Molecular Ecology (2013) 22, 143–156 doi: 10.1111/mec.12085 A geographic mosaic of evolutionary lineages within the insular endemic newt Euproctus montanus ROBERTA BISCONTI,*1 DANIELE CANESTRELLI,*1 DANIELE SALVI† and GIUSEPPE NASCETTI* *Dipartimento di Scienze Ecologiche e Biologiche, Universita` della Tuscia, Viale dell’Universita` s.n.c., I-01100, Viterbo, Italy, †CIBIO, Centro de Investigac¸a˜o em Biodiversidade e Recursos Gene´ticos, Campus Agra´rio de Vaira˜o, 4485-661 Vaira˜o, Portugal Abstract Islands are hotspots of biodiversity, with a disproportionately high fraction of endemic lineages, often of ancient origin. Nevertheless, intra-island phylogeographies are surpris- ingly scarce, leading to a scanty knowledge about the microevolutionary processes induced on island populations by Plio-Pleistocene climatic oscillations, and the manner in which these processes contributed to shape their current genetic diversity. We inves- tigated the phylogeography, historical demography and species distribution models of the Corsican endemic newt Euproctus montanus (north-western Mediterranean). As for many island endemics, the continuous distribution of E. montanus throughout its range has hitherto been considered as evidence for a single large population, a belief that also guided the species’ categorization for conservation purposes. Instead, we found a geographic mosaic of ancient evolutionary lineages, with five main clades of likely Pliocene origin (2.6–5.8 My), all but one restricted to northern Corsica. Moreover, the copresence between main lineages in the same population was limited to a single case. As also suggested by growing literature on intra-island phylogeographic variation, it seems that the extensive use of simplifying assumption on the population structure and historical demography of island populations—both in theoretical and applicative studies—should be carefully reconsidered, a claim that is well exemplified by the case presented here. Keywords: Corsica, diversification, Euproctus montanus, intra-island phylogeography, Island biogeography, Mediterranean basin Received 16 February 2012; revision received 10 September 2012; accepted 13 September 2012 Taberlet et al. 1998; Thompson 2005; Nieberding et al. Introduction 2006; Schmitt 2007; Stock et al. 2008; Papadopoulou In the last three decades, the temperate species of the et al. 2009; La´zaro et al. 2011; Stroscio et al. 2011). Nev- Mediterranean region have been the subject of one of ertheless, there is also increasing realization that there the most intensive phylogeographic surveys (Feliner are fundamental questions about this history still wait- 2011; Hewitt 2011a). As a consequence, there is now ing thorough answers, including how populations ample knowledge of key issues of the Plio-Pleistocene survived climatic oscillations within areas of long-term history of both mainland and island species, including persistence (either glacial refugia or islands), what the location of major glacial refugia, hotspots of genetic microevolutionary processes were induced by these diversity, postglacial re-colonization routes of northern oscillations, and how these processes have shaped the environments, patterns of island occupancy and patterns current genetic structure and diversity within these of subsequent gene exchange among islands and with areas (Hampe & Petit 2005; Gomez & Lunt 2007; the continent (e.g. Hewitt 1996, 2000, 2004, 2011a,b; Canestrelli et al. 2010; Feliner 2011). In fact, these areas have been crucial for the survival of species, of their Correspondence: Daniele Canestrelli, Fax: +039-761-357751; intraspecific diversity and thus of their evolutionary E-mail: [email protected] potential, and they are particularly threatened by 1These authors contributed equally to this work. the recent climate changes and other human-induced © 2012 Blackwell Publishing Ltd 144 R. BISCONTI ET AL. environmental alterations (Hampe & Petit 2005; Arau´ jo islands (Cronk 1997), has not been a prominent feature et al. 2006; Kier et al. 2009). Therefore, a thorough of this island. Indeed, during the last glaciation, north- appreciation of how diversity has been moulded within ern Corsica was substantially invaded by polar air these areas is of primary interest under evolutionary, (Kuhlemann et al. 2008), and thus, climatic oscillations ecological and conservation perspectives (see Hampe & were more pronounced here than in neighbouring areas. Petit 2005; Canestrelli et al. 2010; Feliner 2011 for length- Moreover, extensive glaciers formed on the central ier discussions of these issues). mountain chain during Pleistocene glacial phases To move in this direction, islands appear particularly (Kuhlemann et al. 2005). Finally, as few phylogeographic appealing (see also Thorpe & Malhotra 1998; Emerson studies have been conducted thus far at the intra-island et al. 2006). First, islands have a longstanding and fruit- level on Corsican species (see Discussion), the genetic ful history as natural laboratories for the study of evo- imprints of Plio-Pleistocene climatic oscillations on this lutionary processes (e.g. Darwin 1859; Wallace 1880; island biota are still largely unknown. MacArthur & Wilson 1967; Grant 1998; Grant & Grant The Corsican endemic Euproctus montanus seems an 2007; Whittaker & Ferna´ndez-Palacios 2007). In fact, outstanding model organism to start looking at these several features of islands, particularly that they are imprints. It is a lung-less newt, widely and continu- discrete and isolated spatial entities, allow making a ously distributed on the island, particularly—albeit not number of simplifying assumptions that are useful for exclusively—at intermediate and high altitudes both experimental designs and data interpretation (e.g. (between 600 and 1500 m above sea level; Gasc et al. Frankham 1996a,b; Woolfit & Bromham 2005; Whittaker 1997). It has primarily aquatic habits, living within & Ferna´ndez-Palacios 2007; Vellend & Orrock 2010). mountain streams, brooks (which are abundant on the Second, both at the global level and in the Mediterra- island) or ponds during the aquatic period, or close to nean basin, islands are hotspots of biodiversity, with a them during the terrestrial period (Gasc et al. 1997). disproportionately high fraction of this diversity being Interestingly, as no study has yet investigated the popu- endemic, a feature that is consistent across taxa and lation structure of E. montanus, it has not been given a geographic regions and that makes islands a conserva- high conservation priority in the International Union tion priority (Thompson 2005; Whittaker & Ferna´ndez- for Conservation of Nature (IUCN) Red List in spite of Palacios 2007; Vogiatzakis et al. 2008; Kier et al. 2009; its restricted range, because ‘…although its Extent of Me´dail & Diadema 2009; Blondel et al. 2010). Third, in Occurrence might be <20 000 km2, it is common with a spite of their usefulness as natural laboratories and their presumed large population…’ (IUCN 2011). disproportionate importance as biodiversity hotspots Previous studies of the phylogenetic relationships both at global and at regional levels, <5% of the phylog- and divergence timing of E. montanus indicated that this eographic studies carried out so far (nearly 7100, species started diverging from the closely related Sardi- according to ISI WoK database) presented and discussed nian brook newt Euproctus platycephalus in the Miocene intra-island phylogeographic patterns (Canestrelli & (Caccone et al. 1994; Carranza & Amat 2005; Veith et al. Bisconti in prep.). 2004; see also below). In this study, we analyse the phy- If islands can be used as natural laboratories for the logeographic and historical demographic patterns of study of microevolutionary processes, then Corsica E. montanus. Our aim is to assess whether and how the Island is certainly a good candidate top-level laboratory Plio-Pleistocene climatic oscillations influenced the evo- within the Mediterranean basin. It is a hotspot of Medi- lutionary history of this island endemic species and, terranean biodiversity, with both flora and fauna that more specifically, what microevolutionary processes are rich of endemic species (Thompson 2005; Mouillot they primed, and how these processes contributed et al. 2008). It is the northernmost, the wettest and the shaping its current patterns of population genetic struc- most mountainous island of the Mediterranean basin, ture and diversity. Finally, we will also evaluate the with a central mountain chain having many summits conservation implications of our results. exceeding 2000 m in altitude. Consequently, it has a particularly complex array of landscapes and microcli- Materials and methods matic regions, spanning from Mediterranean climate at low altitudes, to temperate montane climate at interme- Sampling and laboratory procedures diate altitudes, to alpine climate at higher altitudes (see Mouillot et al. 2008 for an extensive description of the We sampled individuals of Euproctus montanus from 15 Corsican environments). Furthermore—and interest- localities spanning the whole species range. Geographic ingly—the marine buffering of climatic oscillations, references for sampling localities and sample sizes are which is thought to contribute to the high endemism shown in Table 1 and Fig. 1A. Tissue samples were richness and to the persistence of old lineages on collected as tail tips after anaesthesia in a 0.1% solution © 2012 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF EUPROCTUS MONTANUS 145 Table 1 Geographic