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Background  One key question in evolutionary biology deals with the mode and rate at which reproductive isolation accumulates during allopatric speciation. Little is known about secondary contacts of recently diverged anuran species. Here we conduct a multi-locus field study to investigate a contact zone between two lineages of green toads with an estimated divergence time of 2.7 My, and report results from preliminary experimental crosses.    Results  The Sicilian endemic  Bufo siculus  and the Italian mainland-origin  B. balearicus  form a narrow hybrid zone east of Mt. Etna. Despite bidirectional mtDNA introgression over a ca. 40 km North-South cline, no F 1  hybrids could be found, and nuclear genomes display almost no admixture. Populations from each side of the contact zone showed depressed genetic diversity and very strong differentiation (F ST  = 0.52). Preliminary experimental crosses point to a slightly reduced fitness in F 1  hybrids, a strong hybrid breakdown in backcrossed offspring (F 1  x parental, with very few reaching metamorphosis) and a complete and early mortality in F 2  (F 1  x F 1 ).    Conclusion  Genetic patterns at the contact zone are molded by drift and selection. Local effective sizes are reduced by the geography and history of the contact zone,  B. balearicus  populations being at the front wave of a recent expansion (late Pleistocene). Selection against hybrids likely results from intrinsic genomic causes (disruption of coadapted sets of genes in backcrosses and F 2 -hybrids), possibly reinforced by local adaptation (the ranges of the two taxa roughly coincide with the borders of semiarid and arid climates). The absence of F 1  in the field might be due to premating isolation mechanisms. Our results, show that these lineages have evolved almost complete reproductive isolation after some 2.7 My of divergence, contrasting sharply with evidence from laboratory experiments that some anuran species may still produce viable F 1  offspring after > 20 My of divergence.

Strong reproductive barriers in a narrow hybrid zone of West-Mediterranean green toads (Bufo viridissubgroup) with Plio-Pleistocene divergence