Physical and ecological isolation contribute to maintain genetic differentiation between fire salamander subspecies.

Landscape features shape patterns of gene flow among populations, ultimately determining where taxa lay along the continuum between panmixia to complete reproductive isolation. Gene flow can be restricted, leading to population differentiation in two non-exclusive ways: "physical isolation", in which geographic distance in combination with the landscape features restricts movement of individuals promoting genetic drift, and "ecological isolation", in which adaptive mechanisms constrain gene flow between different environments via divergent natural selection. In central Iberia, two fire salamander subspecies occur in parapatry across elevation gradients along the Iberian Central System mountains, while in the adjacent Montes de Toledo Region only one of them occurs. By integrating population and landscape genetic analyses, we show a ubiquitous role of physical isolation between and within mountain ranges, with unsuitable landscapes increasing differentiation between populations. However, across the Iberian Central System, we found strong support for a significant contribution of ecological isolation, with low genetic differentiation in environmentally homogeneous areas, but high differentiation across sharp transitions in precipitation seasonality. These patterns are consistent with a significant contribution of ecological isolation in restricting gene flow among subspecies. Overall, our results suggest that ecological divergence contributes to reduce genetic admixture, creating an opportunity for lineages to follow distinct evolutionary trajectories.

Multilocus phylogeography of the common midwife toad, Alytes obstetricans (Anura, Alytidae): Contrasting patterns of lineage diversification and genetic structure in the Iberian refugium.

Recent investigations on the evolutionary history of the common midwife toad (Alytes obstetricans) revealed high levels of geographically structured genetic diversity but also a situation where delineation of major historical lineages and resolution of their relationships are much more complex than previously thought. We studied sequence variation in one mitochondrial and four nuclear genes throughout the entire distribution range of all recognized A. obstetricans subspecies to infer the evolutionary processes that shaped current patterns of genetic diversity and population subdivision. We found six divergent, geographically structured mtDNA haplogroups diagnosing population lineages, and varying levels of admixture in nuclear markers. Given the timeframe inferred for the splits between major lineages, the climatic and environmental changes that occurred during the Pleistocene seem to have shaped the diversification history of A. obstetricans. Survival of populations in allopatric refugia through the Ice Ages supports the generality of the "refugia-within-refugia" scenario for the Iberian Peninsula. However, lineages corresponding to subspecies A. o. almogavarii, A. o. pertinax, A. o. obstetricans, and A. o. boscai responded differently to Pleistocene climatic oscillations after diverging from a common ancestor. Alytes o. obstetricans expanded northward from a northern Iberian refugium through the western Pyrenees, leaving a signal of contrasting patterns of genetic diversity, with a single mtDNA haplotype north of the Pyrenees from SW France to Germany. Both A. o. pertinax and A. o. boscai are widespread and genetically diverse in Iberia, the latter comprising two divergent lineages with a long independent history. Finally, A. o. almogavarii is mostly restricted to the north-eastern corner of Iberia north of the Ebro river, with additional populations in a small region in south-eastern France. This taxon exhibits unparalleled levels of genetic diversity and little haplotype sharing with other lineages, suggesting a process of incipient speciation.

Multilocus species tree analyses resolve the radiation of the widespread Bufo bufo species group (Anura, Bufonidae).

New analytical methods are improving our ability to reconstruct robust species trees from multilocus datasets, despite difficulties in phylogenetic reconstruction associated with recent, rapid divergence, incomplete lineage sorting and/or introgression. In this study, we applied these methods to resolve the radiation of toads in the Bufo bufo (Anura, Bufonidae) species group, ranging from the Iberian Peninsula and North Africa to Siberia, based on sequences from two mitochondrial and four nuclear DNA regions (3490 base pairs). We obtained a fully-resolved topology, with the recently described Bufo eichwaldi from the Talysh Mountains in south Azerbaijan and Iran as the sister taxon to a clade including: (1) north African, Iberian, and most French populations, referred herein to Bufo spinosus based on the implied inclusion of populations from its type locality and (2) a second clade, sister to B. spinosus, including two sister subclades: one with all samples of Bufo verrucosissimus from the Caucasus and another one with samples of B. bufo from northern France to Russia, including the Apennine and Balkan peninsulas and most of Anatolia. Coalescent-based estimations of time to most recent common ancestors for each species and selected subclades allowed historical reconstruction of the diversification of the species group in the context of Mediterranean paleogeography and indicated a long evolutionary history in this region. Finally, we used our data to delimit the ranges of the four species, particularly the more widespread and historically confused B. spinosus and B. bufo, and identify potential contact zones, some of which show striking parallels with other co-distributed species.

