Phylogenetic and coalescent analysis of three loci suggest that the Water Rail is divisible into two species, Rallus aquaticus and R. indicus

Abstract Background Water Rails (Rallus aquaticus) inhabit fragmented freshwater wetlands across their Palearctic distribution. Disjunct populations are now thought to be morphologically similar over their vast geographic range, though four subspecies had been recognized previously. The fossil record suggests that Water Rails (R. aquaticus) were already spread across the Palearctic by the Pleistocene ~2 million years ago, and the oldest fossil remains thought to be closely related to the common ancestor of water rails date from the Pliocene. Results To investigate population structure in Water Rails at the genetic level we sequenced three independent loci: 686 base pairs (bp) of the mitochondrial DNA COI barcode; 618 bp of the intron ADH5; and 746 bp of the exon PTPN12. Phylogeographic analysis revealed that Water Rails breeding in eastern Asia (R. a. indicus, also known as the Brown-cheeked Rail) are strongly differentiated from the Water Rails in Western and Middle Asia and Europe (R. a. aquaticus and R. a. korejewi). The Kimura 3-parameter plus Gamma COI genetic distance between these two geographic groups was > 3%, and they differed by 18 diagnostic substitutions commensurate with differences between recently diverged sister species of birds. In spite of the low number of variable sites, the two nuclear loci supported this split. We estimated the split of the Brown-cheeked Rail and the Water Rail to have occurred ~534,000 years ago (95% CI 275,000-990,000 years ago). Fragmentation of the widespread ancestral population and eventual speciation of water rails is likely attributable to vicariance by a barrier formed by glacial cycles, continuous uplift of the Tibetan Plateau and increased sedimentation in deserts in southern Asia that originated in the Miocene. Conclusions Water Rails from East Asia were genetically differentiated from the ones breeding in Europe and Western to Middle Asia. Most of the genetic signal was from mitochondrial COI, and was corroborated by polymorphic sites in the two nuclear loci we employed. The split between these two lineages was estimated to occur in the Middle Pleistocene, when populations were isolated in disjunct wetlands with little or no gene flow. Independent evidence from differences in morphology and vocalizations in concert with genetic differentiation and a long history of isolation support recognition of the Brown-cheeked Rail breeding in East Asia as a separate species, R. indicus. The use of several independent loci is invaluable in inferring species trees from gene trees and in recognizing species limits