Molecular identification of wild caught phlebotomine sand flies (Diptera, Psychodidae) by mitochondrial DNA barcoding in India

Sand flies are medically important insects with diverse distributions and roles in pathogen transmission. Globally, over a thousand species have been documented, with Indian sand fly fauna currently comprising 71 species. Traditional morphological identification faces challenges due to specimen damage and the presence of cryptic species. This study utilizes DNA barcoding of the mitochondrial marker, cytochrome c oxidase subunit I (COI) to enhance accurate identification of Indian sand flies. A total of 10,456 sand flies, representing 31 species, were collected from 26 districts across six Indian states between 2018 and 2024. Legs from voucher specimens were used to generate ~ 720 bp COI sequences, which were analyzed phylogenetically. In total, 169 COI sequences were generated. A common 570 bp region was selected for final analysis. The gene showed an AT-rich composition with a GC content of 34.8%. Maximum likelihood phylogenetic analysis, supported by ABGD and ASAP species delimitation methods, confirmed the majority of morphological identifications. Species delimitation analyses using ABGD, ASAP, and bPTP grouped the specimens into 32, 34, and 68 clusters, respectively, with bPTP showing evidence of over splitting. Despite this, COI-based classification proved effective in delineating species boundaries and serves as a reliable tool for the DNA barcoding of sand fly species.