Molecular Ecology (2007) 16, 1191–1205 doi: 10.1111/j.1365-294X.2007.03230.x ContrastingBlackwell Publishing Ltd patterns of population subdivision and historical demography in three western Mediterranean lizard species inferred from mitochondrial DNA variation C. PINHO, D. J. HARRIS and N. FERRAND CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661 Vairão, Portugal and Departamento de Zoologia e Antropologia, Faculdade de Ciências da Universidade do Porto, 4099-002 Porto, Portugal Abstract Pleistocene climatic oscillations were a major force shaping genetic variability in many taxa. We analyse the relative effects of the ice ages across a latitudinal gradient in the Western Mediterranean region, testing two main predictions: (i) species with historical distributions in northern latitudes should have experienced greater loss of suitable habitat, resulting in higher extinction of historical lineages than species distributed in southern latitudes, where the effects of the ice ages were not as drastic. This would be reflected in the obser- vation of lower diversity and number of differentiated lineages in northern areas. (ii) a sig- nature of demographic expansion following the climate amelioration should be obvious in northern species, whereas in the south evidence of long-term effective population size stability should be observed. We used as models three species of wall lizards (Podarcis bocagei, Podarcis carbonelli and Podarcis vaucheri) that replace each other along the study area. We investigated the patterns of mitochondrial DNA diversity and subdivision and obtained demographic parameter estimates for each species. Our results suggest that P. bocagei, the northernmost species, bears low genetic diversity, a shallow coalescent history and marks of a demographic expansion. In contrast, P. vaucheri, the species with a south- ernmost distribution, shows deeper coalescence events, complex geographical substructure and no evidence for population growth. The species with an intermediate distribution, P. carbonelli, shows average levels of diversity, substructure and population growth. Taken together, these results conform to our main predictions and are explained by a differential influence of the ice ages on distinct latitudes. Keywords: demography, glaciations, Iberian Peninsula, latitudinal gradient, North Africa, phylo- geography, Podarcis, population structure Received 5 September 2006; revision received 22 October 2006; accepted 6 November 2006 expansions following the retreat of the ice sheets (Taberlet Introduction et al. 1998; Milá et al. 2000; Lessa et al. 2003). Although Pleistocene climatic oscillations are generally believed to this paradigm is one of the most consensual in modern have played a major role in shaping genetic diversity biogeography, some aspects are still under debate, namely across a wide number of taxa (Hewitt 1996, 1999). In recent whether glacial times had an as severe influence on the years, following the rise of phylogeography as a formal historical distributions of nontemperate species as has discipline (Avise et al. 1987; Avise 2000), a large number of been demonstrated for Europe and North America (e.g. surveys of genetic variation have disclosed patterns of Willis & Whittaker 2000). In this context, Lessa et al. (2003) genetic subdivision related to isolation in glacial refugia investigated the effects of the Pleistocene glacial ages during the cold stages and demographic and geographical across the American continent by exploring the genetic signatures of postglacial demographic expansion in mammal Correspondence: Catarina Pinho, Fax: +351252661780; E-mail: populations from both North America and Amazonia. These [email protected] signs of expansion were found to be present in North © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 1192 C. PINHO, D. J. HARRIS and N . FERRAND America, suggesting postglacial colonization of these areas (mtDNA) phylogeny for the Iberian/Maghrebian part of from a reduced source, but absent from tropical species, the genus (Pinho et al. 2006), which major subdivisions are suggesting long-term effective population size stability. corroborated by a study of nuclear markers (Pinho et al. in Although fundamental to understand the relative effects of press). We selected three species that replace each other the ice ages across latitudinal and ecological gradients, along a latitudinal gradient that spreads from northwest- studies such as this have scarcely been repeated across ern Iberia to West-Central Morocco (Fig. 1) [Podarcis bocagei the globe. (Seoane 1884), Podarcis carbonelli Pérez-Mellado 1981, and In Europe, several phylogeographical studies highlighted Podarcis vaucheri (Boulenger 1905)] and investigated