Introduction

Tor putitora (Hamilton,1822), widely known as Golden Mahaseer or Himalayan mahaseer, is an important freshwater fish of the Indian subcontinent occupying mostly the rivers of the Himalayan foothills region1,2. Tor putitora (T. putitora) belongs to the family Cyprinidae under the order Cypriniformes3. It is reported from Pakistan, Bangladesh, Afghanistan, China, Nepal, Bhutan, Myanmar and India. T. putitora is generally distributed all over the major river basins of India viz. Indus, Ganga, Brahmaputra, Krishna, Mahanadi etc.4,5. This species is also promoted as a flagship species due to its ecological, and economic significance6.

T. putitora is a fish of significant economic importance due to its high culinary value, its role in a profitable sport fishery and also its contribution to local employment opportunity5. T. putitora is an omnivore fish that naturally inhabits running water of the rapid stream with rocky substratum and is also found in lakes and reservoirs7,8. T. putitora is the state fish in many states of India viz. Arunachal Pradesh, Himachal Pradesh, Uttrakhand, UT of J&K2. It is a famous game and food fish3 which can reach a maximum length of 275 cm and weigh up to 54 kg9,10.

Due to habitat destruction, loss of breeding grounds, anthropogenic activities such as dam building, pollution, and overfishing, the population of T. putitora has sharply declined, leading to its classification as an “ endangered” species on IUCN Red List11,12,13. Moreover, the numerous dams constructed in the Himalayas region negatively affect the breeding migration of fish species5,14,15. The decrease in the number of individuals in populations living in an area can lead to the extinction of unique genotypes, and once this genetic structure is lost it is almost impossible to restore it16.

Before implementing a successful management or conservation strategy, it is important to understand the population stock of species. In the present era, molecular markers make it easier to understand stock characterisation, as they have been applied in various studies throughout the world to understand population genetics17. Mitochondrial DNA (mtDNA) gene sequences show their ability as a marker in various studies on DNA barcoding, phylogenetics, population and conservation genetics17,18,19. mtDNA, due to its small genome size without intron, maternal mode of inheritance, high rate of evolution, and limited recombination, has been providing an opportunity to analyse the population genetic structure20,21,22. Partial sequence of Cytochrome oxidase subunit I gene (COI), D-loop and Cytochrome b gene (Cyt b) have been employed in several research to identify species, for molecular phylogeny and population genetics18,19,23,24,25. Hence these genes are also appropriate for haplotype and genetic diversity. The small number of mtDNA D-loop sequences for T. putitora population in the NCBI database (GeneBank repository) has led to their exclusion from the current study.

The current study aimed to examine the haplotype diversity, genetic variations and population structure of the mitochondrial (mt) COI & Cyt b gene sequence of T. putitora isolates which had been submitted to GenBank from different regions across countries that might be helpful further in the management and conservation of the species.

Materials and methods

Data collection and alignment

In this study, the sequences of COI and Cyt b gene fragments of T. putitora submitted to the National Center for Biotechnology Information (NCBI) database were used for the in-silico analysis. The data were extracted from the database using specific terms such as “Tor putitora COI” “Tor putitora COX1” and “Tor putitora Cytb”. Sequences submitted up to 1 August 2024 were utilised during the present study. Database search yielded 187 sequences for COI and 239 sequences for the Cyt b gene. Sequences were downloaded in Fasta format, analysed in Bioedit v7.2.526 and MEGA 1127 using ClustalW alignment module. Analysis of the sequences was conducted using multiple sequence alignment tool, taking into account that most sequences were partial so, differ in size. Reference sequences (Accession no. NC021755) and (Accession no. AP011326) were used to align the sequences. After aligning, sequences were trimmed from both ends until the length of the sequences was equalised. After equalising and removing short sequences, 498 bp mt COI (n = 106), and 1140 bp Cyt b (n = 183) with 2 reference sequences were used for bioinformatic analysis. Phylogenetic tree were constructed for the gene region in MEGA 11 software using the neighbour-joining method (NJ) and Juke cantor nucleotide measure with 1000 bootstrap replicates. Phylogenetic tree was constructed using Neolissochilus pnar (Accession no. OQ351362.1 and OQ349707.1) as an outgroup species. The constructed phylogenetic tree were designed using iTol28.

