The dissection of a Pleistocene refugium: phylogeography of the smooth newt, Lissotriton vulgaris, in the Balkans

Aim: To assess the role of the Balkan Peninsula as a Pleistocene refugium for the smooth newt, Lissotriton vulgaris, and to test whether its genetic differentiation is temporally compatible with the southern réfugia model. Location: The Balkan Peninsula. Methods: Phylogeographical analyses were conducted using mitochondrial DNA (mtDNA) sequences sampled from 49 populations of L.vulgaris. A fossil-calibrated estimate of divergence times among major mtDNA clades was obtained. Results: We detected seven parapatrically distributed mtDNA clades with very little admixture among populations. Whereas most clades diverged in the Pleistocene, the earliest splits between Caucasian, Anatolian and Balkan clades occurred in the Pliocene. Clades C, D, K and M have local distributions within the Balkans and have evolved in isolation from other groups. Clade L originated in the Pannonian Basin and northern margin of the Balkan Peninsula and recently expanded across central and western Europe. Clades H and E have recently arrived in the Balkans from source populations in the Apennine Peninsula and Anatolia, respectively. Main conclusions: The history of L. vulgaris involves multiple, independent refugiai populations in the Balkans. Only one of them, located at the northern periphery of the peninsula, showed evidence of post-glacial expansion into western and northern Europe. The Balkans have therefore served as a reservoir of old diversity for L. vulgaris. By contrast, populations at the northern and eastern frontiers of the peninsula have experienced non-equilibrium dynamics. Our dating revealed that very little, if any, pre-Pliocene genetic diversity has survived in Europe, despite an extensive fossil record for this species in the Miocene and Pliocene. Differentiation of the European mtDNA clades thus seems to have been primarily moulded by Pleistocene climate change. None of the currently recognized subspecies present in the Balkans are reciprocally monophyletic in their mtDNA. We hypothesize that incomplete lineage sorting and mtDNA introgression account for the observed discrepancies.