Molecular Identification, Phylogenetic Inference and COI Protein Modelling of Four Bagridae Catfishes from the Beki River Basin, Assam, Northeast India

Background: Mitochondrial cytochrome c oxidase subunit I (COI) gene-based DNA barcoding provides a reliable approach for identification and phylogenetic analysis of Bagridae catfishes, which are often difficult to distinguish morphologically. This study assessed molecular identification and phylogenetic relationships among selected Bagridae species of the Beki River Basin, Assam, along with three-dimensional (3D) structural characterization of the COI protein. Methods: Four species, namely Mystus tengara, Mystus vittatus, Mystus cavasius and Rita rita, were analyzed using PCR-amplified COI gene sequences. Phylogenetic relationships were inferred using the Maximum- Likelihood (ML) method with Kimura 2-parameter (K2P) distance model. COI protein structure was predicted through homology modelling and validated using Ramachandran plot and SASA analysis. Result: COI sequence lengths ranged from 667-685 bp with highest AT content in Rita rita (57.12%) and highest GC content in Mystus tengara (46.71%). Highest pairwise similarity was observed between Mystus tengara and Mystus vittatus (88), while Rita rita showed maximum divergence (74-79). Mean genetic distance among species was 0.01, with greatest divergence between Rita rita and Mystus spp. (0.109-0.228). Phylogenetic analysis placed Rita rita as an outgroup, while Mystus tengara and Mystus vittatus formed a sister clade with strong bootstrap support (92-93%). Predicted COI models showed greater than 91% residues in favored regions with conserved SASA (~11,200-11,400 Å2); active site volume was highest in Mystus vittatus (1485.12 Å3) and lowest in Rita rita (531.33 Å3).