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.

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

With the advent of large multilocus datasets, molecular systematics is experiencing very rapid progress, but important challenges remain regarding data analysis and interpretation. Midwife toads (genus Alytes) exemplify two of the most widespread problems for accurate phylogenetic reconstruction: discerning the causes of discordance between gene trees, and resolving short internodes produced during rapid, successive splitting events. The three species in subgenus Baleaphryne (A. maurus, A. dickhilleni and A. muletensis), the sister group to A. obstetricans, have disjunct and highly restricted geographical ranges, which are thought to result from old vicariant events affecting their common ancestor, but their phylogenetic relationships are still unresolved. In this study we re-address the phylogeny of Alytes with a special focus on the relationships in Baleaphryne with a multilocus dataset including >9000 base pairs of mitochondrial DNA and four nuclear markers (3142bp) in all recognized taxa, including all subspecies of A. obstetricans. Both concatenation and species tree analyses suggest that A. muletensis, endemic to the Balearic island of Mallorca, is the sister taxon to a clade comprising the southeastern Iberian endemic A. dickhilleni and the North African A. maurus. This scenario is consistent with palaeogeological evidence associated with the fragmentation of the Betic-Rifean Massif, followed by the opening of the Strait of Gibraltar. On the other hand, analyses of intraspecific variation in A. obstetricans are inconclusive regarding relationships between major clades and conflict with current subspecific taxonomy.

The common midwife toad (Alytes obstetricans), widely distributed in the northern half of the Iberian Peninsula and part of Western Europe, is currently subdivided into four subspecies: A. o. obstetricans, A. o. boscai, A. o. pertinax and A. o. almogavarii. However, the delimitation of these subspecies and their ranges are still under discussion because strong discordances have been found between morphological and molecular data, and especially among different genetic markers. Here, we screen a set of novel microsatellite loci and mtDNA sequences of A. obstetricans populations representative of all currently recognized subspecies to investigate the correspondence between genetic groupings inferred from clustering analysis of microsatellite genotypes and the described subspecies and test whether patterns of mtDNA variation are concordant with those genetic clusters. Our results confirm previous expectations of extremely high intraspecific diversity in A. obstetricans in Iberia. Analyses of microsatellite and mtDNA data were concordant in recovering five well‐defined groups, of which three correspond to previously defined subspecies, while the two additional clusters correspond to populations of subspecies A. o. boscai separated by the Douro River. Our results suggest the occurrence of two distinct genetic units within A. o. boscai that likely result from a long independent evolutionary history, thus deserving special attention from a conservation point of view.

Context Mediterranean wetland ecosystems are in continuous decline due to human pressure. Amphibians are key elements of biotic communities of Mediterranean temporary ponds and streams, and their persistence depends on the availability and inter-connectivity of breeding sites. Objectives We investigated the role of different factors potentially driving functional connectivity patterns in two amphibian species at the landscape and local scales. We focused on two Mediterranean endemic pond-breeding amphibians inhabiting semi-arid landscapes of central Spain, the common parsley frog (Pelodytes punctatus) and the common midwife toad (Alytes obstetricans). Methods We genotyped 336 individuals of P. punctatus and 318 of A. obstetricans from 17 and 16 breeding populations at 10 and 17 microsatellite loci, respectively. We used remotely sensed vegetation/moisture indices and land use/cover data to derive optimized resistance surfaces and test their association with estimates of gene flow and migration rates across populations. Results We found evidence for higher population connectivity in common midwife toads than in common parsley frogs, with a strong effect of water availability in patterns of population connectivity of both species. However, the two species differ in the role of landscape features on population connectivity, with the distance and spatial distribution of artificial land-use types positively influencing connectivity in A. obstetricans and meadows/pastureland favouring P. punctatus. This is in line with reported breeding site preferences for the two species, with A. obstetricans successfully breeding in artificial water bodies that P. punctatus generally avoid. Conclusions This study highlights the importance of assessing species–habitat relationships shaping connectivity when developing and implementing conservation and management actions to benefit fragmented amphibian populations in the Mediterranean region. Our results show that amphibian species respond differently, even contrastingly to landscape features and thus require alternative, complementary strategies to improve population connectivity and ensure long-term viability.