Genetic structure of natural populations of endangered Tor mahseer, Tor tor (Hamilton, 1822) inferred from two mitochondrial DNA markers

Journal Pre-proof Genetic structure of natural populations of endangered Tor mahseer, Tor tor (Hamilton, 1822) inferred from two mitochondrial DNA markers

Priyanka Sah, Sangeeta Mandal, Rajeev K. Singh, Raj Kumar, Abhinav Pathak, Nimisha Dutta, J.K. Srivastava, Ved Prakash Saini, Kuldeep K. Lal, Vindhya Mohindra PII:

S2214-5400(19)30094-5

DOI:

https://doi.org/10.1016/j.mgene.2019.100635

Reference:

MGENE 100635

To appear in:

Meta Gene

Received date:

12 August 2019

Revised date:

21 October 2019

Accepted date:

11 November 2019

Please cite this article as: P. Sah, S. Mandal, R.K. Singh, et al., Genetic structure of natural populations of endangered Tor mahseer, Tor tor (Hamilton, 1822) inferred from two mitochondrial DNA markers, Meta Gene(2018), https://doi.org/10.1016/ j.mgene.2019.100635

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© 2018 Published by Elsevier.

Journal Pre-proof Genetic structure of natural populations of endangered Tor mahseer, Tor tor (Hamilton, 1822) inferred from two mitochondrial DNA markers Priyanka Sah1,

2

, Sangeeta Mandal1 , Rajeev K. Singh1 , Raj Kumar1 , Abhinav Pathak1 ,

Nimisha Dutta1 , J.K. Srivastava2 , Ved Prakash Saini3 , Kuldeep K Lal1 and Vindhya Mohindra1 Affiliations and addresses of the authors: 1 ICAR- National Bureau of Fish Genetic Resources (NBFGR), Lucknow, India. 2

Amity Institute of Biotechnology, Amity University, Lucknow Campus, Lucknow, Uttar

Aquaculture Research and Seed Unit, Maharana Pratap University of Agriculture and

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Pradesh, India.

Technology, Udaipur, Rajasthan, India

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Corresponding author:

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Vindhya Mohindra ICAR-National Bureau of Fish Genetic Resources, Canal Ring Road, PO Dilkusha, Lucknow, Uttar Pradesh, India [email protected], [email protected] Telephone number-+91 9415234371

Conflict of Interest:

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The authors declare that they have no conflict of interest.

S.NO Author(s) details 1 Authors Email Name 2 Co-authors Email Name Institutional e-mail Research gate profile 3 Co-authors Email Name Institutional e-mail 4 Co-authors Email Name Research gate profile 5 Co-authors Email Name 6 Co-authors Email Name

[email protected] Priyanka Sah [email protected] Dr. Sangeeta Mandal [email protected] https://www.researchgate.net/profile/Sangeeta_Mandal [email protected] Dr. Rajeev K Singh [email protected] [email protected] Raj Kumar https://www.researchgate.net/profile/Raj_Kumar49 [email protected] Nimisha Dutta [email protected] Dr. Abhinav Pathak

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[email protected] Prof.(Dr.) J.K. Srivastava [email protected] [email protected] Ved Prakash Saini https://www.researchgate.net/profile/V_P_Saini [email protected] Dr. Kuldeep K Lal [email protected] https://www.researchgate.net/profile/Kuldeep_Lal [email protected] Dr. Vindhya Mohindra [email protected] https://www.researchgate.net/profile/Vindhya_Mohindra

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Co-authors Email Name Institutional e-mail Co-authors Email Name Institutional e-mail Co-authors Email Name Institutional e-mail Research gate profile Authors Email Name Institutional e-mail Research gate profile

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Abstract:

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Tor tor, Tor mahseer, an endangered cyprinid species, is important as sport fish. Genetic divergence in natural populations of T. tor was investigated using two mitochondrial genes,

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Cytb (Cytochrome b) and ATPase6/8. Analysis of 140 sequences of Cytb (1121 bp) and

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ATPase6/8genes (842 bp) genes revealed 12 and 7 haplotypes respectively, whereas 23 haplotypes were found in concatenated sequences (1963 bp). Sequence analysis of mitochondrial regions revealed balancing selection and displayed low nucleotide and

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moderate haplotype diversities. Mantel tests identified a positive relationship between

