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The use of hyper-variable markers across species is often hindered by low cross-species amplification success, a reduced level of polymorphism or a high frequency of null alleles. However, optimizing sets of reliable and informative markers that can be consistently amplified and scored across taxa is key to address questions about patterns of genetic diversity and structure, hybridization and speciation. Here we present 14 newly developed microsatellite markers in the Spined toad ( Bufo spinosus ), assess their polymorphism in two Iberian populations and test for cross-species amplification in the closely related Common toad ( Bufo bufo ). We then use the 12 loci co-amplifying in both species to the study of a morphologically intermediate population (Moyaux) from the contact zone in northwest France as well as reference populations of the two species from both sides of the contact zone. Individuals from Moyaux had mtDNA haplotypes of the two species and were identified as hybrids in analyses with software NewHybrids. These results provide solid evidence for ongoing hybridization between  B. bufo  and  B. spinosus , with no apparent restrictions to gene flow.

Morphological and molecular data to describe a hybrid population of the Common toad (Bufo bufo) and the Spined toad (Bufo spinosus) in western France