DNA Barcoding and Phylogenetic Analysis of Fishes of Pokhara Valley

Abstract

Despite extensive taxonomic studies, identification of fishes can be problematic often even to the experts due to various reasons. In this context, DNA barcoding can be a promising tool for species identification and biodiversity surveys through the use of short, standardized gene targets, ~652 bp of mitochondrial DNA. This tool can be more broadly applied if a comprehensive reference sequence library for all fish species can be constructed. Here, we make a small contribution to this grand challenge by barcoding some freshwater fishes from Pokhara. The standard barcode fragment of COI was used to barcode 14 individuals, representing 14 taxonomically recognized species in 13 genera, 7 families and 5 orders. A 99% sequence similarity threshold was employed as a matching criterion for specimen identification to the species level. After editing all obtained sequences using Codon Code Aligner 4.0 program, specimens and sequence data were archived and investigated using analytical tools available on BOLD and MEGA. The GC content was 44.97% on average. Mean genetic distance between families was 18.7%. The synonymous changes were much greater than the non-synonymous changes, especially in the 3rd codon position where variation is dominated. There were 174 conserved, 43 variable, 14 parsimony-informative and 29 singleton amino acid sites; while 367 conserved, 285 variable and 218 parsimony-informative sites were present out of 652 bp nucleotides. The NJ, ML and MP analysis indicated different clades corresponding to the recognized groupings; members of same families clustered together. Molecular species identification was in concordance with current taxonomical classification in all cases achieving success rate of ~94%. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers and COI sequences. This work highlights the functional utility of barcodes for the discrimination of diverse ichthyofauna. We infer that DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of taxonomic assessment in biodiversity studies. Finally, our study constituted an important contribution to the iBOL, providing barcode sequences for use in identification of the species by experts and non-experts, and allowing them to be available for use in other applications. Further research is needed to verify the deeper divergence within species and genera with larger sample size.

Keywords: Mitochondria, Cytochrome oxidase, Cytochrome oxidase subunit I (COI) gene, Taxonomy, DNA sequencing, Species identification, GenBank, BOLD.