Molecular forms of Anopheles subpictus s.l. and Anopheles sundaicus s.l. in the Indian subcontinent

Abstract BackgroundAnopheles subpictus s.l. and Anopheles sundaicus s.l. are closely related species, each comprising of several sibling species. Ambiguities exist in the classification of these two nominal species and the specific status of members of An. subpictus complex. Identifying fixed molecular forms and mapping their spatial distribution will help in resolving the taxonomic ambiguities and understanding their relative epidemiological significance. MethodsDNA sequencing of Internal Transcribed Spacer-2 (ITS2), 28S-rDNA (D1-to-D3 domains) and cytochrome oxidase-II of morphologically identified specimens of two nominal species, An. subpictus s.l. and An. sundaicus s.l. collected from the Indian subcontinent, was performed and subjected to genetic distance and molecular phylogenetic analyses. ResultsMolecular characterization of mosquitoes for rDNA revealed the presence of two molecular forms of An. sundaicus s.l. (identified as An. epiroticus s.s. and An. sundaicus D) 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, and molecular forms 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. ConclusionA 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. Presence of An. epiroticus in Indian territory is recorded for the first time.