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pairwise geographical and genetic distances for the study region. Results of AMOVA based on genes pointed out that the genetic variations were mainly due to variation in within

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populations (ATPase6/8: 62.73%, Cytb: 42.13%, concatenated sequences: 48.31%). The total Fst was found to be significant in both genes with a value of 0.5787 (p<0.05) and 0.3726 (p<0.05) for Cytb and ATPase6/8genes respectively, whereas 0.51687 (p<0.05) was found in combined sequences, which revealed sub-structuring in the T. tor natural populations. Population pair-wise Fst ranged from 0.00901 - 0.85631 for Cytb and 0.09910 - 0.64731 for ATPase6/8genes, whereas 0.02039 - 0.85436 in combined sequences. Results indicated the presence of four genetic stocks in the populations studied. The baseline information about stock characterisation in this study would be applicable for planning strategies for conservation, management and sustainable fisheries of this species.

Keywords: Tor tor, Cytb, ATPase 6/8, mtDNA, Polymorphism, Genetic variation Introduction

Journal Pre-proof The iconic mahseer, Tor tor (Hamilton, 1822), a native of the trans-Himalayan region (Shrestha, 1994), is found in fast-running rivers and streams with rocky bottoms in Nepal, Bangladesh, India, Bhutan and Pakistan (Desai, 2003, Khare et al., 2014). In India, T. tor is known from the Ganga (including sub-Himalayan range), Indus, Brahmaputra and Narmada river systems (Shrestha, 1994) and thus T. tor contributes significantly to the capture fisheries of cold as well as warm waters, however, there is no farming of this fish species Lal et al. (2013) reported the extended distribution of T. tor in the peninsular rivers, Godavari and Krishna, which poses an interesting question about its existence as a species native to these rivers ( Pinder et al., 2019). The wild population of T. tor is reported to have declined in

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current years, as it has been facing numerous challenges in its natural range of distribution, due to anthropogenic activities and/or natural causes (Pinder and Raghavan, 2013). As a consequence, it was given a status of ‘Endangered’ species in 2010, however currently, the

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species is assessed as data deficient under IUCN, 2018 (Rayamajhi, et al., 2010, 2018).

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For the scientific management of a species to avoid population decline and ensure resource sustainability, prior information about its genetic variability and stock structure is

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important. Due to absence of introns in mitochondrial DNA and its efficient repair mechanisms, the mutation rate in mtDNA is much more frequent than that of nuclear genes

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(Andalib et al., 2017). The faster evolutionary rate, which tend to enhance recent demographic events; and maternal inheritance make mitochondrial DNA a potential genetic

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marker for analyzing intraspecific variability as well as phylogenetic studies (William et al.,

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2005). In addition, mitochondrial genomes have 1/4 effective population sizes (Ne) than that of nuclear genes (Hard & Clark, 1989), which helps in the detection of selection pressure, and it may have bearing on the population structure of a species (Nekrutenko

et al., 2002).

However, the genetic level studies in T. tor are very limited. Pasi et al. (2013) employed, cytb (967 bp) to reveal the population structure of T. tor from three locations of Madhya Pradesh, India, whereas Sharma et al. (2016) observed no significant genetic distances in T. tor from wild and cultures conditions with dominant RAPD markers. Thus, choice of suitable markers is very important for a particular species, for genetic divergence studies. A combination of markers

is

preferable for resolving genetic connectivity and

historical demography,

particularly in fish species (Mohindra et al., 2019). Role of natural selection on evolution of the mitochondrial genome in vertebrates may be hinted by the analysis of mitochondrial genome (Consuegra et al., 2015).

Journal Pre-proof In the present study, two full length genes from mitogenome, i.e., cytb, ATPase6/8 and concatenated sequences were used to resolve genetic variation and divergence in T. tor from six locations of four rivers (Berach, Narmada, Penganga and Godavari), which will be helpful in developing management strategies in order to conserve for sustainable fisheries of this depleting species. Our aim was to determine spatial structure in the mtDNA diversity over the landscape of the species studied and

demographic history for status of species T. tor,

intraspecific

population structure and gene flow patterns in 6 populations of Tor tor, which belong to

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different river basins and status of T. tor in the peninsular river Godavari.

Materials and Methods: Design of the study

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In this study, 140 samples of T. tor were collected from 6 river locations (Table 1).