Data analysis

Equally aligned sequences were exported to nexus format29. Haplotypes analysis was carried out using DnaSP6 software30 and Haplotype network was generated using PopART (Population Analysis with Reticulate Trees) software31Haplotype, Haplotype number, Polymorphic sites, Haplotype diversity, Nucleotide diversity, Gene Flow, and Neutrality indices were determined using DnaSP6 software. Minimum Spanning Networks were used to visualise relationships between haplotypes in PopART.

Result

During the present study, we analyse 289 gene sequences of T. putitora from the NCBI database. 106 mtCOI gene and 183 Cyt b gene sequences with 2 reference sequences were used in the present study. Sequences belonged to the Indian major riverine systems such as Indus, Ganga, Brahmaputra, Mahanadi, and Krishna, Kosi River of Nepal, Ravi River of Pakistan, Kabul River of Afghanistan and the Tanguar Haor and Someshwari River of Bangladesh (Table 1).

Table 1 Accession number of mt COI and Cyt b gene fragments of T. putitora isolates with their origin used in this study.
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Polymorphism analysis

After analysing 106 sequences of the COI gene (498 sites) and 2 reference sequences using DnaSP software, mutations were observed in 44 different points. In COI gene sequences, 41 polymorphic sites (segregating sites) with 31 parsimony informative sites were identified and the longest conserved area was detected between 368–410 bp. Moreover, 108 T. putitora COI gene isolate sequences were classified into 18 haplotypes (h). COI gene region also showed 0.630 Haplotype diversity (Hd), 0.00662 Nucleotide diversity (Pi) and 3.295 average number of nucleotide differences (k) (Table 2). The highest number of haplotypes was found in the Brahmaputra (India) riverine population with 0.83 haplotype diversity (Table 3). Recombination analysis shows 4 minimum number of recombination events (Rm: 4). Recombination has been detected between the following sites: (93,108) (108,129) (195,234) (234,240). Neither of the analysed datasets contained any protein coding region.

Table 2 Analysis of diversity and neutrality indices.
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Table 3 Haplotype of Tor putitora isolate in different countries from different river population based on Coi and cyt B gene.
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Similarly, analyses of 185 Cyt b gene sequences (1140 sites) show 160 Polymorphic and 61 parsimony informative sites. Within Cyt b, mutations were identified at 173 different sites and the most conserved region was observed between 113–417 bp. Sequence analysis of Cyt b gene showed 85 haplotypes (h) with 0.804 haplotype diversity (Hd), 0.00582 nucleotide diversity (Pi) and 6.444 average number of nucleotide differences (k) (Table 2). The highest number of haplotypes was found in the Ganga riverine population (h = 46), followed by the Brahmaputra (India) river population (h = 18). Haplotype diversity was found to be higher in Brahmaputra (1.0000) and Kosi river (1.0000) followed by Ganga (0.96961) and then Krishna (0.74167) river population (Table 3).

Recombinant Analysis revealed 15 minimum number of recombination events (Rm). Recombination has been found between the following sites (15,16) (16,18) (45,92) (145,445) (445,633) (633,730) (730,732) (733,781) (781,819) (864,892) (892,914) (914,950) (971,974) (974,997) (1038,1098).

Haplotype network analysis and phylogenetic analysis

During the present study, analysis of the COI gene revealed 18 haplotypes (Table 4) whereas Cyt b gene sequences revealed 85 haplotypes (Table 5). In the COI gene, haplotype 4 was the main haplotype with 64 sequences (Fig. 1). Haplotype 4 comprises 59.25% (64/108) of the entire haplotype network followed by haplotype 9, accounting for 11% (12/108). In the COI gene sequences, unique single haplotypes were 66.66% (12/18) of the total haplotypes. Unique single haplotypes were from Ganga (India), Indus (India) and Brahmaputra (India) riverine populations. Unique haplotypes, Hap 01, 07, 14, 15 were from Ganga, Hap 05, 06, 10 were from Indus and Hap 11,12,13,17,18 were from Brahmaputra.

