Genus Puntius is a group of freshwater fish that are widely distributed in Southeast Asia. The phylogenetic relationships inferred from DNA sequences of the two mitochondrial genes (Cytochrome oxidase b (633 bp), Cytochrome oxidase c (501 bp) and two nuclear genes, Recombination activating gene 2 (860 bp) and Beta actin (896 bp). The topologies resulting from maximum parsimony, maximum likelihood and Bayesian analyses of combine genes are broadly congruent with each other. Two distinct clades within the genus were obtained with high bootstrap values, namely Clade I (P. schwanenfeldii, P. bulu, P. gonionotus, and P. daruphani) and Clade II (P. fasciatus, P. hexazona, P. tetrazona, P. lateristriga, P. binotatus and P. everetti). Our evaluation on the efficiency of two mitochondrial and two nuclear genes for animal barcoding has focused on Puntius genus. For a DNA barcode to be effective, interspecific differences should be clearly and preferably significant. A wide gap exists between intra and interspecific divergences showed by Cox1 and Cytb genes only. Hence both genes can be effective as suitable DNA barcodes. The results showed that this gap is less apparent for two nuclear genes indicating that they are less suitable for species delimitation in DNA barcoding.

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