DNA Barcoding of Sri Lankan Anopheline Mosquitoes Reveals Its Usefulness in Identifying Sibling Species

Abstract

“DNA barcoding” is a powerful tool for species identification and used in studying evolutionary relationships among species. The objective of this study was to characterize Cytochrome c Oxidase subunitI (COI) and Internal Transcribed Spacer 2 (ITS2) sequences of morphologically identified Anopheles species collected from two different areas in Sri Lanka and to distinguish sibling species using DNA barcodes. Anopheles peditaneatus, An. nigerrimus, An. jamseii, An. tessellatus, An. annularis, An. pallidus, An. barbirostris, An. vagus, An. subpictus, An. culicifacies and An. varuna were collected from Jaffna in the Northern (N) Province and Wariyapola in the North Western (NW) Province. COI sequences (52) of 3,3,7,1,2,6,1,6,8,8, and 6 and, ITS2 sequences (21) 4,3,6,0, 1,5,0,2,0,0,0 were respectively generated from the PCR positive samples of DNA extracted from the above species (10 individuals from each species).The COI sequences showed high genetic variation compared to that of ITS2. Two of the morphologically identified An. peditaneatus specimens had sequences similar to that of An. nigerrimus which show high resemblance in morphological features to An. peditaneatus. Based on COI sequences, An. subpictus samples from N Province were identified as An. subpictus B and samples of An. subpictus from NW Province had sequences similar to sibling species A. Analysis of COI and ITS2 sequences confirmed the presence of An. annularis sibling species A in the N Province. An. barbirostris sequence obtained from the N Province was different from the previously described sequences of An. barbirostris complex suggesting the existence of a different molecular type. Sequences of morphologically identified other anopheline species confirmed their identity with GenBank entries from South and Southeast Asian countries. However cytologically identified sibling species B and E of An. culicifacies could not be separated using COI and ITS2 markers. The present study indicates that DNA barcoding is useful in identifying sibling species and proves that Sri Lankan Anopheles species have a close evolutionary relationship with the South and Southeast Asian counterparts.