D-Loopis highly mutable and a specific non-coding region (compared with nDNA) in the mtDNA genome due to its fast rate of evolution . The D-loop region (~ 770 bps) was identified by comparing with mitochondrial reference gene sequences from NCBI as per Lalitha and Chandavar . All the D-Loop sequences were subjected to nucleotide BLAST with S. plagiostomus, showed the maximum similarity (E-value is less than or equal to 0) authenticating to rule out the risks of numts (nuclear copies of mitochondrial origin). The numts are actually shuffling of mtDNA fragments into nuclear genome . Sorenson and Quinn  also suggested to apply newly designed primers using reference sequences available instead of using universal primers. Accordingly, we designed and apply the new primers from the reference sequences of S. plagiostomus.
The sequences of D-Loop region of S. plagiostomus were conserved with the no deletion/insertion however 1.28% of variable sites were found. The overall nucleotide composition was 32.6% (A), 33.6% (T), and 19.8% (C), and 14.0% (G), emphasizing AT-rich contents of animal mitochondrial genome. The best-fit ML model for control region was found to be T92 based on lowest AIC (Akaike Information Criterion, corrected) criterion values . Althoughthe control region is highly mutable and rapidly evolving portion of mtDNA , structurally, it consist of three domains like, TAS domain, central CSB domain and CSB domain, as found in freshwater turtles of order- Geoemydidae  and Trionychidae . The TAS domain with the sequence of -TACAT- and its RC sequence -ATGTA- near 5` end were also reported in Cryptodiran and Pleurodiran turtles . These sequences were also observed in current study. Brzuzan and Ciesielski , also reported these TAS motifs in coregonid species and involved in termination of replication process.
Present data revealed that among these CSB, the CSB-3 (GTCAAACCCCTAAA) at position 738–751 show the mutation only in S. plagiostomus (KT833100) of China. The S. plagiostomus (KT833100) showed two transitions at position 739 (T to C) and position 750 (A to G) and 2 transversions (C to A) at position 740 and (A to T) at 749 position. While the rest of the CSBs, TAS and pyrimidine block were same in all the sequences. Zeng and Liu  and Guo et al  identified only the central conserved sequence block (CSB-F, CSB-E and CSB-D) in fishes. While, incurrent study the central CSB block domain (CSB-D, CSB-E, CSB-F) and conserved sequence block domain (CSB 1–3) were also identified in S. plagiostomus. CSB-F was used toseparate the central CSB domain from the TAS domain. The relative position of these regions has been reported also in some other vertebrates [37, 38]. The consensus sequence of CSB-F, CSB-E and CSB-D in S. plagiostomus was highly conserved and consistent with those described in other fishes studied [39, 40]. The CSBs and TAS were also identified in S. esocinus of Pakistan . A GTGGG-box (common to euteleosts), next to CSB-D was also identified, and also reported by Syed et al  in Indian Schizothoracinae. Moreover, a pyrimidine block (TTTTTTCCTTTTTTC) consists of 15 bps was also detected between the TAS-4 and CSB-1 regions. This pyrimidine motif is similarly described in Indian Schizothoracinae .
In the primitive Schizothoracinae, the genetic divergance time was estimated through mitochondrial genome. Li et al  reported that specifically Schizothorax species radiated from Early Pleistocene to Late Miocene (1.0–10.2 Ma) notably the time periodof uplifting of Plateau . Possibly the ancestors of Schizothorax species were separated through this tectonic unrest, and cause the successive speciation. Present study reported that S. plagiostomus were radiated from other Schizothorax species about 0.91 mya while this species itself radiate in 0.05 mya due to the geological event that causes the uplifting of Himalaya.
In this study, a relatively high haplotype diversity (0.961) and low nucleotide diversity (0.013) were observed. The combination of high haplotype diversity and low nucleotide diversity also reported from previous studies [45, 46, 47, 48]. This is likely due to rapid demographic expansion from a small effective population size [49, 50]. Most of the haplotypes were shared between population 1 and population 2. The haplotype sharing and its connection with other lowest frequencies indicated that the population undergone a series of expansion event in recent time .
The deviation from neutrality estimates through the neutrality tests with Tajima’s D and Fu’s Fs statistics, that is based on the expectation of a constant population size at mutation-drift equilibrium. Here, a negative Tajima’s D (-1.566) indicates an excess of low rate polymorphisms relative to expectation, indicating population size expansion or positive selection . The Tajima’s D was also used to estimates of selective neutrality, population bottlenecks and range expansion. The overall Tajima’s D value was negative with an insignificant p-value, indicating deviation from evolutionary neutrality. Similarly, the Fu’s Fs test, indicating the rare mutations in the populations compared to what is expected under a neutral model of evolution. The significant negative Fu’s Fs statistical value provides strong support for previous population expansion, and exclude the possibility of background selection and evolutionary forces that fabricate a pattern to population expansion [27, 48].
Many fish species are threatened by different factors like, introduction of invasive-exotic species and human activities . These constitute serious challenges which are threatening viability of many fish species, including those of endemic species. The phylogenetic, systematic and taxonomic affinities of cyprinids species of Pakistan, and those of indigenous taxa in particular, are still poorly resolved and highly fragmentary. Due to lack of reliable management plan in this region, natural populations of this species are exposed to overfishing by fishermen. It is almost impossible to bring them back when they are lost. This is the first study to report genetic data of S. Plagiostomus from AJK state, where there is a need for devise conservation and management plans for the exploited cold-water fish species. We report the genetic data and phylogenetic relationships among cyprinids, and especially of the S. plagiostomus. It is mandatory to prevent overfishing, particularly to prohibit fishing throughout reproductive season.