An ancient lineage of slow worms, genus Anguis (Squamata: Anguidae), survived in the Italian Peninsula Václav Gvoz ˇdík a,b , Norbert Benkovsky ´ c , Angelica Crottini d , Adriana Bellati e , Jir ˇí Moravec a , Antonio Romano f , Roberto Sacchi e , David Jandzik c,g,⇑ a Department of Zoology, National Museum, Cirkusová 1740, 193 00 Prague, Czech Republic b Department of Environmental Sciences, Biogeography, University of Basel, Klingelbergstrasse 27, 4056 Basel, Switzerland c Department of Zoology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska dolina B-1, 842 15 Bratislava, Slovakia d CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, R. Padre Armando Quintas, 4485-661 Vairão, Vilo do Conde, Portugal e Department of Earth and Environmental Science, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy f Istituto di Biologia Agro-ambientale e Forestale (IBAF), Consiglio Nazionale delle Ricerche, Via Salaria km, 29.300, 00015 Monterotondo scalo (Roma), Italy g Department of Ecology and Evolutionary Biology (EBIO), University of Colorado, Ramaley N122, Campus Box 334, Boulder, CO 80309-0334, USA article info Article history: Received 19 September 2012 Revised 1 April 2013 Accepted 8 May 2013 Available online xxxx Keywords: Cryptic diversity Miocene Southern refugia Morphological differentiation Phylogeny Phylogeography abstract Four species of legless anguid lizard genus Anguis have been currently recognized: A. fragilis from western and central Europe, A. colchica from eastern Europe and western Asia, A. graeca from southern Balkans, and A. cephallonica from the Peloponnese. Slow worms from the Italian Peninsula have been considered conspecific with A. fragilis, despite the fact that the region served as an important speciation center for European flora and fauna, and included some Pleistocene glacial refugia. We used mitochondrial and nuclear DNA sequences to investigate the systematic and phylogenetic position of the Italian slow-worm populations and morphological analyses to test for phenotypic differentiation from A. fragilis from other parts of Europe. Our phylogenetic analyses revealed that Italian slow worms form a distinct deeply dif- ferentiated mtDNA clade, which presumably diverged during or shortly after the basal radiation within the genus Anguis. In addition, the specimens assigned to this clade bear distinct haplotypes in nuclear PRLR gene and show morphological differentiation from A. fragilis. Based on the differentiation in all three independent markers, we propose to assign the Italian clade species level under the name Anguis veron- ensis Pollini, 1818. The newly recognized species is distributed throughout the Italian Peninsula to the Southern Alps and south-eastern France. We hypothesize that the Tertiary Alpine orogeny with subse- quent vicariance might have played a role in differentiation of this species. The current genetic variability was later presumably shaped in multiple glacial refugia within the Italian Peninsula, with the first split- ting event separating populations from the region of the Dolomite Mountains. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Due to the complex geological history and habitat diversity, the Mediterranean region encompassing the Balkan, Italian, and Ibe- rian peninsulas played a crucial role for the origin of the biodiver- sity of the European fauna and its subsequent diversification. For many taxa the peninsulas represent the radiation centers, areas with the highest in-group diversity and centers of endemism (Blondel et al., 2010; Hidalgo-Galiana and Ribera, 2011). The origin of many of the oldest extant lineages of terrestrial animal groups dates back to the Oligocene, while the origin of the younger lin- eages could be related to the Late Oligocene–Early Miocene separa- tion of Tethys and Paratethys with the diversification being mainly driven by subsequent vicariance during the Miocene (Oosterbroek and Arntzen, 1992). Later, in the Quaternary, climate changes had strong effect on European fauna and caused extinctions or repeated range contractions and expansions in many species or their popu- lations. While populations survived glaciations in refugia situated primarily within the three main Mediterranean peninsulas, they expanded to the northern areas during the warmer interglacial periods and particularly after the last glaciation (e.g. Feliner, 2011; Hewitt, 1996, 1999; Taberlet et al., 1998; Weiss and Ferrand, 2007). Within vertebrates, genetic patterns of less vagile taxa, such as amphibians and reptiles, were particularly influenced by popu- lation contractions and expansions and their assemblages thus of- ten better reflect location of glacial refugia than contemporary climate (Araújo and Pearson, 2005; Araújo et al., 2006). Slow worms, legless lizards of the genus Anguis Linnaeus, 1758, inhabit a large territory of the Western Palearctic region including 1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2013.05.004 ⇑ Corresponding author. Address: Department of Zoology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska dolina B-1, 842 15 Bratislava, Slovakia. Fax: + 421 2 60296 333. E-mail address: davidjandzik@gmail.com (D. Jandzik). Molecular Phylogenetics and Evolution xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Please cite this article in press as: Gvoz ˇdík, V., et al. An ancient lineage of slow worms, genus Anguis (Squamata: Anguidae), survived in the Italian Pen- insula. Mol. Phylogenet. Evol. (2013), http://dx.doi.org/10.1016/j.ympev.2013.05.004