Recent studies have shown that Anopheles oswaldoi sensu lato comprises a cryptic species complex in South America. Anopheles konderi, which was previously raised to synonymy with An. oswaldoi, has also been suggested to form a species complex. An. oswaldoi has been incriminated as a malaria vector in some areas of the Brazilian Amazon, Colombia, Peru and Venezuela, but was not recognized as a vector in the remaining regions in its geographic distribution. The role of An. konderi as a malaria vector is unknown or has been misattributed to An. oswaldoi. The focus of this study was molecular identification to infer the evolutionary relationships and preliminarily delimit the geographic distribution of the members of these complexes in the Brazilian Amazon region. The specimens were sampled from 18 localities belonging to five states in the Brazilian Amazon and sequenced for two molecular markers: the DNA barcode region (COI gene of mitochondrial DNA) and Internal Transcribed Spacer 2 (ITS2 ribosomal DNA). COI (83 sequences) and ITS2 (27 sequences) datasets generated 43 and 10 haplotypes, respectively. Haplotype networks and phylogenetic analyses generated with the barcode region (COI gene) recovered five groups corresponding to An. oswaldoi s. s., An. oswaldoi B, An. oswaldoi A, An. konderi and An. sp. nr. konderi; all pairwise genetic distances were greater than 3%. The group represented by An. oswaldoi A exhibited three strongly supported lineages. The molecular dating indicated that the diversification process in these complexes started approximately 2.8 Mya, in the Pliocene. These findings confirm five very closely related species and present new records for these species in the Brazilian Amazon region. The paraphyly observed for the An. oswaldoi complex suggests that An. oswaldoi and An. konderi complexes may comprise a unique species complex named Oswaldoi-Konderi. Anopheles oswaldoi B may be a potential malaria vector in the extreme north of the Brazilian Amazon, whereas evidence of sympatry for the remaining species in other parts of the Brazilian Amazon (Acre, Amazonas, Para A and Rondo A nia) precluded identification of probable vectors in those areas.