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Cytochrome b and ATPase 8 and 6 genes of these samples were amplified and sequenced. These sequences were analysed to determine spatial structure in the mtDNA diversity over

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the landscape, demographic history, population structure and gene flow patterns in these

Sample collection:

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populations of T. tor.

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Tissue samples (muscles and fin) from a total of 140 individuals of T. tor were

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collected, from commercial riverine catches of six different geographical sites, i.e, Berach (17), Narmada (3 locations; Tawa, Khargaon and Kolar ) (84), Penganga (18) and Godavari (21) (Table 1, Fig. 1) and fixed in 95% ethanol and stored at 4o C until use. River Berach is a seasonal tributary of river Banas and Chambal under Ganga river basin. The samples were collected from Madar dam, located at Salumber in district Udaipur of western Rajasthan, is an irrigation dam on river Berach River Narmada is one of only three major rivers in peninsular India and is the longest west flowing river originates near Anuppur district, Madhya Pradesh state, from Amarkantak Plateau, which flows westwards and drains into the Arabian Sea.The samples were collected from Tawa reservoir, Hoshangabad; Khargone and Kolar dam of Kolar tributary on the northern side of River Narmada. The Penganga River, a tributary of Godavari Basin, originates in the Ajantha ranges in Aurangabad district in Maharashtra state and after travelling 676 km confluence into Pranhita

Journal Pre-proof River. The Godavari is India's second longest river after the Ganga, which originates in Trimbakeshwar, Maharashtra state and flows eastward to drain into the Bay of Bengal. Mitochondrial Gene Amplification Tissue samples were used for total genomic DNA extraction using a modified PhenolChloroform method (Ruzzante et al., 1997). Cytb region was amplified with primers, L14724 and H15915 (Xiao et al., 2001) and ATPase8 and ATPase6 genes with primers, ATP8.2L8331 and COIII.2H9236 (Sivasundar et al., 2001). Primer sequences are given in Supplementary Table 1. Polymerase chain reactions (PCR) were carried out in 50 μl reaction mixtures, following conditions of Mandal et al., (2016) and checked on 2 % agarose gels.

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The products were purified and sequenced bi-directionally using an automated capillary

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sequencer (ABI 3730Xl) following the manufacturer’s recommendations. Consensus DNA sequence synthesis and genotyping

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The sequences generated from both forward and reverse primers (Suppl. Fig. 1a and

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1b) were aligned using Clustal Omega (Thompson et al., 1997) and consensus sequences were synthesised manually. Differences in the nucleotide sequences were identified and

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haplotypes were designated as unique sequences that differed from all others by at least one nucleotide change. Haplotypes were identified from

populations studied were identified

Statistical analysis

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through by DnaSP (Librado and Rozas, 2009).

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Three data sets were generated (1) Cytochrome b (cytb) (2) ATPase 6 and 8

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(ATPase6/8) and (3) concatenated sequences of cytb ATPase6/8. The use of 3rd dataset was validated through phylogeny analysis of haplogroups by MEGAX (Tamura et al., 2007) with 1000 boot-strappings. Mantels test (Mantel 1967) was used to assess the presence of significance levels of genetic distance across the distribution range by overlaying on lands cape co-ordinates and their 3-D pattern of distribution plots through genetic landscape shapes (AIS, Miller, 2005), with parameters of a 50 × 50 grid and a distance weighting parameter (a) of 1.

Ka_Ks Calculator (Zhang et al., 2006) was used tocalculate the ratio of non-

synonymous substitutions (KA) to synonymous substitutions (KS) for identification of the selective pressures occurring in protein-coding genes and best model for it was identified through MEGA. MEGA were used to determine sequence composition and variability parameters. Haplotype diversity (h), nucleotide diversity (π), AMOVA and Fst values were calculated using Arlequin 3.11 (Excoffier et al., 2005). Gene flow estimates, i.e, number of migrants,

Journal Pre-proof (Nm) by the permutation tests with 1000 replicates (Librado et al., 2009) with Jukes and Cantor correction, was calculated by DnaSP (Librado and Rozas, 2009). The median-joining haplotype network was created in Network 4.6.1.1 (Bandelt et al., 1999) from haplotype data of mitochondrial genes from all data sets. For each population of T. tor, demographic history was assessed using neutrality tests and mismatch distributions of pair-wise differences in Arlequin. Two widely used statistical tests Fu’s Fs (Fu, 1997), Tajima’s D (Tajima, 1989) and raggedness index were calculated by Arlequin to determine the goodness of fit to a unimodal distribution (Harpending, 1994).

