Mitochondrial DNA detects a complex evolutionary history with Pleistocene Epoch divergence for the neotropical malaria vector Anopheles nuneztovari sensu lato.

Cryptic species and lineages characterize Anopheles nuneztovari s.l. Gabaldón, an important malaria vector in South America. We investigated the phylogeographic structure across the range of this species with cytochrome oxidase subunit I (COI) mitochondrial DNA sequences to estimate the number of clades and levels of divergence. Bayesian and maximum-likelihood phylogenetic analyses detected four groups distributed in two major monophyletic clades (I and II). Samples from the Amazon Basin were clustered in clade I, as were subclades II-A and II-B, whereas those from Bolivia/Colombia/Venezuela were restricted to one basal subclade (II-C). These data, together with a statistical parsimony network, confirm results of previous studies that An. nuneztovari is a species complex consisting of at least two cryptic taxa, one occurring in Colombia and Venezuela and the another occurring in the Amazon Basin. These data also suggest that additional incipient species may exist in the Amazon Basin. Divergence time and expansion tests suggested that these groups separated and expanded in the Pleistocene Epoch. In addition, the COI sequences clearly separated An. nuneztovari s.l. from the closely related species An. dunhami Causey, and three new records are reported for An. dunhami in Amazonian Brazil. These findings are relevant for vector control programs in areas where both species occur. Our analyses support dynamic geologic and landscape changes in northern South America, and infer particularly active divergence during the Pleistocene Epoch for New World anophelines.