1 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany; 2 Museum of Zoology (Museum fu¨r Tierkunde), Senckenberg Dresden, Germany; 3 Observatorio de Herpetologı´a, Aranzadi Society of Sciences, Donostia-San Sebastia´n, Gipuzkoa, Spain Gene flow across secondary contact zones of the Emys orbicularis complex in the Western Mediterranean and evidence for extinction and re-introduction of pond turtles on Corsica and Sardinia (Testudines: Emydidae) Inken Pedall 1 ,Uwe Fritz 2 ,Heiko Stuckas 2 ,Aitor Valdeo´n 3 and Michael Wink 1 Abstract European pond turtles represent a phylogeographically deeply structured complex of distinct taxa. Here, we use mitochondrial DNA sequences (cytochrome b gene) and eight polymorphic microsatellite loci to investigate genetic differentiation and gene flow of Sicilian, Corsican and Sardinian pond turtles and of subspecies involved in two secondary contact zones in the Pyrenean region and Southern Italy. Mitochondrial and microsatellite differentiation is largely concordant in populations from the core regions of the distribution ranges of the studied taxa. Both marker systems provide no evidence for gene flow between Sicilian pond turtles (Emys trinacris) and Southern Italian subspecies of E. orbicularis. By contrast, in the contact zones limited gene flow occurs between distinct subspecies of E. orbicularis. Although the Southern Italian contact zone is significantly older than the Pyrenean contact zone of Holocene age, patterns of asymmetric introgression are similar. Introgressive hybridization leads to the exchange of mitochondria, but microsatellite data indicate only a few individuals with mixed ancestry. This suggests that incipient isolating mechanisms maintain largely discrete nuclear genomic gene pools. Furthermore, this implies that Southern Italy acted as a hotspot rather than as a melting pot of genetic diversity during the last glacial. Pond turtles from Corsica and Sardinia are not differentiated from continental populations of the subspecies E. o. galloitalica, neither in the mitochondrial nor in the quickly evolving microsatellite markers. As the fossil record argues for a continuous presence of pond turtles on both islands since the Middle Pleistocene, this suggests that the native island populations became extinct and the extant turtles were later introduced by prehistoric settlers. The lack of genetic differentiation of pond turtles from Corsica and Sardinia supports the view that the subspecies described from these islands are not valid. Key words: Europe – phylogeography – secondary contact zone – hybridization – glacial refugium – speciation – genetic cluster analysis Introduction Secondary contact zones between closely related taxa allow insights into early stages of allopatric speciation. Studies on the genetic structure of contact zones are also particularly important for understanding the complex nature of hybridiza- tion processes (cf. Coyne and Orr 2004; Currat et al. 2008). In the absence of reproductive isolation, secondary contact may lead to complete admixture of parental taxa, while prezygotic (e.g., mating behaviour, ecological adaptations) and post- zygotic (genetic incompatibilities) isolation mechanisms are expected to maintain the distinctiveness of parental gene pools, but may result in relatively narrow hybrid zones characterized by steep allele ⁄ haplotype frequency clines (Barton and Hewitt 1985; Orive and Barton 2002; Ballard and Whitlock 2004; Mallet 2005). However, introgression can reach far beyond such hybrid zones and has often been found to be asymmetric (see review in Currat et al. 2008). Within the framework of the Biological Species Concept (e.g., Mayr 1942, 1963; Coyne and Orr 2004), subspecies may be understood as incipient species that are not yet reproduc- tively isolated, so that in secondary contact zones extensive gene flow occurs and may ultimately lead to complete genetic amalgamation. European pond turtles of the Emys orbicularis complex are an attractive model for the study of gene flow in secondary contact zones. They inhabit one of the largest distribution ranges among all chelonians, extending from North Africa over the Iberian peninsula and parts of western, central, and eastern Europe eastwards to the Aral Sea (Fritz 2003). Previous morphological and genetic surveys demon- strated that pond turtles represent a highly differentiated complex of ten major evolutionary lineages with a clear phylogeographic structure (Lenk et al. 1999; Fritz 2003; Fritz et al. 2004, 2005a,b, 2007, 2009a; Fig. 1). Pond turtles from Sicily, representing the most basal mtDNA lineage and being distinct in nuclear genomic ISSR fingerprint profiles, have recently been removed from E. orbicularis as the distinct species E. trinacris (Fritz et al. 2005b). All other lineages are thought to constitute the polytypic species E. orbicularis with many recognized subspecies. Their subspecies status was inferred from narrow contact zones with morphologically intermediate turtles and sympatric occurrences of distinct mitochondrial lineages, suggestive of extensive gene flow (Fritz 1995, 1996, 2003; Lenk et al. 1999; Fritz et al. 2005a,b, 2007). The phylogeographic structure of pond turtles largely corresponds to general phylogeographic patterns identified for Western Palaearctic biota (Hewitt 1996, 1999, 2000, 2001; Taberlet et al. 1998; Joger et al. 2007; Schmitt 2007). Two to three endemic turtle lineages occur on each of the southern European peninsulas and in Asia Minor. It is generally accepted that most of these lineages diverged during the Pliocene and that their current distribution was shaped during Pleistocene and Holocene range fluctuations (Fritz 1996, 2003; Lenk et al. 1999; Fritz et al. 2007, 2009a). The northern part of the distribution range has been recolonized in post-glacial times from two refuges in the south-eastern Balkans and the Black Sea region (Lenk et al. 1999; Sommer et al. 2009) according to the ÔgrasshopperÕ and Ôbear patternsÕ of Hewitt Corresponding author: Uwe Fritz (uwe.fritz@senckenberg.de) Contributing authors: Inken Pedall (inken.pedall@yahoo.de), Heiko Stuckas (heiko.stuckas@senckenberg.de), Aitor Valdeo´n (emys@ galapagosdenavarra.es), Michael Wink (wink@uni-hd.de) Ó 2010 Blackwell Verlag GmbH Accepted on 5 May 2010 J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2010.00572.x J Zool Syst Evol Res (2011) 49(1), 44–57