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Results: Sequence composition

Cytb gene: From the cytb gene sequences (1121 bp) for 140 T. tor specimens, 12 different

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haplotypes were obtained (NCBI Accession no. MN105957 - 68). Out of the 1121 bases, 12

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were variable with 6 parsimony informative sites and 6 singletons. The average nucleotide frequencies were: A: 30.0%; T: 27.7%; C: 28.5%, G: 13.9% and were found to be A+T rich

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(57.1%).

ATPase6/8 genes: The amplified product contained ATPase 8 and 6 genes of 842 bp.

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ATPase 8 region extended from 1–165 bp of the sequence and ATPase 6 from 159–842 bp with a coinciding region (7 bp) between two genes from 159–165 bp. The two regions were

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analysed together for determining variation in T. tor. A total of 7 different haplotypes were

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obtained (NCBI Accession no.MN105950-56). Out of the 842 bases, 15 were variable with 13 parsimony informative sites and 2 singletons. The average nucleotide frequencies were: A: 31.1%; T: 28.1%; C: 27.7%, G: 13.1% and were found to be A+T rich (57.2%). Concatenated sequences (Cytb + ATPase 6/8 genes): A total of 23 different haplotypes were obtained. Out of the 1963 bases, 18 were variable with 10 parsimony informative sites and 8 singletons. The average nucleotide frequencies were: A: 30.5%; T: 27.8%; C: 28.1%, G: 13.6% and were found to be A+T rich (57.2%). When Cytb and ATPase 6/8 sequences were analysed individually and concatenated for phylogenetic relationships among the haplotypes, two consistent haplogroups were observed in all the three data sets (Suppl. Fig. 2a and 2b, Tree with ATPase not shown.). Ka/Ks ratio on Kimuras- two parameter (K2P) model (0.5831) indicated constraining (purifying) selection. Spatial structure of mtDNA diversity within T. tor

Journal Pre-proof The distribution of mitochondrial sequence variation within the natural range of T. tor was explored to identify potential spatial structure. Mantel tests identified a positive relationship between pairwise geographical and genetic distances for the study region, as a whole (r = 0.3410775634, P=0 .00099900) and pattern of scatter plot is given in Fig.2. The analysis of genetic landscape shape (Fig. 3) strongly indicated a pattern of higher genetic differentiation among populations in the central Indian region of the study area (Suppl. Fig. 3). The positive peaks with high genetic divergence P1 (20.58 77.22) and P2 (21.27 78.195) were located in the regions between the collection points of {2, 3, 4} and {5, 6} (Suppl. Fig.

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Genetic and phylogenetic relationship among haplotypes

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3).

The cytb gene showed among samples from six locations, the most common haplotype to be H02 (Suppl. Table 1), while H04 was the dominant haplotype in Penganga

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and Godavari. Total shared haplotypes were 4, i.e., H02, H03, H04 and H06. Population

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specific haplotypes were found in Berach (1), Penganga (1), Narmada-Tawa (3) and Narmada-Kolar (3). Samples from river Narmada-Tawa exhibited a maximum number of

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haplotypes, i.e., 6. Haplotype network (Fig. 4a) demonstrated the formation of double clades, with H02 and H04 as the ancestral haplotypes, however, no particular clade contained

geographical area.

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haplotypes from the distinct geographical region and were distributed in a different

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For ATPase6/8, haplotype H02 was the most common of the 7 haplotypes and two

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haplotypes were shared among all populations (Suppl. Table 1). Haplotypes H02 was shared between all six sites, whereas H01 was also shared between all sites, except the Godavari. Population specific haplotypes were found in Berach (1), Narmada-Tawa (2), NarmadaKhargone (1) and Narmada-Kolar (1). Haplotype network demonstrated the formation of double clades, with H01 and H02 as the ancestral haplotypes; however, no specific clade contains haplotypes from a single geographical region (Fig. 4b). For concatenated sequences, haplotypes H02, H07 and H09 was the most common haplotype of the 23 haplotypes and a total of six shared haplotypes i.e., H02, H03, H07, H08, H09 and H11 (Suppl. Table 1). Population specific haplotypes were found in Berach (1), Narmada-Tawa (2), Narmada-Khargone (2), Narmada-kolar (1) and Penganga (1). Samples from river Narmada-Tawa exhibited a maximum number of haplotypes, i.e., 10. It was interesting to find single haplotype H9 in Godavari river population. Haplotype network demonstrated the formation of two clades, with H02 with H07 in one cluster and H09 in

