Phylogenetic, phylogeographic and divergence time analysis of Anopheles subpictus species complex using ITS2 and COI sequences.

Objective: To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles (An.) subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2 (ITS2) and cytochrome C oxidase I (COI) sequences deposited at the GenBank. Methods: Seventy-five ITS2, 210 COI and 26 concatenated sequences available in the NCBI database were used. Phylogenetic analysis was performed using Bayesian likelihood trees, whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis. Genetic diversity indices and genetic differentiation were also calculated. Results: Two genetically divergent molecular forms of An. subpictus species complex corresponding to sibling species A and B are established. Species A evolved around 37-82 million years ago in Sri Lanka, India, and the Netherlands, and species B evolved around 22-79 million years ago in Sri Lanka, India, and Myanmar. Vietnam, Thailand, and Cambodia have two molecular forms: one is phylogenetically similar to species B. Other forms differ from species A and B and evolved recently in the above mentioned countries, Indonesia and the Philippines. Genetic subdivision among Sri Lanka, India, and the Netherlands is almost absent. A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances. Genetic diversity indices reveal the presence of a long-established stable mosquito population, at mutation-drift equilibrium, regardless of population fluctuations. Conclusions: An. subpictus species complex consists of more than two genetically divergent molecular forms. Species A is highly divergent from the rest. Sri Lanka and India contain only species A and B. [ABSTRACT FROM AUTHOR]