Phylogenetic taxonomy of the Zambian Anopheles coustani group using a mitogenomics approach

Abstract

Abstract Background Mosquito species belonging to the Anopheles coustani group have been implicated in driving residual malaria transmission in sub-Saharan Africa and are regarded as an established primary vector in Madagascar. The morphological identification of mosquitoes in this group is challenging due to similarity of features and their molecular confirmation is difficult due to a paucity of reference sequence data representing all members of the group. Conventional molecular barcoding with the cytochrome oxidase I (COI) gene and the internal transcribed spacer 2 (ITS2) region targets is limited in their discrimination and conclusive identification of members of species complexes. In contrast, complete mitochondrial genomes (mitogenomes) have demonstrated much improved power over barcodes to be useful in rectifying taxonomic discrepancies in Culicidae. The goal of this study was to characterize the phylogenetic taxonomy of Zambian members of the An. coustani group by generating and then using complete mitochondrial genomes for phylogenetic rectification. Methods A genome skimming approach was utilized via shallow shotgun sequencing on individual mosquito specimens to generate sequence reads for mitogenome assembly. Bayesian inferred phylogenies and molecular dating estimations were perfomed on the concatenated protein coding genes using the Bayesian Evolutionary Analysis by Sampling Trees 2 (BEAST 2) platform. Divergence estimates were calibrated for members of the An. coustani group based on published calucations for Anopheles-Aedes. Results This study generated 17 new complete mitogenomes which were comprable to reference An. coustani mitogenomes in the GenBank repository by having 13 protein coding, 22 transfer RNA and 2 ribosomal RNA genes, with an average length of 15,400 bp and AT content of 78.3%. Bayesian inference using the concatenated protein coding genes from the generated and publicly available mitogenomes yielded six clades: one for each of the four taxa targeted in this study, the GenBank references, and a currently unknown species. Divergence times estimated that the An. coustani group separated from the Anopheles gambiae complex approximately 110 million years ago (MYA), and members within the complex diverged at times points ranging from ~ 34 MYA to as recent as ~ 7 MYA. Conclusions These findings demonstrate the value of mitochondrial genomes in differentiating cryptic taxa and help to confirm morphological identities of An. coustani sensu stricto, Anopheles paludis, Anopheles zeimanni and Anopheles tenebrosus. Divergence estimates within the An. coustani group are similar when compared to species with morphologically distinct features. These analyses also highlight the likely presence of other cryptic An. coustani group members circulating in Zambia.

Keywords

• Anopheles coustani
• Mitochondrial genome
• Phylogeny
• Zambia
• Malaria