Genetic Divergence and Evolution of Reproductive Isolation in Eastern Mediterranean Water Frogs

Water frogs [genus Pelophylax (Rana)] that occur around the eastern Mediterranean Sea provide an opportunity to study early stages of speciation. The geography of the eastern Mediterranean region has changed dramatically since the Middle Miocene as a result of motions of adjoining lithospheric plates and regionalscale vertical crustal motions (uplift and subsidence). For several hundred thousand years between 6 and 5 million years ago (Mya), the Mediterranean basin was isolated from the Atlantic Ocean, and became desiccated (the Messinian Salinity Crisis; MSC). Geological data suggest that the endemic water frog lineage on Cyprus was isolated by the flooding of the Mediterranean basin by salt water at the end of the MSC, circa 5.5–5.3 Mya. This suggests a rate of uncorrected genetic divergence of approximately 1.1% per million years (My). Divergence time estimates based on this rate are in good agreement with the chronology of events in the history of crustal deformation and landscape development in the eastern Mediterranean region. Despite a high similarity in morphology, eastern Mediterranean water frogs show considerable genetic divergence, indicating the existence of several evolutionary species at varied levels of differentiation. Based on two mitochondrial (mt) genes (ND2 and ND3), several lineages have been identified: Pelophylax bedriagae, P. cretensis, P. epeiroticus, P. ridibundus (Europe), six Anatolian lineages, all provisionally subsumed under the name P. cf. bedriagae, and a distinct lineage restricted to Cyprus. Genetic data from transition zones in eastern Greece/western Anatolia, south-western Anatolia, and south-eastern Anatolia, in concert with the results of female choice experiments, indicate that antihybridization mechanisms are only weakly developed in eastern Mediterranean water frogs. Genetic incompatibility, as expressed by average hatching rate of heterospecific crosses, increases with genetic divergence measured by uncorrected distance estimated from mtDNA sequences. Hatching rates of heterospecific crosses show an extremely high variability, however, and viable F1 hybrids originated from almost all crosses. We conclude that speciation in eastern Mediterranean water frogs follows the allopatric model and has been closely associated with the geodynamic evolution of the Mediterranean since the Middle Miocene (i.e., since 11 Mya)