Mitochondrial Cytochrome Oxidase I (mtCOI) Gene Sequence Based Identification of Subterranean Termite Species from the Punjab Province of Pakistan

Correct species identification of subterranean termites is crucial for their effective control. However, achieving species-level identification has been difficult due to the complex biology, caste system and sexual dimorphism inherent to subterranean termites. In this study, worker and soldier castes of termites were collected from various locations across the Punjab province of Pakistan. Species identification was initially performed using the morphometric characteristics of the soldier caste. Subsequently, mitochondrial genes, including COI-5′ (barcode) and COI-3′ genes, were sequenced from both identified soldier and unidentified worker specimens. This molecular approach was employed to confirm species identities and establish phylogenetic relations. Both DNA barcodes and COI-3′ sequences successfully discriminated the analyzed termites to the species level. Cluster analysis showed species-level relationships between studied termite taxa. Disparity index test of species based on barcode data showed significant differences among most species pairs, highlighting the genetic diversity within the dataset. Nucleotide similarity analysis indicated that none of the species showed more than 98% similarity, with most of the species showing less than 89% similarity. Comparative analyses of COI-3′ sequences from the seven termite species, combined with the reference sequences available in the GenBank, consistently grouped the termite species of each genus into distinct, well-defined clusters. In conclusion, COI-5′ barcode and COI-3′ sequences provided robust nucleotide differentiation among the termite species, highlighting their effectiveness as precise molecular tools for species the discrimination and phylogenetic analysis of subterranean termites.