Abstract
The barb Barbus petenyi is a cyprinid widely distributed throughout the mountain regions in the Danube River basin. Phylogenetic analysis of the DNA sequence variation at the mitochondrial cytochrome b gene over much of this range yielded three deep-branching (5.9–9.4% average divergence), well-supported haplotype clades with mutually exclusive geographic distributions and divergence times estimated to be in the Tertiary. The clades did not form an altogether monophyletic group as the most divergent one coalesced more recently with haplotypes of phylogenetically close species than with the other B. petenyi haplotypes. This pattern was supported by bootstrap and log-likelihood Shimodaira–Hasegawa tests. The other two were sister clades, but their distinctiveness was supported by previous allozyme data. Hence, from a taxonomic point of view, the current recognition of B. petenyi is erroneous, as it does not represent a single evolutionary lineage, and we suggest that three species be recognized instead. Substantial phylogeographic differences were evident among the three putative species, the two more southerly ones displaying significant structure, which suggested that they each survived in several glacial refugia throughout the Pleistocene. The phylogeographic pattern of multiple populations of rheophilic barbs with a history of long-term persistence and separation within the Danube River basin is novel within fishes and provides a hypothesis against which phylogeographic patterns among other similarly distributed rheophilic species may be compared.