and Evolution 20: 229–237.

Mallet J. 2007. Hybrid speciation. Nature 446: 279–283.Mallet J. 2008. Hybridization, ecological races and the nature

of species: emperical evidence for the ease of speciation.Philosophical Transactions of the Royal Society of LondonSeries B, Biological Sciences 363: 2971–2986.

Margules CR, Sarkar S. 2007. Systematic conservation plan-ning. Cambridge: Cambridge University Press.

Mayr E. 1942. Systematics and the origin of species. NewYork, NY: Columbia University Press.

Mayr E. 1963. Animal species and evolution. Cambridge, MA:Harvard University Press.

Mayr E. 1982a. The growth of biological thought: diversity,evolution, and inheritance. Cambridge, MA: HarvardUniversity Press.

Mayr E. 1982b. Of what use are subspecies? Auk 99: 593–595.

McDade LC. 1995. Species concepts and problems in prac-tice: insight from botanical monographs. Systematic Botany20: 606–622.

McQuillan PB, Ek CJ. 1997. A biogeographical analysisof the Tasmanian endemic Ptunarra brown butterfly,Oreixenica ptunarra Couchman, 1953 (Lepidoptera:Nymphalidae: Satyrinae). Australian Journal of Zoology44: 21–37.

Mitter KT, Larsen TB, De Prins W, De Prins J, CollinsSC, Vande Weghe G, Sáfián S, Zakharov EV, Haw-thorne DJ, Kawahara AY, Regier JC. 2011. The butter-fly subfamily Pseudopontiinae is not monobasic: markedgenetic diversity and morphology reveal three new speciesof Pseudopontia (Lepidoptera: Pieridae). Systematic Ento-mology 36: 139–163.

Moritz C. 1994. Defining ‘evolutionary significant units’ forconservation. Trends in Ecology and Evolution 9: 373–375.

Moritz C. 2002. Strategies to protect biological diversity andthe evolutionary processes that sustain it. SystematicBiology 51: 238–254.

Neigel JE, Avise JC. 1986. Phylogenetic relationships ofmitochondrial DNA under various demographic models ofspeciation. In: Nevo E, Karlin S, eds. Evolutionary processesand theory. New York, NY: Academic Press.

New TR. 1984. Insect conservation – an Australian perspec-tive. Dordrecht: Dr W. Junk Publishers.

New TR. 1999. The evolution and characteristics of theAustralian butterfly fauna. In: Kitching RL, Jones RE,Scheermeyer E, Pierce NE, eds. Biology of Australianbutterflies. Monographs of Australian lepidoptera, Vol.6. Melbourne: CSIRO Publishing, 33–52.

New TR, Field RP, Sands DPA. 2007. Victoria’s butterfliesin a national conservation context. The Victorian Naturalist124: 243–249.

Nixon KC, Wheeler QD. 1990. An amplification of the phy-logenetic species concept. Cladistics 6: 211–223.

O’Brien SJ, Mayr E. 1991. Bureaucratic mischief: recognis-ing endangered species and subspecies. Science 251: 1187–1188.

Paetkau D. 1999. Using genetics to identify intraspecificconservation units: a critque of current methods. Conserva-tion Biology 13: 1507–1509.



Pearse FK. 1978. Geographic variation and natural selectionin the Common Brown butterfly Heteronympha merope.PhD Thesis, La Trobe University.

Pearse FK, Murray ND. 1981. Clinal variation in theCommon Brown butterfly Heteronympha merope merope(Lepidoptera: Satyrinae). Australian Journal of Zoology29: 631–642.

Petit RJ, Excoffier L. 2009. Gene flow and species delimi-tation. Trends in Ecology and Evolution 24: 386–393.

de Queiroz K. 1998. The general lineage concept of species,species criteria, and the process of speciation: a conceptualunification and terminological recommendations. In:Howard DJ, Berlocher SH, eds. Endless forms: species andspeciation. Oxford: Oxford University Press, 57–75.

de Queiroz K. 2007. Species concepts and species delimita-tion. Systematic Biology 56: 879–886.

Ryder OA. 1986. Species conservation and systematics: thedilemma of subspecies. Trends in Ecology and Evolution 1:9–10.

Sands DPA. 2009. Stretching the trinomials: what are limitsfor recognizing a subspecies? News Bulletin of the Entomo-logical Society of Queensland 37: 65–66.