Table 4 Haplotypes of COI sequences of Tor putitora and accession numbers of isolates forming group.
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Table 5 Haplotypes of Cyt b sequences of Tor putitora and accession numbers of isolates forming group.
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Fig. 1
figure 1

Haplotype network based on COI gene sequences of T. putitora isolates. The size of the circle represents haplotype frequency and hatch marks indicate mutation number that discriminate haplotypes. Here, INDIA_BRMPT: Brahmaputra; INDIA_INDS: Indus; INDIA_GNG: Ganga; INDIA_KRSNA: Krishna; INDIA_MHND: Mahanadi River. PAKISTAN represents the Ravi River; Nepal represents the Kosi River and BANGLADESH represents the Tanguhar and Someshwari Rivers.(Created with PopART Qt version 4.8.4).

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COI gene haplotype network comprised 18 haplotypes, which were connected by 1 to 20 mutation steps corresponding to 8 populations. The maximum mutation in nucleotide was found between Hap 08 and Hap 03. Haplotype (Hap) 4 was found to be the most common and ancestral haplotype due to its maximum abundance in the studied population (Fig. 1). Haplotype 4 shares populations from major Indian riverine systems (Brahmaputra, Indus, Ganga, Krishna, and Mahanadi), as well as from Pakistan (Ravi River) and Bangladesh (Tanguar Haor basin river). Hap1, Hap 5, Hap 6, Hap 8, Hap 12, Hap 14 and Hap 15 were considered as budding haplotypes and found periphery in the network. Hap 02, Hap 03 and Hap 04 share sequences of more than one population. The sequence of the Kosi River (Nepal) shares haplotype (Hap 02) with the population of the Brahmaputra River (India) and Bangladesh River. Hap 3 shares the population of the Brahmaputra River (India) and the Tanguar Haor River of Bangladesh.

Similarly, within the Cyt b gene sequence analysis haplotype 37 was the main haplotype (Fig. 2), comprising 43.78% (81/185) of the haplotype network, followed by haplotype 38 which accounts for 7.56% (14/185). Unique single haplotypes contribute 92.94% (79/85) of the total haplotypes. The Ganga riverine population contains 43 (Hap 1- Hap 36, Hap 67, Hap 68, Hap 70, Hap 71, Hap 72, Hap 73, and Hap 85) unique haplotypes, followed by the Brahmaputra riverine population having 17 unique haplotypes (Hap 49- Hap 53, Hap 55- Hap 66). Indus population have 12 unique haplotypes (Hap 39, Hap 41- Hap 48, Hap 80, Hap 81, Hap 82) whereas the Kosi river of Nepal (Hap 84) and Kabul river of Afghanistan (Hap 83) contain a single unique haplotype.

Fig. 2
figure 2

Haplotype network based on Cyt b gene sequences of T. putitora isolates. The size of the circle represents haplotype frequency and hatch marks indicate mutation number that discriminate haplotypes. Here, INDIA_INDS: Indus; INDIA_GNG: Ganga; INDIA_KRSNA: Krishna; INDIA_MHND: Mahanadi River. Afghanistan represents the Kabul River and Nepal represents the Kosi River. (Created with PopART Qt version 4.8.4).

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Cyt b gene haplotype network comprised 85 haplotypes connected by 1 to 18 mutation steps corresponding to seven populations. The maximum mutation in nucleotide (18) was found between Hap 61 and Hap 51 and Hap 61 and Hap 54. Hap 37 was the main haplotype having population from Indus (India), Ganga (India), Krishna (India) and Kabul River (Afghanistan). Hap 54, Hap 38 and Hap 37 share sequences of more than one haplotype. Hap 54 share population of Brahmaputra and Kosi River. Similarly, Hap 38 share the population of Ganga and Mahanadi River systems. The phylogenetic tree also reveals the evolutionary relationships between several isolates of T. putitora . Phylogenetic analysis of COI gene and Cyt b are shown in Fig. 3 and Fig. 4. COI gene sequence analysis revealed one main clade in phylogenetic tree whereas Cyt b analysis revealed two main clades. The phylogenetic tree results were rational with the haplotype network.

Fig. 3
figure 3

Phylogenetic relationship of T. putitora isolates of COI gene sequences. MEGA 11 was used to construct a Neighbour-joining tree based on Juke cantor nucleotide measure with 1000 bootstrap replications. For the construction of phylogentic tree, Neolissochilus pnar (Accession no. OQ351362.1) was used as an outgroup. (Created with iTol :https://itol.embl.de/itol.cgi).