Journal Pre-proof another cluster, as the central haplotypes (Fig. 4c)., however, no particular clade contained haplotypes from the distinct geographical region and were distributed in a different geographical area. Nucleotide and haplotype diversity T. tor mitochondrial sequences revealed absence of haplotype and nucleotide diversities, as in Godavari samples, only one haplotype was found. Rest of the populations exhibited moderate haplotype and low nucleotide diversities for all populations (table 3). In cytb, haplotype diversity (h), except ranged 0.0000 (Godavari) to 0.6691 (Berach) and nucleotide diversity (π) was low, ranged from 0.0000 (Godavari) to 0.00114 (Penganga). In

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ATPase6/8, h varied between 0.0000 (Godavari) to 0.5903 (Narmada-Tawa) and π between 0.0000 (Godavari) to 0.0079 (Narmada-Tawa) (Table 2). In concatenated sequences, h varied between 0.0000 (Godavari) to 0.7768 (Narmada-Tawa) and π between 0.0000 (Godavari) to

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0.000756 (Penganga) (Table 2).

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Genetic variability and differentiation

AMOVA results for hierarchical partitioning of variation for cytb. ATPase and

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concatenated sequences are shown in Table 3. For cytb, out of total variation, 54.26% was contributed by among groups, while 42.13% to within populations. The mean F st 0.5787

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(p<0.05) was found to be significant. For ATPase6/8, out of total variation, only 37.59% was contributed by among groups and 62.73% to within populations. The mean F ST 0.3726 (p<

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0.05) was found to be significant. Whereas for concatenated sequences, 49.25% was

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contributed by among groups while 48.31% to within populations. The mean F st 0.51687 (p<0.05) was found to be significant. The

pairwise

Fst

between population samples using Cytb,

ATPase6/8

and

concatenated sequences revealed that out of 15 population pairs, significant divergent between 12,10 and 10,

population pairs, respectively. The population pairs which did not

show significant divergence with ATPase and concatenated are Berach-Kolar and PengangaGodavari. The genetic relationship between populations (FST values) is presented in Table 4 based on two markers. Neutrality and mismatch distribution In cytb, the population Narmada-Kolar showed negative significant D and Fu’s values with a unimodal pattern of mismatch distribution. However, none of the population showed significant values with ATPase6/8. However, significant Fu’s values were found for three populations, Narmada- Tawa, Khargone and Kolar with unimodal distribution. In overall

Journal Pre-proof population. Negative D was not significant; Fu’s significant while raggered index was also not significant, with unimodal distribution (Fig. 4). Gene flow estimates Gene flow estimates (number of migration, N m) in 4 rivers, also showed (Table 6) very high values between pairs of Narmada- Tawa, Khargone and Kolar populations, while low to moderate for rest of the population pairs. Discussion: The present study characterized T. tor from four Indian rivers and six collection sites with two mitochondrial markers cytb and ATPase6/8genes. Nucleotide sequences of cytb and

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ATPase6/8genes were rich in A+T, which showed conformity with previous studies for fish (Luhariya et al., 2012; Mandal et al., 2016). Congruence of phylogenetic pattern observed for individual cytb and ATPase6/8 genes and concatenated genes supports the analysis of

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concatenated sequences for the present study (Theirgart et al., 2014). Presence of purifying

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selection pressure on mitochondrial genes studied may be due to strong functional constraints in mitochondria and deleterious non-synonymous mutations accumulations are removed by

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effective purifying selection (Sun et al., 2011).

From our studies in T. tor from natural populations studied, a pattern of isolation by

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distance (IBD) suggests the influences of gene flow and drift on the distribution of its genetic

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variability. The scatterplot pattern revealed isolation of the samples, where genetic drift has occurred independently of geographic distance (Hutchison and Templeton, 1990). With the

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highest genetic divergence found be in central Indian region, it may be speculated to be the region of origin of the species and from there, it may have migrated to other regions/rivers. A highly negative value of Fu’s for overall population depicts alleles in excess, may be owing to recent population expansion or genetic hitchhiking. Non-significant raggedness values and unimodal pattern of mismatch distribution also supports the genetic signal of sudden population expansion for overall T. tor under study (Joshi et al., 2013). Haplotype network with two clusters, in which each displayed star like topology also indicated recent expansion of population, following population bottleneck (Slatkin and Hudson, 1991). Thus, T. tor as an endangered species (IUCN 2010), in isolated populations with genetic drift, may face inbreeding depression, thus calls for study of its population structure analysis, for implementation of conservation measures.