mtDNA the importance of southern Peninsulas as glacial refugia variation and patterns of population subdivision and his- for many taxa, functioning as survival pockets from which torical demography in each of them. These three species northern areas were later colonized (Hewitt 1996, 1999, display similar morphological and ecological character- 2000; Taberlet et al. 1998). Recent studies have also dis- istics: they are small (average snout-vent length of about closed complex phylogeographical patterns within these 6–7 cm), tend to avoid arid habitats, prey mainly on southern regions, consistent with the isolation of populations invertebrates and show reduced dispersal capabilities. during Pleistocene glaciations, leading to the acknowledge- Although such a comparative phylogeographical approach ment of these regions as hotspots of diversification instead could be performed on any nonrelated organisms, working of mere historical survival areas (e.g. Alexandrino et al. with closely related species makes the inferences simpler 2000; Gómez & Lunt 2007). Furthermore, the biogeograph- and more straightforward, since general ecological require- ical history of Western Europe is undoubtedly linked to ments, dispersal abilities and important quantities for the that of North Africa. Although these two regions have estimation of demographic parameters (such as mutation been separated by the Strait of Gibraltar for over 5 million rates or generation times) can be treated as similar without years (Duggen et al. 2003), resulting on vicariant differen- strongly biasing the analyses. tiation of several Iberian and North African taxa (e.g. Castella et al. 2000; Harris et al. 2004a; Pinho et al. 2006; Materials and methods Vasconcelos et al. 2006), recent studies have documented cases of natural trans-Gibraltar colonization of either Iberia Sampling and mtDNA sequencing or the Maghreb, suggesting some degree of permeability of this barrier (Harris et al. 2002, 2004b; Carranza et al. 2004; We sampled a total of 247 individuals from 57 localities Cosson et al. 2005; Carranza et al. 2006; Guillaumet et al. across the Iberian Peninsula and Morocco. Three species 2006). All these features contribute to establish the bio- were collected: Podarcis bocagei (N = 82), an endemism of geography of the Western Mediterranean region as actually the northwestern corner of the Iberian Peninsula, Podarcis very complex and difficult to interpret in the light of simple carbonelli (N = 84), endemic to Central and Southern Iberia patterns (de Jong 1998). and Podarcis vaucheri (N = 81), distributed both in the Nevertheless, we expect some common patterns to Maghreb and in southern Spain. According to the biogeo- emerge from studies on different organisms. In this con- graphical scenario suggested by Pinho et al. (2006), it is text, we were interested in testing two main predictions: (i) likely that P. bocagei and P. carbonelli speciated in the Iberian in northern latitudes, where the effects of glaciations were Peninsula, whereas P. vaucheri originated in North Africa, more severe, fewer and smaller patches of suitable habitat having colonized southern Spain c. 2.8 million years were left for the survival of populations across multiple ago (Ma). Although the distributions of P. bocagei and glaciation cycles, thus leading to a higher degree of extinc- P. carbonelli are fairly well known (Pleguezuelos et al. 2002; tion of historical lineages; this should be reflected in overall A. Loureiro, personal communication), the distribution of lower diversity, and number of differentiated lineages P. vaucheri is still far from being completely described, both in in northern than in southern areas (as in for example in the Iberian Peninsula and in North Africa (e.g. it is unknown Michaux et al. 2005; Deffontaine et al. 2005); (ii) the effects whether Algerian forms of Podarcis are representative of climatic changes on the effective population sizes were of this or of other species). Sampling details are given in more dramatic in northern than in southern regions, mean- Table 1 and Fig. 1. Sequences from 10 individuals were ing that northern populations should bear the signature analysed in a previous study (Pinho et al. 2006; GenBank of a rapid demographic expansion following the climate Accession nos DQ081151–6, DQ081172, DQ081174–6). amelioration (as in Lessa et al. 2003), whereas southern Samples consisted of a portion of tail muscle, using the populations should evidence marks of more stable long- lizards’ natural autotomy capacity. Samples were stored in term effective population size. 96% ethanol. Total genomic DNA was extracted following To test these predictions, we focused on wall lizards standard methods (Sambrook