Persistence across Pleistocene ice ages in Mediterranean and extra-Mediterranean refugia: phylogeographic insights from the common wall lizard

Abstract

Background: Pleistocene climatic oscillations have played a major role in structuring present-day biodiversity. The southern Mediterranean peninsulas have long been recognized as major glacial refugia, from where Northern Europe was post-glacially colonized. However, recent studies have unravelled numerous additional refugia also in northern regions. We investigated the phylogeographic pattern of the widespread Western Palaearctic lizard Podarcis muralis, using a range-wide multilocus approach, to evaluate whether it is concordant with a recent expansion from southern glacial refugia or alternatively from a combination of Mediterranean and northern refugia.

Results: We analyzed DNA sequences of two mitochondrial (cytb and nd4) and three nuclear (acm4, mc1r, and pdc) gene fragments in individuals from 52 localities across the species range, using phylogenetic and phylogeographic methods. The complex phylogeographic pattern observed, with 23 reciprocally monophyletic allo- parapatric lineages having a Pleistocene divergence, suggests a scenario of long-term isolation in multiple ice-age refugia across the species distribution range. Multiple lineages were identified within the three Mediterranean peninsulas - Iberia, Italy and the Balkans - where the highest genetic diversity was observed. Such an unprecedented phylogeographic pattern - here called "refugia within all refugia" - compasses the classical scenario of multiple southern refugia. However, unlike the southern refugia model, various distinct lineages were also found in northern regions, suggesting that additional refugia in France, Northern Italy, Eastern Alps and Central Balkans allowed the long-term persistence of this species throughout Pleistocene glaciations.

Conclusions: The phylogeography of Podarcis muralis provides a paradigm of temperate species survival in Mediterranean and extra-Mediterranean glacial refugia. Such refugia acted as independent biogeographic compartments for the long-term persistence of this species, for the differentiation of its genetic lineages, and for the short-distance post-glacial re-colonization of neighbouring areas. This finding echoes previous findings from recent phylogeographic studies on species from temperate ecoregions, thus suggesting the need for a reappraisal of the role of northern refugia for glacial persistence and post-glacial assembly of Holarctic biota.

Figure 1

Sampling localities, phylogenetic relationships, and distribution of mitochondrial haplotypes and haploclades. Maximum Likelihood (ML) trees depicting the phylogenetic relationships among 75 combined mitochondrial sequences (cytb and nd4) of Podarcis muralis from 52 localities (A) and among 185 cytb sequences of Podarcis muralis obtained in this study (N=82) and from GenBank, (N=103) (see Additional file 1) (B). ML bootstrap values over 1000 replicates are reported in correspondence to the nodes; black stars indicate BA posterior probabilities = 1.00; Podarcis liolepis was used as an outgroup. In the combined cytb/nd4 ML tree specimen codes are reported (see Table 1 for details) and main mitochondrial clades numbered from 1 to 17; in the cytb ML tree tip nodes are collapsed and main mitochondrial clades are numbered from 1 to 23 according to the combined cytb/nd4 ML tree and named according to previous studies. Samples used in this study and geographic distribution of mitochondrial clades are reported in the map: big circles show the 53 localities sampled in this study and triangles show the geographic origin for the cytb sequences obtained from GenBank, coloured according to mitochondrial clades defined by the ML trees (black triangles indicate admixed populations for which the frequency of haplotypes belonging to each clades is shown by the pie diagrams; grey circles indicate samples for which only nuclear sequences were available).

Figure 2

Phylogenetic relationships among nuclear haplotypes. Haplotype median joining networks based on sequences of the nuclear genes acm4, mc1r, and pdc. Haplotypes are represented by circles with area proportional to their frequency and coloured according to mitochondrial clades as defined by the Maximum Likelihood tree (see Figure 1).