Amphibian phylogeography in the Antipodes: Refugia and postglacial colonization explain mitochondrial haplotype distribution in the Patagonian frog Eupsophus calcaratus (Cycloramphidae) José J. Nuñez a,⇑ , Nicole K. Wood b , Felipe E. Rabanal a , Frank M. Fontanella b , Jack W. Sites Jr. b a Instituto de Zoología, Universidad Austral de Chile, Casilla 567, Valdivia, Chile b Department of Biology and Bean Life Science Museum, Brigham Young University, Provo, UT 84602, USA article info Article history: Received 7 September 2010 Revised 22 November 2010 Accepted 28 November 2010 Available online 8 December 2010 Keywords: Phylogeography Last glacial maximum Recolonization Valdivian forest Eupsophus calcaratus mtDNA abstract Climatic oscillations, heterogeneity in elevation, topographical position, and isolation time in southwest- ern Patagonia have been important in promoting diversification of the biota. Geological studies have shown that this region had wide ice-free areas during periods of the last glacial maximum and provided forested refugia for the biota during Pleistocene glaciations. In this study, we sampled the endemic frog Eupsophus calcaratus from 20 localities, covering most of its distribution and including glaciated and non- glaciated regions. We collected DNA sequences for three mitochondrial regions (D-loop, cyt b, 16S), and describe patterns of variation consistent with a history of both the displacement to glacial refugia and recent recolonization to extensively glaciated regions. The inferred demographic history and divergence times of the lineages of E. calcaratus suggest that the Pleistocene had profound effects on the genetic pat- terns within this taxon in which some populations were able to survive in refugia within colder regions followed by demographic increases but without evidence of significant range expansion. The mtDNA gene tree recovers six major haploclades of E. calcaratus, which we consider diagnostic of species lineages. These results contribute to our understanding of how geological events, predominately glacial oscilla- tions, have influenced current population structure of a broad-ranging, ectothermic vertebrate in the Val- divian Forest region of southern South America. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction The Andean and adjacent regions in southern South America have a rich history of geological and climatic changes along broad altitudinal and latitudinal gradients, and the highly endemic biota has distributional patterns strongly linked to these histories (Hinojosa and Villagrán, 1997; Veblen, 2007). Although the Andes began to rise at the end of Cretaceous, more than 65 million years ago (Ma), it was not until the middle Miocene (11–14 Ma) that elevations exceeded 1000 m above sea level (a.s.l.; Potts and Behrensmeyer, 1992; Gregory-Wodzicki, 2000). Major orogenies occurred in the last 10 Ma, causing uplift to current elevations of greater than 4000 m a.s.l. (Gregory-Wodzicki, 2000), and forming geographic barriers with pronounced effects on the distribution and diversification of flora and fauna (Smith-Ramírez, 2004; Muell- ner et al., 2005; Ruzzante et al., 2006; Yoke et al., 2006). On a more recent time scale, glaciations in southernmost Patagonia began as early as the Middle Pliocene (Rabassa, 2008), in which a series of glacial advances and retreats characterized the Patagonian land- scape. During the Early Pleistocene, a single, continuous montane ice sheet developed for the first time and covered broad areas of Andean South America from 36°S to 56°S(Hulton et al., 1994; McCulloch et al., 2000). However, parts of the coastal range of wes- tern Chile remained ice free. The dates of the recent Pleistocene glaciations in South America are well known (Rabassa and Clapperton, 1990; Ruzzante et al., 2008), and include: (1) the most extensive Andean glaciation (1.1 Ma); (2) the coldest Pleistocene glaciation (0.7 Ma); (3) the last southern Patagonian glaciation (180 kya); and (4) the last glacial maximum (LGM, 25–23 Ka). These glaciers changed drain- age patterns, lake distributions, and even the position of the conti- nental divide, displacing plant and animal populations and thus providing a natural theater for examining the effects of glaciers on biodiversity patterns in this region. For example, strong genetic structure concordant with paleoclimatic shifts has been reported for endemic freshwater crabs (Xu et al., 2009), while repeated expansion and contraction events and subsequent population mix- ing were detected in percichthyid and galaxiid fishes (Ruzzante et al., 2006; Zemlak et al., 2008). Historical signatures are also 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.11.026 ⇑ Corresponding author. E-mail addresses: jjnunezn@gmail.com (J.J. Nuñez), npkoontz@gmail.com (N.K. Wood), feliperabanal@gmail.com (F.E. Rabanal), fonta@byu.edu (F.M. Fontanella), jack_sites@byu.edu (J.W. Sites). Molecular Phylogenetics and Evolution 58 (2011) 343–352 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev