Delineating species boundary through DNA barcodes ofsome fishes inhabiting river Murti in the foothills of Darjeeling Himalayas

Abstract

The aim of the present study was to establish a global bioidentification system in the form of a reference sequencelibrary where fish species inhabiting river Murti were delineated by a particular sequence denoted as genetic‘barcode’.40 species representing 27 genera, 16 families and 5 orders were collected along different altitudinal gradients if river Murti. Mitochondrial DNA was isolated from the samples followed by amplification of COI gene, alignment by CLUSTAL W and submission to GenBank. The average nucleotide frequencies were calculated whereall the fishes showed relatively higher GC content at second codon position. Haplotypes showed gradually decreasinggenetic distance within orders, families, genera with very low genetic distance within species indicating markedgenetic divergence beyond species boundaries. The average transitional pairs were found more frequent than averagetransversional pairs. The Neighbour Joining tree revealed distinct clusters formed by members of order Cypriniformes, Siluriformes, Perciformes, Synbranchiformes and Beloniformes, where the conspecific individualswere always found to cluster under the same node supported by high bootstrap value, while dissimilar species wereclustered under separate nodes, ensuring unambiguous identification of species. The research work suggests that COI barcoding can be taken up as a sensible approach to eliminate obscurity in the identification of the fish fauna withapplications in its management and conservation of the species inhabiting in specific microhabitats. Keywords: COI gene, DNA Barcode, sequence library, unambiguous identification, conservation.