A multigenic perspective on phylogenetic relationships in the largest family of salamanders, the Plethodontidae David R. Vieites a , Sandra Nieto Román a,b , Marvalee H. Wake c,d , David B. Wake c,d,⇑ a Museo Nacional de Ciencias Naturales, Consejo Superior de Investigaciones Científicas (CSIC), C/ José Gutierrez Abascal 2, 28006 Madrid, Spain b Departamento de Ecología y Biología Animal, Facultad de Ciencias, Universidad de Vigo, 36200 Vigo, Spain c Department of Integrative Biology, University of California Berkeley, CA 94720-3140, USA d Museum of Vertebrate Zoology, University of California Berkeley, CA 94720-3160, USA article info Article history: Received 28 August 2010 Revised 4 March 2011 Accepted 9 March 2011 Available online 15 March 2011 Keywords: Mitochondrial genomes Nuclear loci Lungless salamanders Phylogeny New taxonomy Amphibians abstract Despite several recent studies, the phylogeny of plethodontid salamanders is not yet fully resolved and the phylogenetic positions of several key genera, especially Aneides, Hemidactylium, Hydromantes and Kar- senia, are contentious. Here we present a combined dataset of complete mitochondrial genomes and three nuclear loci for 20 species (16 genera) of plethodontids, representing all major clades in the family. The combined dataset without mitochondrial third codon positions provides a fully resolved, statistically well-supported tree. In this topology two major clades are recovered. A northern clade includes Aneides, Desmognathus, Ensatina, Hydromantes, Karsenia, Phaeognathus and Plethodon, with Plethodon being the sis- ter taxon to the rest of the clade. Hydromantes and Karsenia are sister taxa, and Aneides is recovered as the sister taxon to Ensatina. Desmognathus + Phaeognathus form the sister taxon to Aneides + Ensatina. An eastern/southern clade comprises two subclades. One subclade, the spelerpines (Eurycea, Gyrinophilus, Pseudotriton, Stereochilus, Urspelerpes) is the sister taxon to a subclade comprising Hemidactylium, Batra- choseps and the tropical plethodontids (represented by Bolitoglossa, Nototriton and Thorius). In this topol- ogy Hemidactylium is well-supported as the sister taxon to Batrachoseps. Only when mitochondrial third codon positions are included using maximum likelihood analysis is Hemidactylium recovered as the sister taxon to Batrachoseps + tropical genera. Hypothesis testing of alternative topologies supports these con- clusions. On the basis of these results we propose a conservative taxonomy for Plethodontidae. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Resolving phylogenies for very old (more than 50 million years [myr] old) and large clades is challenging. Lineage origins can be concentrated in particular short spans of time, producing short internodes and ambiguous resolution, especially when the nodes are old. In this paper we examine the evolutionary history of such a clade, the salamander family Plethodontidae, the largest of the ten families of salamanders, which contains approximately two- thirds of the roughly 610 living species forming the order Caudata. The phylogenetic analysis of the Plethodontidae by Wake (1966) led to a taxonomic revision that recognized two subfami- lies, a very large Plethodontinae and a much smaller Desmognathi- nae. The Plethodontinae included three tribes, Bolitoglossini (with three supergenera, Batrachoseps including only Batrachoseps, Boli- toglossa including all of the tropical genera, and Hydromantes including European and American species), Hemidactyliini (Hemidactylium, which was not clearly associated with the remain- der of the genera in the group, currently assigned as spelerpines, see below), and Plethodontini (Aneides, Ensatina, Plethodon). This taxonomy, based on analysis of comparative osteology and life his- tory, was almost universally accepted until the publication of large molecular (e.g., Mueller et al., 2004) and combined molecular and morphological (e.g., Chippindale et al., 2004) datasets. Analyses of these databases showed that taxonomic revision was necessary. In particular, one of the two subfamilies, Desmognathinae, was dee- ply nested within the other, Plethodontinae. Organization of the Plethodontinae into three clades, corresponding mainly to the existing Bolitoglossini, Hemidactyliini, and Plethodontini, was gen- erally supported, but with some important exceptions. Hydroman- tes, placed by Wake (1966) in the Bolitoglossini, was nested within the Plethodontini (Mueller et al., 2004), and Hemidactylium was recovered as the sister taxon of Batrachoseps (ML and BI analysis of complete mitochondrial [mt] genomes by Mueller et al., 2004) or as the sister taxon to all other plethodontids (MP analysis of data of Mueller et al., 2004, by Macey, 2005; topology found in MP anal- ysis including third codon positions but not without them by 1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.03.012 ⇑ Corresponding author. E-mail address: wakelab@berkeley.edu (D.B. Wake). Molecular Phylogenetics and Evolution 59 (2011) 623–635 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev