, Allan J Baker 1,3† 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 ~534,00
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