A Contradiction in Genes: Phylogeography of the Black Mountain Salamander (Desmognathus welteri Barbour 1950) Jessica A. Wooten1, Zachary I. Felix2, and Carlos D. Camp3 1Department of Biology, Centre College, Danville, Kentucky 2Department of Biology, Reinhardt College, Waleska, Georgia 3Department of Biology, Piedmont College, Demorest, Georgia RESULTS ABSTRACT INTRODUCTION 78 / 81 96 / 98 Modern molecular techniques have enabled the discovery of numerous cryptic evolutionary lineages, many of which have been recognized as distinct species. This is true for the salamander family Plethodontidae, a taxon characterized by morphological conservatism and rampant homoplasy. Previous studies have identified cryptic lineages within recognized species using electrophoretic extraction of allozymes (e.g., Highton 1989) and, more recently, sequencing of mtDNA (e.g., Beamer & Lamb 2008). Recent investigations, however, have revealed that, at least at the generic level, phylogenetic relationships determined by nDNA sequences may disagree with those based on mtDNA (Fisher-Reid & Wiens 2011). Conflicting phylogenies can result a taxonomy that fails to reflect underlying evolutionary patterns. Therefore, we investigated the phylogeography of a single species of lungless salamander to determine if patterns agree between mtDNA and nDNA. The development of modern molecular techniques has led to the discovery of previously unsuspected levels of genetic variation, particularly within taxa characterized by morphological conservatism and/or rampant homoplasy. The lungless-salamander family Plethodontidae is characterized by both, and molecular-based investigations of phylogeography have uncovered a number of cryptic lineages, including many that have subsequently been described as species. An important phylogeographic tool continues to be the sequencing of mitochondrial DNA (mtDNA), which is especially useful for analyses at the specific and generic levels because of its rapid rate of nucleotide substitution relative to nuclear genes (nDNA). MtDNA has been criticized as a phylogenetic marker, however, because it represents only a small fraction of an organism’s genome and is inherited strictly maternally. In fact, recent studies have indicated that mitochondrial-based and nuclear-based conclusions regarding phylogeny may sharply disagree. We used both mitochondrial (ND2, CO1, 12S) and independent nuclear (introns of ILF3 and GAP3) genes to investigate the phylogeography of the Black Mountain Salamander (Desmognathus welteri Barbour 1950). We found that both nuclear introns exhibited considerable genetic structure among populations but that mtDNA sequences showed hardly any variation across the entire geographic range. The most parsimonious explanation is that the mitochondrial genome has undergone a relatively recent selective sweep in this species. Our results underscore the need to use caution when drawing phylogenetic conclusions from mtDNA sequences. 86 / 82 99 / 94 96 / 94 96 / 98 88 / 88 91 / 86 100 / 98 91 / 93 82 / 80 96 / 98 PURPOSE 99 / 98 We investigated the phylogeography of a species of plethodontid to determine whether nDNA and mtDNA patterns of relationship agree at the level of population divergence. Because mtDNA mutates faster than nDNA (Ballard & Whitlock 2004), we hypothesized that mitochondrial genes exhibit greater diversity and structure than nuclear genes. Maximum likelihood (ML) topology for populations of D. welteri and selected Desmognathus lineages based on mtDNA (2350 bp). First number is the maximum-likelihood bootstrap and the second is the posterior probability. Values < 75 were excluded from the figure. METHODS Maximum likelihood (ML) topology for populations of D. welteri compared with southern D. quadramaculatus based on two nuclear introns (780 bp). Individuals of D. welteri were collected from Kentucky, Tennessee, and West Virginia. WV3 WV2 WV1 TN2 TN3 TN1 KY1 KY3 KY2 KY4 KY8 KY9 KY6 KY7 KY5 DISCUSSION The lack of mitochondrial variation relative to nuclear introns in D. welteri is surprising given that mtDNA is believed to evolve faster than nDNA (Ballard & Whitlock 2004). There are three possible explanations for this apparent anomaly. Repeated, male-biased hybridization with one or more other species has continually introduced new nuclear genes via introgression. However, D. welteri does not appear closely related to any other known species (Kozak et al. 2005; Beamer & Lamb 2008); therefore, repeated introgression seems unlikely. Nuclear introns evolve more rapidly than mtDNA. This contradicts current understanding of relative mutation rates. Moreover, our results for D. quadramaculatus show that these introns have less variation than mitochondrial genes. A positive mutation ─ along with linked genes via hitch-hiking ─ has recently swept through the species, erasing genetic variation in the mitochondrial genome. While the frequency of selective sweeps is controversial (Brown et al. 1979; Ballard & Whitlock 2004), this is the most parsimonious explanation for our results. Literature Cited 12S, CO1, and ND2 portions (2350 bp) of the mtDNA and nuclear introns (780 bp) for ILF3 and GAPD were sequenced. Comparisons were made with the large species of Desmognathus, especially southern D. quadramculatus (sensu Kozak et al 2005). Separate phylogenies for mtDNA and nDNA genes were reconstructed using maximum-likelihood methods with models of evolution. Ballard & Whitlock 2004. Mol Ecol 13:729─744. Beamer & Lamb 2008. Mol Phylogenet Evol 47:143─153. Brown, George, & Wilson 1979. Proc Natl Acad Sci 76:1967─1971. Highton 1989. Illinois Biol Monogr 57:1─78. Fisher-Reid & Wiens 2011. BMC Evol Biol 11:300. Kozak, Larson, Bonett, & Harmon 2005. Evolution 59:2000─2016.