DNA barcoding of freshwater fish species from Upper Lake: A Ramsar site, Bhopal, India: Implications for species authentication and reference barcode database development

Upper Lake, Bhopal, a Ramsar designated wetland and one of the largest natural freshwater lakes in central India, supports rich fish diversity and provides important ecological, economic, and fisheries resources. Accurate identification is expected to make proper conservation of any animal species, yet traditional morphometric methods are challenging and time consuming. To address these limitations, this study employed DNA barcoding using the mitochondrial cytochrome-c oxidase subunit I (mtcox1) gene to generate DNA barcodes for 24 fish specimens representing 20 species from 7 orders collected from Upper Lake, Bhopal. We generated sequences ranging from 560 - 640 bp and deposited them in GenBank and the Barcode of Life Data System (BOLD Systems). The mean intraspecific divergence was extremely low (0.58%), exhibiting a significant separation as a clear barcode gap from the minimum interspecific divergence at the genus level (10.24%). Nearest-neighbor method supported species delimitation with a mean interspecific distance (14.7%) 122.5- fold greater than the mean intraspecific distance (0.12%). However, the Barcode Index Number (BIN) analysis revealed notable instances of taxonomic discordance with 24 sequences generating 18 BINs. Nucleotide composition analysis showed a mean GC content of 45.38% with a characteristic positional bias, highest at the first codon position (mean 56.25%) and lowest at the third (37.32%). This study established a novel genetic reference dataset for the fish fauna of this Ramsar designated wetland and demonstrated the utility of the mtcox1 gene for species identification, biodiversity monitoring, and conservation management of freshwater ecosystems.