Multiplex PCR assay and phylogenetic analysis of sequences derived from D2 domain of 28S rDNA distinguished members of the Anopheles culicifacies complex into two groups, A/D and B/C/E.

A multiplex PCR assay was developed using the sequences of the D2 region of 28S ribosomal DNA (rDNA) to discriminate the five members of the Anopheles culicifacies complex provisionally designated as species A, B, C, D and E. Two minus strand primers derived from sequence differences in the D2 variable region and a universal plus strand primer derived from the conserved 28S (rDNA) has delimited five members into species A and D (group 1) and species B, C and E (group 2) in a PCR diagnostic assay. The complete 28S rDNA-D2 region sequence of A. culicifacies sibling species is reported for the first time. Inter-specific sequence divergence was greater than the intra-specific divergence. The phylogenetic relationships inferred from maximum likelihood, maximum parsimony and the neighbor joining analysis confirmed the presence of two unambiguous monophyly clades one consisting of species A and D and the other of species B, C and E and that the A. culicifacies sibling species diverged relatively recently in evolutionary terms despite their considerable differences in bionomics.