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The 'sinistral Phalloceros group' consists of three species, P. aspilos , P. leptokeras and P. tupinamba , that belong to Phalloceros , a genus of freshwater fish endemic to South America. They inhabit the Paraíba do Sul Basin and coastal drainages in south-eastern Brazil. This group is diagnosed by large hooks in the medial portion of the gonopodial appendices in males and the sinistral direction of the urogenital papilla in females. We conducted a phylogeographic analysis of mitochondrial (COI and Cytb) and nuclear (RAG1) haplotypes of 36 individuals sampled from 11 localities to test the hypothesis that the biogeographic history of sinistral Phalloceros was mediated by connections of coastal basins caused by lowered sea-levels during the Quaternary. We evaluated the taxonomic status of these nominal taxa by integrating molecular species delimitation methods and morphological data. Our results suggest that the three nominal taxa are synonyms, and P. leptokeras is designated as the valid species name. The geographic expansion of the P. leptokeras lineage began in the coastal region (~2.3 Mya). The phylogeographic relationships among populations partially corroborate the palaeodrainage model, but also suggest that P. leptokeras colonized the inland Paraíba do Sul drainage, overcoming the Serra do Mar mountains. [ABSTRACT FROM AUTHOR]