Results and Discussion
The taxonomic classification of Mahseer species has often led to ambiguity; sequencing of the COI gene region of Tor torwill provide valuable genetic information that can be used to solve the ambiguity. Mitochondrial DNA (mtDNA), owing to its unique attributes such as uniparental inheritance, exclusive presence of displacement loop, lack of recombination, high mutation rate, small size, and constant gene content, has recently come to the foreground in the filed of evolutionary genomics. Furthermore, Kraytsberg et al. (2004) stated that mtDNA recombinants fall into two structural classes: class 1, with a short paternal sequence inserted into a mostly maternal molecule, and class 2, with a maternal sequence flanked by paternal sequences. Therefore, mitochondrial DNA can play a pivotal role in exploring population structure and phylogenetic relationship within Tor tor , which could be of great assistance in species identification and conservation.
It was observed that of the 32 specimens collected, nine specimens from two sites exhibited close association and the other 23 specimens collected from the other six sites exhibited similar mitochondrial DNA sequences, based on the COI gene sequence analysis for species identification and phylogenetic relationship. Analysis of the alignedCOI gene sequences identified 3 haplotypes (Hap_1 to Hap_3); the 3 haplotypes were shown in all of the populations collected from eight sites. Number of specimens belonging to each haplotype is shown in table 1. Specimens from five sites, Chambal river (CR), Rana Pratap Sagar (RPS), Bassi dam (BD), Kali Sindh river (KSR), and Parwan river (PR), exhibited Hap_1; Hap_2 was observed only in specimens obtained from Badi lake; Hap_3 was observed in specimens obtained from Daya dam and Madar tank. Haplotype network analysis (Fig. 2) shows three distinct clades originating from a common ancestral haplotype. They were named as group I, group II and group III, which correlate to Hap_1, Hap_2, and hap_3, respectively. Group I and group II sequences were found to be closely associated. Group III sequences showed divergence from group I and group II sequences. This is also depicted graphically in figure 3, which shows evolutionary divergence over sequence pairs within groups. Haplotype/nucleotide diversity parameters are depicted in Fig.4. Phylogenetic analysis shows that the fish population collected from Madar tank showed distinct genetic diversity compared to the fish populations collected from the other six sites.
Phylogenetic analysis of the mitochondrial sequences using the Neighbour-Joining (NJ) method strongly supported the reciprocally monophyletic status between Tor tor populations. The Neighbour-joining tree constructed using COI gene data showed that the sequences were clustered into two major clades. Different environmental conditions can lead to the emergence of the same species in different geographic locations with different genetic variations. Present-day climate change issues have widespread effects on organisms (Rocha et al.,  2017; Wu et al.,  2017).
Evolutionary relationship of the sequenced COI genes of the specimens is shown in Fig. 5. The phylogenetic tree was constructed using the UPGMA method and the optimal tree with the sum of branch length (0.02044436). The highest genetic distance was observed between specimens from Bassi Dam and Chambal River (0.0202-2.02%) followed by that between MT and RPS (0.0199–1.99%). The lowest genetic distance was observed between specimens from Bassi Dam and Badi Lake (0.0026-0.26%). The Daya Dam has the “closest” genetic distance from Badi Lake as compared to Chambal river, Rana Pratap Sagar Dam, Kali Sindh River, Bassi Dam and Parwan River. This Comparison clearly indicates that fish population of Daya Dam and Madar tank are genetically different from the other six groups. Most of the specimens from different sites were clustered together, which reflects the earlier morphology-based taxonomic assignment.