Abstract

Paper wasps (Hymenoptera: Vespidae: Polistinae) are ecologically significant insects; however, their species-level identification is often challenging due to morphological similarities and the scarcity of regional molecular data, particularly from northern Pakistan. This study aimed to confirm species identity and reconstruct phylogenetic relationships of Polistes species from District Shangla, Khyber Pakhtunkhwa, Pakistan, using an integrative taxonomic approach combining morphological examination and DNA barcoding. A total of 393 specimens of the genus Polistes (Latreille 1802) were collected from multiple localities and identified using morphological characteristics. Molecular validation was performed using the mitochondrial cytochrome c oxidase subunit I (COI) gene. BLAST analysis revealed high sequence similarity for Polistes perplexus (99.84%) and Polistes stigma (100%) with reference sequences available in GenBank. Phylogenetic analysis using the Maximum Likelihood method (Kimura 2-parameter model, 1,000 bootstrap replicates) showed strong bootstrap support (95–100%) for distinct species-level clades. Polistes perplexus showed very low genetic divergence, indicating genetic homogeneity across populations, whereas Polistes stigma showed relatively higher intraspecific variation, suggesting population differentiation and potential population structuring. This study provides the first phylogenetic data for Polistes species from District Shangla and contributes reference sequences to the global barcode database. These findings show the effectiveness of COI barcoding for species identification and support the use of integrative taxonomy in resolving species boundaries within the genus Polistes.