Journal Pre-proof Moderate haplotype and low nucleotide diversity was found to be characteristics of the populations of T. tor, exception of absence of genetic diversity in Godavari population. All populations, except Godavari, are with moderate haplotype and low nucleotide diversity values suggest existence of small populations, which have undergone recent population growth (Grant and Bowen, 1998). This is indicative of recent population bottleneck or founder effect. However, only Narmada-Kolar population possesses genetic signals of demographic expansion, inferred from unimodal pattern of mismatch distribution (Rogers and Harpending 1992), along with negative significant values for Fu’s F and non-significant Raggedness index.

This demographic expansion may result in an excess of rare

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polymorphisms, as this population has several private haplotypes. Moderate level of diversity may also be due to constraining selection detected. In general, population decline for all the populations, except Narmada-Kolar population, was suggested by the neutrality tests.

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The AMOVA results as well as total Fst values revealed sub-structuring in the T. tor

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natural populations. A majority of variation observed in within population as well as among groups, while low among populations. For relatively high levels of within-population variations between population is expected

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variation but limited

in migratory fishes

(Vrijenhoek, 1998) and T. tor being a migratory fish species (Talwar and Jhingran, 1991), its

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genetic variation showed a similar pattern. Migratory behaviour also indicates that gene flow can offset the pattern of genetic differentiation among the population (Nguyen et al., 2006).

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Pairwise Fst comparison based on cytb as well ATPase6/8 genes showed that lack of

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genetic divergence among the three locations of Narmada and may be considered a single genetic stock. It may be due to a high level of genetic connectivity because of interconnected tributaries of Narmada river system. Thus, overall, there is the evidence of the existence of four genetic stocks, Berach, Narmada, Penganga and Godavari populations. River Berach is significantly divergent from other rivers, as it is a separate river system and there is very low gene flow with other rivers. It is important to note here that the one of the dominant haplotype observed in Berach is also unique and not present in any collection from other location. The haplotype diversity observed in Berach sample is comparable to that found in other populations including River Narmada. This indicates that the dam harbours native population of the river Berach and is differentiated from its conspecifics from the rivers of other basins. Such differentiated populations with restricted distribution are important from conservation point of view. The low genetic divergence between the three collection locations in River Narmada is expected. The sites of collection in Narmada are

Journal Pre-proof interconnecting and as T. tor has migratory nature. (Desai 2003) and thus genetic mixing is possible. However, the small divergence between the three locations within Narmada and private haplotypes found in small frequency, indicate differentiating populations existing in the tributaries. Penganga is a tributary of Godavari, there may be events of population mixing in the past and this might have resulted in low divergence, though significant, than with other populations. However,It is surprising to observe the absence of genetic variability (only one haplotype) in the main river Godavari population in contrast to its northern tributary Penganga. The presence of T. tor in Godavari river basin, including river Penganga, was first

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reported by Lal et. al. (2012), as a new extended distribution in peninsular India. The haplotype network for both genes suggests a common ancestor for the lineages of samples from rivers Godavari and Penganga. The samples from river Penganga show diversity and on

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the other hand the samples of river Godavari are homozygous. This could be possible that the

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river Goadavri has restricted habitats, suitable for existence of mahseer and possibly have small populations. However, based on the present data it cannot be said that this is

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consequence of an introduced population or the populations have constricted and leading to restricted gene flow and homozygosity. It is well known that small constricted population of

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a few individuals, possibly due to migration or introduction, would result in inbreeding, following the small population founding effect (Chen et al., 2012), which would in turn give

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rise to a highly homozygous population.

