Frequent mitochondrial gene introgression among high elevation Tibetan megophryid frogs revealed by conflicting gene genealogies.

Historical mitochondrial introgression causes differences between a species' mitochondrial gene genealogy and its nuclear gene genealogy, making tree-based species delineation ambiguous. Using sequence data from one mitochondrial gene (cytochrome b) and three nuclear genes (introns), we examined the evolutionary history of four high elevation Tibetan megophryid frog species, Scutiger boulengeri, Scutiger glandulatus, Scutiger mammatus and Scutiger tuberculatus. The three nuclear genes shared a similar history but the mitochondrial gene tree suggested a drastically different evolutionary scenario. The conflicts between them were explained by multiple episodes of mitochondrial introgression events via historical interspecific hybridization. 'Foreign' mitochondrial genomes might have been fixed in populations and extended through a large portion of the species' distribution. Some hybridization events were probably as old as 10 Myr, while others were recent. An F(1) hybrid was also identified. Historical hybridization events among the four species appeared to be persistent and were not restricted to the period of Pleistocene glaciation, as in several other well-studied cases. Furthermore, hybridization involved several species and occurred in multiple directions, and there was no indication of one mitochondrial genome being superior to others. In addition, incomplete lineage sorting resulting from budding speciation may have also explained some discrepancies between the mitochondrial DNA and nuclear gene trees. Combining all evidences, the former 'Scutiger mammatus' appeared to be two species, including a new species. With the availability of a wide range of highly variable nuclear gene markers, we recommend using a combination of mitochondrial gene and multiple nuclear genes to reveal a complete species history.