A molecular phylogeny of mosquitoes in the Anopheles barbirostris Subgroup reveals cryptic species: implications for identification of disease vectors.

The Barbirostris Subgroup of the genus Anopheles includes six mosquito species that are almost identical in adult morphology, but differ in their roles in the transmission of malaria and filariasis within Southeast Asia. The lack of robust, diagnostic morphological characters in adults has contributed to extensive misidentification of the species. Mosquitoes were collected from localities in Thailand and Indonesia, with an emphasis on specimens identified in the field as An. barbirostris and An. campestris. A 754 bp COI mitochondrial gene fragment was sequenced from 136 specimens and the rDNA ITS2 region (c.1600-1800 bp) from 51 specimens. Phylogenetic analyzes based on Bayesian methods, distance measures and Maximum-parsimony produced five clades (I-V) that are congruent between the nuclear and mitochondrial data sets. Based on adult female morphology, it is deduced that three of these clades, I-III, are members of the Barbirostris Complex whereas Clade V is An. campestris. The identity of Clade IV is as yet unknown. Using a haplotype network analysis, Clade III was found to have a star-like genealogy, suggesting population expansion. There were no shared haplotypes between clades. In Afrotropical anopheline mosquitoes, speciation has been linked to the expansion of human populations and the development of agriculture. We postulate that the radiation of species within the Barbirostris Subgroup in Southeast Asia may similarly be linked to human population expansion and the agrarian revolution. The development of a propensity for feeding on the blood of humans in some species of the Subgroup would have led to the transmission of malaria protozoa and filarial nematodes.