Multilocus Phylogeography and Species Delimitation in the Cumberland Plateau Salamander, Plethodon kentucki: Incongruence among Data Sets and Methods

RESEARCH ARTICLE Multilocus Phylogeography and Species Delimitation in the Cumberland Plateau Salamander, Plethodon kentucki: Incongruence among Data Sets and Methods Shawn R. Kuchta1*, Ashley D. Brown1, Paul E. Converse1, Richard Highton2 1 Department of Biological Sciences, Ohio Center for Ecology and Evolutionary Studies, Ohio University, Athens, Ohio, United States of America, 2 Department of Biology, University of Maryland, College Park, Maryland, United States of America * Abstract OPEN ACCESS Citation: Kuchta SR, Brown AD, Converse PE, Highton R (2016) Multilocus Phylogeography and Species Delimitation in the Cumberland Plateau Salamander, Plethodon kentucki: Incongruence among Data Sets and Methods. PLoS ONE 11(3): e0150022. doi:10.1371/journal.pone.0150022 Editor: Robert Guralnick, University of Colorado, UNITED STATES Received: August 22, 2015 Accepted: February 8, 2016 Published: March 14, 2016 Copyright: © 2016 Kuchta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Sequence data generated in the present study has been deposited in Genbank. Details regarding the sampling of populations and loci, geographic coordinates, and GenBank accession numbers are presented in the S1 Appendix. Species are a fundamental unit of biodiversity, yet can be challenging to delimit objectively. This is particularly true of species complexes characterized by high levels of population genetic structure, hybridization between genetic groups, isolation by distance, and limited phenotypic variation. Previous work on the Cumberland Plateau Salamander, Plethodon kentucki, suggested that it might constitute a species complex despite occupying a relatively small geographic range. To examine this hypothesis, we sampled 135 individuals from 43 populations, and used four mitochondrial loci and five nuclear loci (5693 base pairs) to quantify phylogeographic structure and probe for cryptic species diversity. Rates of evolution for each locus were inferred using the multidistribute package, and time calibrated gene trees and species trees were inferred using BEAST 2 and *BEAST 2, respectively. Because the parameter space relevant for species delimitation is large and complex, and all methods make simplifying assumptions that may lead them to fail, we conducted an array of analyses. Our assumption was that strongly supported species would be congruent across methods. Putative species were first delimited using a Bayesian implementation of the GMYC model (bGMYC), Geneland, and Brownie. We then validated these species using the genealogical sorting index and BPP. We found substantial phylogeographic diversity using mtDNA, including four divergent clades and an inferred common ancestor at 14.9 myr (95% HPD: 10.8–19.7 myr). By contrast, this diversity was not corroborated by nuclear sequence data, which exhibited low levels of variation and weak phylogeographic structure. Species trees estimated a far younger root than did the mtDNA data, closer to 1.0 myr old. Mutually exclusive putative species were identified by the different approaches. Possible causes of data set discordance, and the problem of species delimitation in complexes with high levels of population structure and introgressive hybridization, are discussed. Funding: Funding was provided to SRK by Ohio University. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0150022 March 14, 2016 1 / 25 Species Delimitation in Plethodon kentucki Introduction As species are fundamental units in ecology, biodiversity, conservation, and evolutionary biology, accurate species delimitation is of critical importance. Nonetheless, the diagnosis of species has a contentious history, with many biologists advocating alternative species concepts and conflicting taxonomies [1–5]. Work on species delimitation has historically focused on morphology and patterns of reproductive isolation [6–8], but molecular techniques are increasingly used to clarify species boundaries [9–13]. Using molecular markers, many morphologically cryptic taxa have been identified, and deep divergences among allopatric components quantified [14–17]. While species delimitation remains a challenging, philosophically rich topic [3,5], increasing numbers of biologists—especially systematists—are conceptualizing species as segments of independently evolving metapopulation-level evolutionary lineages, a perspective known as the general lineage species concept [4,18,19]. In particular, this perspective has been widely applied to molecular systematic investigations of complexes characterized by allopatry, parapatry, and morphological stasis [3,20–22]. In conjunction with advances in developing a unified concept of species [23], multispecies coalescent models, which lie at the interface of modern population genetic and phylogenetic methods, are revamping the science of species delimitation [24–26]. The development of these models was spurred by the observation that genealogies estimated from different genes can be discordant simply because the coalescence of genealogical lineages is a stochastic process. Unlike concatenated phylogenetic analyses, which assume a single tree underlies all loci, the multispecies coalescent accounts for gene tree conflict by modeling coalescent stochasticity [24,27]. Thus, evolutionary lineages can be diagnosed, and a reliable estimate of a species tree made, in the absence of monophyly, or when the information content of many loci is weak [28,29]. On the other hand, serious complications such as introgressive hybridization, high levels of population structure, isolation by distance, and phylogenetic estimation error present analytical challenges for genetic data, and if not accounted for can mislead inferences [25,30,31]. Species limits are notoriously difficult to identify in plethodontid salamanders, which can exhibit a high degree of phenotypic and ecological conservatism [32–35], yet commonly harbor extraordinary levels of genetic variation, including isolation by distance and deep population structure [9,36–41]. Objectively defining and delimiting plethodontid species is therefore a challenging task [3,42–44]. Nonetheless, largely as a result of studies using allozymes, the number of species in the genus Plethodon has increased from 16 in 1962 [45] to 55 today (AmphibiaWeb: http://amphibiaweb.org), including numerous cryptic and allopatric species [9,37,46]. Species complexes in Plethodon are thus eminent examples of a non-adaptive radiation, whereby an ancestral source taxon disintegrates into a complex of isolated lineages [46–49]. The Cumberland Plateau Salamander, P. kentucki, is an example of a species of Plethodon that harbors a high degree of genetic stru (...truncated)