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.