Sands DPA, New TR. 2002. The action plan for Australianbutterflies. Canberra: Environment Australia.

Santos H, Burban C, Rousselet J, Rossi J-P, Branco M,Kerdelhué C. 2011. Incipient allochronic speciation in thepine processionary moth (Thaumetopoea pityocampa, Lepi-doptera, Notodontidae). Journal of Evolutionary Biology 24:146–158.

Sarkar S, Pressey RL, Faith DP, Margules CR, Fuller T,Stoms DM, Moffett A, Wilson KA, Williams KJ, Will-iams PH, Andelman S. 2006. Biodiversity conservationplanning tools: present status and challenges for the future.Annual Review of Environment and Resources 31: 123–159.

Schmidt DJ, Hughes JM. 2006. Genetic affinities amongsubspecies of a widespread Australian lycaenid butterfly,Ogyris amaryllis (Hewitson). Australian Journal of Zoology54: 429–446.

Sibatani A, Grund RB. 1978. A revision of the Theclinesthesonycha complex (Lepidoptera: Lycaenidae). Transactions ofthe Lepidopterological Society of Japan 29: 1–34.

Sites JW, Crandall KA. 1997. Testing species boundaries inbiodiversity studies. Conservation Biology 11: 1289–1297.

Sites JW, Marshall JC. 2003. Delimiting species: a renais-sance issue in systematic biology. Trends in Ecology andEvolution 18: 462–470.

Sperling FAH. 1993. Mitochondrial DNA variation andHaldane’s rule in the Papilio glaucus and P. troilus speciesgroups. Heredity 71: 227–233.

Sperling FAH. 2003. Butterfly molecular systematics: fromspecies definitions to higher-level phylogenies. In: Boggs CL,Watt WB, Ehrlich PR, eds. Butterflies: ecology and evolutiontaking flight. Chicago, IL: University of Chicago Press,431–458.

Stanford CB. 2001. The subspecies concept in primatology:the case of mountain gorillas. Primates 42: 309–318.

Tobias JA, Seddon N, Spottiswoode CN, Pilgrim JD,Fishpool LDC, Collar NJ. 2010. Quantitative criteria forspecies delimitation. Ibis 152: 724–746.

Vane-Wright RI. 1996. Systematics and the conservation ofbiological diversity. Annals of the Missouri BotanicalGarden 83: 47–57.

Vane-Wright RI. 2003. Indifferent philosophy versusalmighty authority: on consistency, consensus and unitarytaxonomy. Systematics and Biodiversity 1: 3–11.

Vane-Wright RI, Tennent J. 2011. Colour and size inJunonia villida (Lepidoptera, Nymphalidae): subspecies orphenotypic plasticity? Systematics and Biodiversity 9: 289–305.

Vogler AP, DeSalle R. 1994. Diagnosing units of conserva-tion managment. Conservation Biology 8: 354–363.

Waples RS. 1995. Evolutionary significant units and theconservation of biological diversity under the EndangeredSpecies Act. In: Nielsen J, ed. Evolution and the aquaticecosystem. Bethesda, MD: American Fisheries Society, 8–27.

Waterhouse GA. 1914. A monograph of the genus TisiphoneHübner. Australian Zoologist 1: 15–19.

Waterhouse GA. 1928. A second monograph of the genusTisiphone Hübner. Australian Zoologist 5: 217–240.

Waterhouse GA, Lyell G. 1914. The butterflies of Australia.A monograph of the Australian Rhopalocera. Sydney: Angusand Robertson.

Wiens JJ. 2007. Species delimitation: new approaches fordiscovering diversity. Systematic Biology 56: 875–878.

Wilson EO, Brown WL. 1953. The subspecies concept and itstaxonomic application. Systematic Zoology 2: 97–111.

Wüster W, Bush B, Keogh JS, O’Shea M, Shine R.2001. Taxonomic contributions in the ‘amateur’ literature:comments on recent descriptions of new genera and speciesby Raymond Hoser. Litteratura Serpentium 21: 67–79,86–91.

Yeates DK, Seago A, Nelson L, Cameron SL, Joseph L,Trueman JWH. 2011. Integrative taxonomy, or iterativetaxonomy? Systematic Entomology 36: 209–217.

Zink RM. 2004. The role of subspecies in obscuring avianbiological diversity and misleading conservation policy. Pro-ceedings of the Royal Society of London Series B, BiologicalSciences 271: 561–564.



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