Phylogeographical analysis of DNA sequence data has been routinely used to test species boundaries using the monophyly criterion; however, a complementary criterion, reproductive isolation, is often ignored. We used a combination of phylogenetic and population genetic approaches to determine species boundaries among stream salamanders in the genus Batrachuperus. First, cytochrome b sequence data from 174 Batrachuperus individuals, sampled from 78 populations, were used to reconstruct historical relationships within the genus. Second, allozyme data for 14 presumptive nuclear loci, from 463 individuals sampled from 60 populations, were collected and analysed to assess population similarity or disparity, as well as potential reproductive isolation. The DNA sequence data grouped all populations into seven major monophyletic groups, and the allozyme data provided evidence for reproductive isolation among four of the seven groups, thereby supporting the species status of these groups. The allozyme data suggested that two of the other groups share the same gene pool, and therefore belong to a single species. Finally, the allozyme data revealed two reproductively isolated units within the seventh group, which we suggest represents a case of ‘budding speciation’ based on the DNA gene tree. In total, seven species of the genus Batrachuperus were defined, two of which were previously unknown. The phylogeographical analysis also revealed that vicariance events might have dominated the evolutionary history of this group, but the speciation events might precede the formation of the existing mountain topology. This study demonstrates the importance of including frequency data from multiple nuclear gene loci in determining species boundaries.
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