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The phylogenetic relationships within the piranhas were assessed using mitochondrial sequences with the aim of testing several hypotheses proposed to explain the origin of Neotropical diversity (palaeogeography, hydrogeology and museum hypotheses). Sequences of the ribosomal 16S gene (510 bp) and control region (980 bp) were obtained from 15 localities throughout the main South American rivers for 21 of the 28 extant piranha species. The results indicate that the genus Serrasalmus is monophyletic and comprises three major clades. The phylogeographical analyses of these clades allowed the identification of five vicariant events, extensive dispersal and four lineage duplications suggesting the occurrence of sympatric speciation. Biogeographical patterns are consistent with the prediction made by the museum hypothesis that lineages from the Precambrian shields are older than those from the lowlands of the Amazon. The vicariant events inferred here match the distribution of the palaeoarches and several postdispersal speciation events are identified, thereby matching the predictions of the palaeogeography and hydrogeology hypotheses, respectively. Molecular clock calibration of the control region sequences indicates that the main lineages differentiated from their most recent common ancestor at 9 million years ago in the proto Amazon-Orinoco and the present rate of diversification is the highest reported to date for large carnivorous Characiformes. The present results emphasize that an interaction among geology, sea-level changes, and hydrography created opportunities for cladogenesis in the piranhas at different temporal and geographical scales.

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