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Conclusion:

The results demonstrated the utility of mitochondrial cytb and ATPase6/8to determine the demographic history and intra-specific genetic diversity and to discriminate genetic stocks in the wild population of T. tor. There is an evidence of distinct genetic structure in the wild populations of T. tor. The populations where decline in population size is observed, there is a possibility of inbreeding and genetic drift to further reduce the genetic diversity in these populations. Based on the results of the present study, development of conservation strategies to maintain the natural genetic diversity is suggested. Acknowledgment: The authors are grateful to Director, ICAR-NBFGR for providing facilities for this research. This work was a part of the ICAR plan funded project entitled, Outreach Activity on Fish Genetic Stocks, Phase II. Mr. R.S. Sah, Mr. Rajesh Kumar and Mr. Pradipta Paul are

Journal Pre-proof acknowledged for their role in sampling and other technical assistance. Mr. Kantharajan G. is acknowledged for his help in the location maps.

Conflict of interest: The authors declare that there is no financial or non-financial conflict of interest.

Compliance with Ethical Standards: Funding: This study was funded by, ICAR- National Bureau of Fish Genetic Resources (ICAR-NBFGR), Lucknow, India.

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Ethical approval:

The sampling protocols followed in the current study were approved by the Institutional Animal

Ethical

Committee

(IAEC),

ICAR-NBFGR,

India

vide

No.

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G/CPCSEA/IAEC/2015/2 dated 27 October, 2015.

Lucknow,

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No studies on live animals were performed, in the present investigation.

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Rayamajhi, A., Jha, B.R., Sharma, C.M., Pinder, A., Harrison, A., Katwate, U. & Dahanukar, N. 2018. Tor tor. The IUCN Red List of Threatened Species 2018: e.T166534A126321898. http://dx.doi.org/10.2305/IUCN.UK.2018-2.RLTS. T166534A126321898.en. Downloaded on 04 August 2019. Rogers, A.R. and Harpending, H., 1992. Population growth makes waves in the distribution of pairwise genetic differences. Molecular Biology and Evolution, 9(3), pp.552-569. Ruzzante, D.E., Taggart, C.T., Cook, D. and Goddard, S.V., 1997. Genetic differentiation between inshore and offshore Atlantic cod (Gadus morhua) off Newfoundland: A test and evidence of temporal stability. Canadian Journal of Fisheries and Aquatic Sciences, 54(11), pp.2700-2708. Sharma,J., Alka, P., Pratibha, B. and Jitendra, K., 2016. Assessment of genetic diversity of mahseer (Tor tor) using RAPD markers from wild and cultured conditions in Madhya Pradesh. Journal of Experimental Zoology, India, 19(1), pp.23-29. Shrestha, T.K., 1994. Migration and spawning of golden mahseer in Himalayan waters of Nepal. Journal of Freshwater Biology, 6(1), pp.71-77.

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William J, Ballard O, Rand DM. 2005. The population biology of mitochondrial DNA and its phylogenetic implications. The Annual Reveiw of Ecology, Evolution and Systematics, 36 ,pp. 621–42. Xiao, W., Zhang, Y. and Liu, H., 2001. Molecular systematics of Xenocyprinae (Teleostei: Cyprinidae): taxonomy, biogeography, and coevolution of a special group restricted in East Asia. Molecular Phylogenetics and Evolution, 18(2), pp.163-173. Zhang, Z., Li, J., Zhao, X.Q., Wang, J., Wong, G.K.S. and Yu, J., 2006. KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genomics, Proteomics & Bioinformatics, 4(4), pp.259-263.

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Fig. 1 Map showing sample collection sites for natural population of Tor tor for present study (refer table 1). Mapped in ArcMap V. 10.6 platform using the India-river basin data sets obtained from NRSC-India: Water Resources Information System (http://www.india-wris.nrsc.gov.in/)''.

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Mantel Test Results 0.004 0.003 0.003 0.003 0.003 0.003

0.002 0.002 0.002 0.002 0.001 0.001 0.001 0.001 0.001 0.000 0.000 0 1

2

3

4 5 Geographical Distance

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Figure 2. Scatter plot of Mantel’s test depicting ccorrelation of genetic and geographical distances. Pattern revealed isolation of the samples, where genetic drift has occurred independently of geographic distance.

