Molecular Ecology 2011 20 2771 2786 doi 10 1111 j 1365 294X 2011 05124 x An integrative approach to delimiting species in a rare but widespread mycoheterotrophic orchid C R A I G F B A R R E T T and J O H N V F R E U D E N S T E I N 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 USA Abstract In the spirit of recent calls for species delimitation studies to become more pluralistic incorporating multiple sources of evidence we adopted an integrative phylogeographic approach to delimiting species and evolutionarily significant units ESUs in the Corallorhiza striata species complex This rare North American mycoheterotrophic orchid has been a taxonomic challenge regarding species boundaries displaying complex patterns of variation and reduced vegetative morphology We employed plastid DNA nuclear DNA and morphometrics treating the C striata complex as a case study for integrative species delimitation We found evidence for the differentiation of the endangered C bentleyi eastern USA C striata var involuta Mexico from the remaining C striata C striata s s USA Canada Mexico Corallorhiza striata involuta and C bentleyi disjunct by thousands of kilometres Mexico Appalachia were genetically identical but morphologically distinct Evidence suggests the C striata complex represents three species C bentleyi C involuta and a widespread C striata s s under operational criteria of diagnosability and common allele pools In contrast Bayesian coalescent estimation delimited four species but more informative loci and a resultant species tree will be needed to place higher confidence in future analyses Three distinct groupings were identified within C striata s s corresponding to C striata striata C striata vreelandii and Californian accessions but these were not delimited as species because of occupying a common allele pool Each comprises an ESU warranting conservation considerations This study represents perhaps the most geographically comprehensive example of integrative species delimitation for any orchid and any mycoheterotroph Keywords integrative taxonomy morphology phylogeography population Received 29 November 2010 revision received 31 March 2011 accepted 13 April 2011 Introduction Explicit species delimitation studies represent an essential component of systematics yet they are currently underrepresented relative to phylogenetic studies Wiens 2007 This is unfortunate because delimiting species represents a crucially important first step in framing studies of conservation ecology evolution Correspondence Craig F Barrett Fax 607 255 5407 E mail cfb66 cornell edu 2011 Blackwell Publishing Ltd phylogenetics and population genetics However there is no universal consensus on how this should be accomplished although numerous methods have been proposed e g Davis Nixon 1992 Baum Shaw 1995 Doyle 1995 Wiens Penkrot 2002 reviewed in Sites Marshall 2004 Knowles Carstens 2007 Yang Rannala 2010 Despite important advances in sequencing technology and coalescent theory coalescent gene species tree approaches e g Yang Rannala 2010 there exists a strongly professed need to utilize multiple forms of evidence Many researchers favour a multifaceted 2772 C F B A R R E T T and J V F R E U D E N S T E I N integrative approach to delimiting species e g Wiens Penkrot 2002 Sites Marshall 2004 Dayrat 2005 Will et 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 Bayesian coalescent molecular approach At a minimum species delimitation should rely on many kinds of data such as morphological behavioural and geographic evidence Yang Rannala 2010 We adopt a pluralistic integrative approach to delimiting species and ESUs in a group of fungus eating orchids the Corallorhiza striata complex We utilize 14 quantitative morphological characters eight morphological landmarks two plastid DNA loci and three nuclear loci to investigate the patterns of variation and delimit species across populations spanning the entire geographic range A case study in a widespread variable orchid Morphology behaviour phylogeography and population genetics play important often crucial roles in taxonomic and speciation studies and thus are paramount in conservation biology Rangewide investigation is the ideal method for informing species decisions which in turn provides the information necessary to best preserve variation within the resultant taxa The geographic ubiquity and unmatched species richness of orchids among plants combined with their tendency to be rare and endangered Dressler 1981 1993 illustrate the need for these types of studies The Corallorhiza striata species complex is rare but widespread highly variable and has historically been a taxonomically challenging group Freudenstein 1997 owing to its severely reduced vegetative and root morphology Thus it will be especially crucial to examine multiple sources of data in highly reduced parasitic and mycoheterotrophic species augmenting morphology with DNA sequences Barrett Freudenstein 2009 identified four plastid DNA clades within the C striata complex Corallorhiza striata var involuta Mexico and the endangered C bentleyi eastern USA were virtually identical for the loci examined forming a distinct clade and were together highly divergent relative to the rest of the complex These two small flowered apparently self pollinating taxa were also morphologically distinct from the remainder of the complex and furthermore distinct from one another suggesting their recognition as two separate species The remaining populations of C striata hereafter termed C striata sensu stricto s s also formed a clade Nested within this clade were three subclades each significantly differentiated in terms of floral size The large flowered C striata var striata northern USA and Canada was sister to the smaller flowered C striata var vreelandii and these in turn were sister to an intermediate flowered clade endemic to California Despite these preliminary findings numerous questions remain regarding the patterns of variation in the C striata complex especially as they relate to species boundaries Questions addressed here are as follows i In the context of
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