Molecular forms of Anopheles subpictus and Anopheles sundaicus in the Indian subcontinent

BACKGROUND: Anopheles subpictus and Anopheles sundaicus are closely related species, each comprising several sibling species. Ambiguities exist in the classification of these two nominal species and the specific status of members of these species complexes. Identifying fixed molecular forms and mapping their spatial distribution will help in resolving the taxonomic ambiguities and understanding their relative epidemiological significance. METHODS: DNA sequencing of Internal Transcribed Spacer-2 (ITS2), 28S-rDNA (D1-to-D3 domains) and cytochrome oxidase-II (COII) of morphologically identified specimens of two nominal species, An. subpictus sensu lato (s.l.) and An. sundaicus s.l., collected from the Indian subcontinent, was performed and subjected to genetic distance and molecular phylogenetic analyses. RESULTS: Molecular characterization of mosquitoes for rDNA revealed the presence of two molecular forms of An. sundaicus s.l. and three molecular forms of An. subpictus s.l. (provisionally designated as Form A, B and C) in the Indian subcontinent. Phylogenetic analyses revealed two distinct clades: (i) subpictus clade, with a single molecular form of An. subpictus (Form A) prevalent in mainland India and Sri Lanka, and (ii) sundaicus clade, comprising of members of Sundaicus Complex, two molecular forms of An. subpictus s.l. (Form B and C), prevalent in coastal areas or islands in Indian subcontinent, and molecular forms of An. subpictus s.l. reported from Thailand and Indonesia. Based on the number of float-ridges on eggs, all An. subpictus molecular Form B were classified as Species B whereas majority (80%) of the molecular Form A were classified as sibling species C. Fixed intragenomic sequence variation in ITS2 with the presence of two haplotypes was found in molecular Form A throughout its distribution. CONCLUSION: A total of three molecular forms of An. subpictus s.l. and two molecular forms of An. sundaicus s.l. were recorded in the Indian subcontinent. Phylogenetically, two forms of An. subpictus s.l. (Form B and C) prevalent in coastal areas or islands in the Indian subcontinent and molecular forms reported from Southeast Asia are members of Sundaicus Complex. Molecular Form A of An. subpictus is distantly related to all other forms and deserve a distinct specific status.