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Fig. 4
figure 4

Phylogenetic relationship of T. putitora isolates of Cyt b gene sequences. MEGA 11 was used to construct a Neighbour-joining tree based on Juke cantor nucleotide measure with 1000 bootstrap replications. For the construction of phylogenetic trees, Neolissochilus pnar (Accession no. OQ349707.1) was used as an outgroup. (Created with iTol :https://itol.embl.de/itol.cgi).

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Diversity, gene flow and neutrality analysis

The diversity and Neutrality indices of COI and Cyt b gene are given in Table 2. Tajima’s D and Fu’s Fs neutrality statistics are generally used in population genetics to examine evolutionary processes, such as to ascertain the presence of selection pressure of DNA sequences in the population. The measurements of Tajima’s D and Fu’s Fs indicate significant negative values in both COI and Cyt b, suggesting the presence of an excess number of rare nucleotide site variants. The negative value of Tajima,s D was also statically significant for both COI and Cyt b gene sequences.

Discussion

The assessment of genetic diversity represents an essential component in diversity assessment and is progressively used for the conservation and management purposes of species32. Genetic diversity and population structure of endangered fish species play an important role in their conservation and management19. mtDNA genes were the most widely used DNA marker for Population genetics11. For this reason, the genetic diversity and population structure of T. putitora were investigated through Insilico analysis using two mtDNA genes (COI and Cyt b). A total of 106 mt COI (498 bp) and 183 Cyt b (1140 bp) gene sequences of T. putitora isolate already submitted in Genebank (NCBI) were utilised in the present study. Overall, the analysis of 289 samples identified 18 haplotypes using mt COI gene and 85 Haplotypes using Cyt b genes in different populations of T. putitora. Haplotype difference is mainly corresponded to the gene length analysed. Therefore, gene fragments with longer length have more haplotypes24.

The result of the present study reveals high haplotypic diversity in T. putitora using the Cyt b gene whereas COI gene has moderate haplotype diversity. Similarly, Sati et al., (2015) also, identify high haplotypic diversity in T. putitora using Cyt b gene11. Genetic variability (h and Pi) of the COI gene is lower because of few polymorphic sites as compared to the Cyt b gene. Earlier based on Cyt b gene, Yadav et al., (2020) identified 38 haplotypes from Himalayan rivers such as Bhagirathi, Alaknanda, Ganga and Yamuna19 and Sati et al., (2015) identified 47 haplotypes from rivers like Jia Bhoreli, Satluj, Ravi, Beas, Chenab, Kosi and Indrayani11. Our data supplement this information by adding information from other rivers from geographically different locations. There was a high haplotype (COI = 0.83175, Cyt b = 1.00000 0.01012) and nucleotide diversity (COI = 0.01012, Cyt b = 0.00696) in the Brahmaputra River population. The high genetic diversity (COI and Cyt b) in the Brahmaputra River population may be due to more segregating sites than other riverine populations. Substantial population sizes may facilitate the maintenance of elevated haplotype diversity within the population33. Sati et al., (2015) also found high genetic variability due to the large number of polymorphic sites11. Similarly, the population from Bangladesh’s rivers also has high genetic diversity (COI), with haplotype diversity = 0.83333 and nucleotide diversity = 0.01640. The high haplotype diversities suggest a rapid population expansion, which is accompanied by the accumulation of new mutations following a period of low effective population size34. The migratory behaviour may be the primary cause of genetic variation within a population, rather than among the population35,36. The present study reveals less genetic diversity in the Indus riverine population. Inbreeding in the population may be responsible for low genetic diversity11,37.

Our analysis of the mt COI gene identified a total of 18 haplotypes. The predominant haplotype (Hap 4) comprises 59.25% of the total network and there were 12 unique haplotypes. Similarly, Cyt b gene analysis reveals 85 haplotypes with the dominance of Hap 37 which contributes 43.78% of the total haplotype network. These predominant haplotypes are likely the ancestors from which other haplotypes may have come directly or through mutation11. Unique haplotypes originated from mutational events denoted by hatch marks in the branches of the haplotype network38. Recently emerged haplotypes show restricted geographical distribution, whereas ancestral haplotypes show their presence in various geographical regions39,40. The Kosi River (Nepal) population share the haplotype with the Brahmaputra River population in both COI and Cyt b gene Haplotype network analysis. In Cyt b gene haplotype analysis, Hap 84 which represents Kosi riverine population show their ancestral linkage with Hap 54. Hap 54 shares the population of the Kosi and Brahmaputra Rivers. This sharing can be attributed to ecological and genetic factors. T. putitora is widely distributed in the Himalayan River system including Indus, Ganga, Brahmaputra etc. and their tributaries which extend into Nepal 5,41. Historical connectivity among these river networks facilitates gene flow and genetic exchange among populations. Additionally, habitat fragmentation due to human activity, such as river modification and dam construction also affects the genetic diversity. Such fragmentation can lead to localised populations that may still retain connection through migration, therefore maintaining haplotype similarity42.

