A new genus of Anostomidae (Characiformes) is described to include ten valid extant species previously classified in Leporinus or Hypomasticus and distributed throughout most major river basins in South America: L. brinco, L. conirostris, L. elongatus, H. garmani, L. macrocephalus, L. muyscorum, L. obtusidens, L. piavussu, L. reinhardti, and L. trifasciatus. The monophyly of Megaleporinus is well-supported in a phylogenetic analysis based on two mitochondrial and three nuclear genes, as well as its sister group relationship to Abramites. Megaleporinus is diagnosed by having the exclusive combination of three unicuspid teeth on each premaxillary and dentary bone and a color pattern composed of one to four dark midlateral blotches. Additional distinguishing features and possible synapomorphies include a unique ZZ/ZW sex chromosome system confirmed for six congeners and a drumming apparatus wherein the first rib is elongated and associated with hypertrophied intercostal muscles, which was confirmed for three congeners as exclusive to mature males. Furthermore, our study identified at least four undescribed cryptic species, emphasizing the need for further taxonomic work and genetic analyses. A time-calibrated phylogenetic and biogeographical analysis of the new genus suggests that speciation in the proto-Amazon-Orinoco lineage was primarily driven by paleogeographic processes, such as the formation of the Orinoco and Tocantins basins. Dispersal and diversification of the genus in coastal basins draining the Eastern Brazilian Shield appears to have been facilitated by connections between paleo-basins during low sea level periods and headwater captures between coastal and inland watersheds. The present contribution demonstrates the importance of integrating data from morphology, DNA sequences and cytogenetics to advance the taxonomy and systematics of any complex species group.
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