High levels of population subdivision in a morphologically conserved Mediterranean toad (Alytes cisternasii) result from recent, multiple refugia: evidence from mtDNA, microsatellites and nuclear genealogies.

Pleistocene glaciations often resulted in differentiation of taxa in southern European peninsulas, producing the high levels of endemism characteristic of these regions (e.g. the Iberian Peninsula). Despite their small ranges, endemic species often exhibit high levels of intraspecific differentiation as a result of a complex evolutionary history dominated by successive cycles of fragmentation, expansion and subsequent admixture of populations. Most evidence so far has come from the study of species with an Atlantic distribution in northwestern Iberia, and taxa restricted to Mediterranean-type habitats remain poorly studied. The Iberian Midwife toad (Alytes cisternasii) is a morphologically conserved species endemic to southwestern and central Iberia and a typical inhabitant of Mediterranean habitats. Applying highly variable genetic markers from both mitochondrial and nuclear genomes to samples collected across the species' range, we found evidence of high population subdivision within A. cisternasii. Mitochondrial haplotypes and microsatellites show geographically concordant patterns of genetic diversity, suggesting population fragmentation into several refugia during Pleistocene glaciations followed by subsequent events of geographical and demographic expansions with secondary contact. In addition, the absence of variation at the nuclear beta-fibint7 and Ppp3caint4 gene fragments suggests that populations of A. cisternasii have been recurrently affected by episodes of extinction and recolonization, and that documented patterns of population subdivision are the outcome of recent and multiple refugia. We discuss the evolutionary history of the species with particular interest in the increasing relevance of Mediterranean refugia for the survival of genetically differentiated populations during the Pleistocene glaciations as revealed by studies in co-distributed taxa.

Mitochondrial DNA phylogeography of Lissotriton boscai (Caudata, Salamandridae): evidence for old, multiple refugia in an Iberian endemic.

In Europe, southern peninsulas served as refugia during cold periods in the Pleistocene, acting both as centres of origin of endemisms and as sources from which formerly glaciated areas were recolonized during interglacial periods. Previous studies have revealed that within the main refugial areas, intraspecific lineages often survived in allopatric refugia. We analysed two mitochondrial markers (nad4, control region, approximately 1.4 kb) in 103 individuals representing the entire distribution of Lissotriton boscai, a newt endemic to the western Iberian Peninsula. We inferred the evolutionary history of the species through phylogenetic, phylogeographic and historical demographic analyses. The results revealed unexpected, deep levels of geographically structured genetic variability. We identified two main evolutionary lineages, each containing three well-supported clades. The first historical split involved populations from central-southwestern coastal Portugal and the ancestor of all the remaining populations around 5.8 million years ago. Both lineages were subsequently fragmented into different population groups between 2.5 and 1.2 million years ago. According to nested clade analysis, at lower hierarchical levels the patterns suggest restricted gene flow with isolation by distance, whereas at higher levels the clades exhibit signatures of contiguous range expansion. Bayesian Skyline Plots show recent bottlenecks, followed by demographic expansions in all lineages. The significant genetic structure found is consistent with long-term survival of populations in allopatric refugia, supporting the 'refugia-within-refugia' scenario for southern European peninsulas. The comparison of our results with other co-distributed species highlights the generality of this hypothesis for the Iberian herpetofauna and suggests that Mediterranean refuges had more relevance for the composition and distribution of present biodiversity patterns than currently acknowledged. We briefly discuss the taxonomic and conservation implications of our results.