Neutral theory served has served as a foundation framework for understanding genetic diversity. This theory suggests that the accumulation of genetic differences over time in populations is primarily due to random fixation of these neutral mutations, rather than adaptive change driven by natural selection. However, this theory faces challenges as studies found that many mutations do not evolve neutrally, and selection appears to play a significant role in shaping genome43,44. Donati et al. (2021) found that the spatial component of genetic diversity in tropical reef fishes did not align with the expectation of neutral theory. Their finding indicated a negative relationship between genetic diversity and regional abundance, contradicting the theory’s prediction that higher abundance should correlate with increasing genetic diversity45. Human mtDNA genome is predominantly functional rather than neutral, thereby directly refuting the neutral theory46. Similarly, STR repeats were previously considered neutral; however recent finding indicates their ability to bind transcription factors47. Also, the maximum genetic diversity theory (MGD) stands in contrast to the neutral theory. This theory emphasizes that higher genetic diversity enhances a population’s ability to adapt to environmental changes, thereby increasing its chance of survival and evolutionary success48,49.

Neutral theory faces challenges which led to the development of the neutrality test. These neutrality tests are statistical tools designed to evaluates whether observed genetic variation fits neutral theory’s expectations or if selective forces are also at play. Neutrality tests are essential tools for assessing genetic variation and population dynamics50. In this study, we have performed neutrality test to decipher the nature of evolutionary pressure acting on concerned mitochondrial gene, which in turn helped to infer the selective advantages of specific genotype over others, as indicated by their prevalence within population. Tajima’s D test compares the number of segregating sites (polymorphic sites) to the average pairwise nucleotide differences among sequences33,51. Using the Tajima’s D test, populations are evaluated for their deviation from the neutral modal. A positive Tajima’s D value signifies heterozygosity which is indicative of selective advantage. On the other hand, a negative Tajima’s D value is indicative of selective advantage of a particular allele over another allele and signifies the rapid increase in population24. Fu’s Fs test is particularly sensitive to recent population expansions52. A positive Tajima’s D and Fu’s Fs indicate an excess of intermediate frequency allele, which may suggest a decline in population33. During the present study, the value of Tajima’s D and Fu’s Fs test for both COI and Cyt b were negative, suggesting an expansion of the population which could be attributed to habitat destruction53. Earlier, Yadav et al. (2020) reported the negative value of Tajima’s D and Fu’s Fs for T. putitora from Himalayan rivers19. As shown by the finding obtain in this study maximum genetic variation were observed in the Cyt b gene as compared to the COI gene, also reject the neutral theory and suggested Cyt b gene has considerable role in the evolution and adaptability of newly evolved genotype.

Conclusion

T. putitora is an endangered freshwater fish species restricted to the cold-water habitat having their distribution in the rivers of India, Pakistan, Nepal, Bangladesh, Afghanistan, Bhutan and Myanmar. Habitat destruction, introducing exotic species and other anthropogenic activities result in the declining T. putitora population in their natural habitat. Mitochondrial DNA genes such as COI, and Cyt b are the gene loci that are applicable in fish species identification, phylogenetics, population and conservation genetics. All previous research on the haplotype diversity and population study of T. putitora has been conducted within the Indian riverine system. This investigation will serve as the basis for subsequent extensive investigation into the population structure and geographical distribution of T. putitora on a global scale. The genetic diversity pattern analyses in this study give us information that is helpful in developing conservation and management strategies for T. putitora population. To ensure the continuity of T. putitora populations, preservation of natural habitat, establishing protected areas in critical habitat, community-based conservation initiatives, and regular monitoring of population dynamics are essential. By implementing these strategies, the long-term continuity of T. putitora population can be better supported, preventing further decline and promoting recovery.