Molecular and morphological evidence for sibling species within Anopheles (Anopheles) lindesayi Giles (Diptera: Culicidae) in Bhutan

This paper reports the results of a comparative molecular and morphological study of An. lindesayi collected from various districts of Bhutan and An. l. cameronensis from Thailand, compared with GenBank accessions and publications for An. l. japonicas from Japan, South Korea and China, An. l. pleccau from Taiwan, and An. lindesayi from India. Phylogenetic analyses based on ribosomal (ITS2) and mitochondrial (COI) DNA sequences using the Maximum Likelihood method revealed five genetically distinct clades (A, B, C, D and E) in Bhutan. Specimens in Clade A correspond to the original description of An. lindesayi, particularly in wing markings, the pattern of basal pale scales on the hindfemur and the single seta 4-C of larvae, and their COI sequences were closely related to one Indian sequence. Larvae of Clades B, C, D and E are similar in having seta 4-C branched rather than single. The adults of Clades C, D and E (B not available) are distinguishable from those of Clade A and other subspecies. Specimens of Clade C are unique in having a long pale spot on wing vein R and the subcosta, scattered pale scales on several veins and a dark spot at the tip of vein R-2. The adults of Clades D and E are similar in having a dark spot at the tip of vein R-2 and no scattered pale scales on all other veins. We provisionally recognize mosquitoes of Clades A, B, C, D and E as species A, B, C, D and E, respectively, of the Lindesayi Complex. Species A is An. lindesayi sensu stricto and the others are unnamed species. Concomitantly, the previous concept of the "Lindesayi Complex", which included An. lindesayi, An. menglangensis, An. nilgiricus and An. wellingtonianus, is now recognized as the Lindesayi Subgroup of the Lindesayi Group (Anopheles Series, subgenus Anopheles) with the five sibling species of An. lindesayi comprising a more apposite Lindesayi Complex within the subgroup.