Aim  To devise a robust phylogenetic hypothesis for midwife toads (Alytes: Anura: Discoglossidae) and to discuss its implications for the reconstruction of the biogeographical history of the group. Location  Western Palearctic. Methods  Analysis of sequences of mitochondrial DNA (cytochrome b and 16S RNA, 861 bp) and 29 characters of cranial osteology of all species and subspecies within Alytes. Results  Phylogenetic analyses support a sister group relationship between Alytes dickhilleni and A. muletensis, and of this clade with A. maurus. The monophyly of A. obstetricans is controversial; in particular, the phylogenetic position of A. obstetricans almogavarii is uncertain. The estimated dates for the cladogenetic events within Alytes are congruent with those derived from independent analyses (allozymes), except for the differentiation of A. o. almogavarii and the split between A. dickhilleni and A. muletensis. Main conclusions  The phylogeny based on the analysis of morphological and mtDNA data differs from previous hypotheses in the positions of A. o. almogavarii and A. maurus. Events associated with the radiation of Alytes are the formation of large inland saline lakes in Iberia c. 16 Ma and the 11 ° C dramatic decrease in average annual temperature during the Middle–Late Badenian transition c. 14–13.5 Ma (A.cisternasii vs. the ancestor of the remaining clades), the structuring of the Neo‐Pyrenees and the reopening of the Betic Strait c. 10–8 Ma(A. o. almogavarii vs. A. obstetricans and vs. the subgenus Baleaphryne), the opening of the Gibraltar Strait at the end of the Messinian Salinity Crisis at c. 5.3 Ma (A. maurus vs. the ancestor of the A. dickhilleni A. muletensis clade) and a transmarine colonization event at c. 3 Ma (the ancestor of A. muletensis vs. A. dickhilleni). Following the new hypothesis, A. maurus, previously considered a subspecies of A. obstetricans, deserves species status. Second, A. o. almogavarii is a well‐differentiated lineage that was isolated from other A. obstetricans more than c. 5 Ma, but later lost its genetic and specific identity following secondary contact, hybridization and introgression with the main stock. The presence of a marked morphological and genetic diversity within A. obstetricans renders reconstruction of the evolutionary history of the genus more complicated than previously appreciated.

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.

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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.

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Aim: Understanding the roles of allopatric fragmentation and niche divergence in intraspecific lineage diversification is central to our comprehension of how new species arise and evolve. Here we integrate an extensive multilocus dataset with species distribution models to address these topics in genetically differentiated population lineages of the common midwife toad (Alytes obstetricans).Location: Iberian Peninsula, Western Europe. Methods:We genotyped 965 individuals from 142 localities at 12 microsatellite loci to finely delineate the ranges of population lineages in A. obstetricans. We then built ecological niche-based models for each lineage and tested for niche overlap, equivalency and similarity based on PCA-env analyses. We also investigated the relationship between genetic and environmental distances across lineages. Results:We recovered seven differentiated genetic clusters, with mostly different (sometimes nonoverlapping) niches. However, niches are not more divergent than expected given the largely allopatric ranges of population lineages. We found no significant isolation by environment within lineages, with genetic distances mostly explained by geographical distances.Main conclusions: Allopatric fragmentation in A. obstetricans resulted in (sometimes highly) different niches in intraspecific population lineages. Within lineages, isolation by distance is the major driver of patterns of genetic variation. Our study highlights the importance of incorporating intraspecific genetic structure into species distribution models to explore spatial patterns of genetic diversity in terms of their underlying processes. K E Y W O R D Sdiversification, ecological niche-based modelling, intraspecific lineage, microsatellites, niche conservatism, niche divergence

We report new primers for the amplification and sequencing of 11 nuclear markers in squamate reptiles and anuran amphibians (five in squamates, six in anurans). Ten out of the 11 loci are introns (three of which are linked) that were amplified using an exon-primed, introncrossing (EPIC) PCR strategy, whereas an eleventh locus spans part of a protein-coding gene. Squamate and anuran primers were initially developed for Lacerta schreiberi (Squamata: Lacertidae) and Pelodytes spp. (Anura: Pelodytidae), respectively. Cross-species amplification of the squamate markers was evaluated in four genera representing two additional families, whereas for anurans three genera corresponding to three additional families were tested. Three out of the five loci were successfully sequenced in all squamate taxa tested. Cross-amplification of the six anuran markers had lower, but still significant, success. We predict these markers will be of great utility for both population genetics and phylogenetic studies.