Agradecimentos: We thank Alessandra Ferreira da Costa for technical assistance and the Laboratory for Integrative and System Biology (LaBIS) for the use of the Thunder Cluster funded by Fapesp grants 2011/00417-3 and 2015/50612-8. CH and GA are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a research fellowship. DB is grateful to FONCyT (PICT 2011/1524, 2011/1895, 2012/2687, 2013/0404, 2014/1343, 2014/1930, 2014/2035, 2015/0813, 2015/0820, 2015/2381) and CONICET (PIP 112201101/00875) for financial support. JN is grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES) for PhD studentship Ver menos

Abstract: Previous cytogenetic and phylogenetic analyses showed a high variability in the frog taxa Physalaemus cuvieri and Physalaemus ephippifer and suggested the presence of undescribed diversity in this species complex. Here, by 1) adding specimens from the Brazilian Amazon region, 2) employing sequence-based species delimitation approaches, and 3) including RADseq-style markers, we demonstrate that the diversity in the P. cuvieri-P. ephippifer species complex is even greater than previously suspected. Specimens from Virua and Western Para, located at the Guiana Amazonian area of endemism, were recovered as distinct from all previously identified lineages by the phylogenetic analyses based on mitochondria! DNA and RAD markers, a PCA from RAD data, and cytogenetic analysis. The sequence-based species delimitation analyses supported the recognition of one or two undescribed species among these Amazonian specimens and also supported the recognition of at least three other species in the P. cuvieri-P. ephippifer species complex. These new results reinforce the need for a comprehensive taxonomic revision Ver menos