An integrative approach to delimiting species in a rarebut widespread mycoheterotrophic orchidCRAIG F. BARRETT* and JOHN V. FREUDENSTEIN†*L. H. Bailey Hortorium and Department of Plant Biology, 412 Mann Library, Cornell University, Ithaca, NY 14850, USA,†Department of Evolution, Ecology, and Organismal Biology, The Ohio State University Museum of Biological Diversity,1315 Kinnear Road, Columbus, OH 43212, USAAbstractIn the spirit of recent calls for species delimitation studies to become more pluralistic,incorporating multiple sources of evidence, we adopted an integrative, phylogeographicapproach to delimiting species and e volutionarily significant units (ESUs) in theCorallorhiza striata species complex. This rare, North American, mycoheterotrophic orchidhas been a taxonomic challenge regarding species boundaries, displaying complex patternsof variation and reduced vegetative morphology. We employed plastid DNA, nuclear DNAand morphometrics, treating the C. striata complex as a case study for integrative speciesdelimitation. We found evidence for the differentiation of the endangered C. bentleyi(eastern USA) + C. striata var. involuta (Mexico) from the remaining C. striata (= C. stri-ata s.s.; USA, Canada, Mexico). Corallorhiza striata involuta and C. bentleyi, disjunct bythousands of kilometres (Mexico-Appalachia), were genetically identical but morpholog-ically distinct. Evidence suggests the C. striata complex represents three species:C. bentleyi, C. involuta and a widespread C. striata s.s under operational criteria ofdiagnosability and common allele pools. In contrast, Bayesian coalescent estimationdelimited four species, but more informative loci and a resultant species tree will be neededto place higher confidence in future analyses. Three distinct groupings were identifiedwithin C. striata s.s., corresponding to C. striata striata, C. striata vreelandii, andCalifornian accessions, but these were not delimited as species because of occupying acommon allele pool. Each comprises an ESU, warranting conservation considerations. Thisstudy represents perhaps the most geographically comprehensive example of integrativespecies delimitation for any orchid and any mycoheterotroph.Keywords: integrative taxonomy, morphology, phylogeography, populationReceived 29 November 2010; revision received 31 March 2011; accepted 13 April 2011IntroductionExplicit species delimitation studies represent an essen-tial component of systematics, yet they are currentlyunderrepresented relative to phylogenetic studies(Wiens 2007). This is unfortunate, because delimitingspecies represents a crucially important first step inframing studies of conservation, ecology, evolution,phylogenetics and population genetics. However, thereis no universal consensus on how this should be accom-plished, although numerous methods have been pro-posed (e.g. Davis & Nixon 1992; Baum & Shaw 1995;Doyle 1995; Wiens & Penkrot 2002; reviewed in Sites &Marshall 2004; Knowles & Carstens 2007; Yang & Rann-ala 2010). Despite important advances in sequencingtechnology and coalescent theory [coalescent gene ⁄ spe-cies tree approaches (e.g. Yang & Rannala 2010)], thereexists a strongly professed need to utilize multiple formsof evidence. Many researchers favour a multifaceted,Correspondence: Craig F. Barrett, Fax: (607) 255-5407;E-mail: [email protected] 2011 Blackwell Publishing LtdMolecular Ecology (2011) 20, 2771–2786 doi: 10.1111/j.1365-294X.2011.05124.xintegrative approach to delimiting species (e.g. Wiens &Penkrot 2002; Sites & Marshall 2004; Dayrat 2005; Willet al. 2005; Roe & Sperling 2007; Groenveld et al. 2009;Leache´et al. 2009; Padial & de la Riva 2010; Padial et al.2010; Schlick-Steiner et al. 2010; Weisrock et al. 2010),including the architects of a recently published Bayesiancoalescent molecular approach: ‘‘At a minimum, speciesdelimitation should rely on many kinds of data, such asmorphological, behavioural, and geographic evidence(Yang & Rannala 2010).’’ We adopt a pluralistic, integra-tive approach to delimiting species and ESUs in a groupof fungus-eating orchids, the Corallorhiza striata complex.We utilize 14 quantitative morphological characters,eight morphological landmarks, two plastid DNA lociand three nuclear loci to investigate the patterns of varia-tion and delimit species across populations spanning theentire geographic range.A case study in a widespread, variable orchidMorphology, behaviour, phylogeography and popula-tion genetics play important, often crucial, roles in taxo-nomic and speciation studies, and thus are paramountin conservation biology. Rangewide investigation is theideal method for informing species decisions, which inturn provides the information necessary to best pre-serve variation within the resultant taxa. The geo-graphic ubiquity and unmatched species richness oforchids among plants, combined with their tendency tobe rare and endangered (Dressler 1981, 1993), illustratethe need for these types of studies. The Corallorhiza stri-ata species complex is rare but widespread, highly vari-able and has historically been a taxonomicallychallenging group (Freudenstein 1997), owing to itsseverely reduced vegetative and root morphology.Thus, it will be especially crucial to examine multiplesources of data in highly reduced parasitic and myco-heterotrophic species, augmenting morphology withDNA sequences. Barrett & Freudenstein (2009) identi-fied four plastid DNA clades within the C. striata com-plex. Corallorhiza striata var. involuta (Mexico) and theendangered C. bentleyi (eastern USA) were virtuallyidentical for the loci examined, forming a distinct clade,and were together highly divergent relative to the restof the complex. These two small-flowered, apparentlyself-pollinating taxa were also morphologically distinctfrom the remainder of the complex, and furthermoredistinct from one another, suggesting their recognitionas two separate species. The remaining populations ofC. striata, hereafter termed C. striata sensu stricto(= s.s.), also formed a clade. Nested within this cladewere three subclades, each significantly differentiated interms of floral size. The large-flowered C. striata var.striata (northern USA and Canada) was sister to thesmaller-flowered C. striata var. vreelandii, and these inturn were sister to an intermediate-flowered clade ende-mic to California.Despite these preliminary findings, numerous ques-tions remain regarding the patterns of variation in theC.
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