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Genetic Distance

0.002

50 48 46 44 42 40 38 36 34 32 30 28 26 24 22 20 18 16 14 12 10 8 Y (Western edge)

6

4

2

5

4550 40 35 30 25 X (Southern edge) 20 15 10

Figure 3. Genetic landscape shape plot showing patterns of spatial genetic distance for 6 populations of Tor tor. X and Y axes correspond to geographic coordinates and the Z axis (height) corresponds to genetic distance between individuals

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a, Cytochrome B

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b. ATPase6/8

c. Concatenated sequences

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Fig.4. Median joining Network obtained from Tor tor from six collection sites. (a) Cyt b haplotypes, (b) ATPase haplotypes (b) and concatenated (c) sequence haplotypes. The size of each haplotype circle denotes the number of observed individuals. Colors correspond to different regions. Values across lines represent base pair at which variability between adjacent haplotypes.

Fig. 4 Unimodal pattern of mismatch distribution and relative haplotype frequency for overall Tor tor under study.

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Journal Pre-proof Table 1: Sample collection sites for natural population of Tor tor, used in present study

River

Tributary/ reservoir

Ganga Basin Narmada Basin

River Berach, Berach Dam Narmada river , Tawa reservoir Narmada river

1

Udaipur, State Rajasthan

17

2

45

Kolar tributary, Kolar Dam Penganga River

4

Godavari River

6

Tawa, State Madhya Pradesh Khargone, State Madhya Pradesh Kolar, State Madhya Pradesh Satnala, Adilabad, State Telangana Nirmal, State Telangana

15 18

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5

24

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3

N

21

latitude and longitude 26.47, 74.63 22.75, 77.72 22.17, 76.07 23.25, 77.40 19.79, 78.67 18.99, 78.37

Year of collectio n 2005 2015, 2017 2015 2003 2015, 2016 2017, 2018

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Godavari Basin

Collection Site

Godavari Overall

ATPase6/8 concatenated sequences 0.3235 ± 0.7353±0.0770 0.1359 0.5903 ± 0.7768±0.0421 0.0370 0.5543 ± 0.7754±0.0665 0.0525 0.5385 ± 0.6476±0.1338 0.1146 0.2092 ± 0.5294±0.1170 0.1163 0.0000 ± 0.0000±0.0000 0.0000 0.8226 +/0.0175

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NarmadaTawa NarmadaKhargone NarmadaKolar Penganga

Cytochrome B 0.6691 ± 0.0591 0.4485 ± 0.0858 0.4819 ± 0.1106 0.3714 ± 0.1532 0.4248 ± 0.0993 0.0000 ± 0.0000

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Berach

Haplotype diversity

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Populations

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Table 2: Haplotype and nucleotide diversities calculated through analysis of Cytochrome B, ATPase6/8 and concatenated sequences in Tor tor

Nucleotide diversity Cytochrome B 0.00073 ± 0.00062 0.00071 ± 0.00058 0.00073 ± 0.00061 0.00036 ± 0.00039 0.00114 ± 0.00084 0.00000 ± 0.00000

ATPase6/8 0.00066 ± 0.00064 0.00079 ± 0.00069 0.00071 ± 0.00066 0.00076 ± 0.00070 0.00025 ± 0.00035 0.00000 ± 0.00000

concatenated sequences 0.00070±0.00051 0.00073±0.00050 0.00072±0.00051 0.00051±0.00041 0.00075±0.00053 0.00000 ± 0.00000 0.000959 +/0.000610

Journal Pre-proof Table 3: AMOVA results for natural population of Tor tor, used in present study.

Source Variation

Of D.F.

Percentage Of Fixation Indices Variation

P Values

36.804

0.45404 Va

54.26

FCT: 0.5425

0.01857

3

3.168

0.03026 Vb

3.62

FSC: 0.0791

0.01173

134

47.235

0.35250 Vc

42.13

FST : 0.5787

0.0000

139

87.207

0.83680

Among groups

2

11.537

0.14486 Va

37.59

FCT: 0.3759

0.01173

Among populations within groups Within Populations Total

3

0.640

-0.00124 Vb

-0.32

FSC: -0.0052

0.4252

134

32.395

62.73

FST: 0.3726

0.0000

139

44.571

Among populations within groups Within Populations Total

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2

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Cytochrome B Among groups

Sum Of Variance Squares Components

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ATPase6/8

0.24175 Vc

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0.38537

2

48.317

0.59840 Va

49.25

FCT: 0.49246

0.02737

Among populations within groups Within Populations Total

3

3.830

0.02967 Vb

2.44

FSC: 0.04811

0.0000

134

78.667

0.58707 Vc

48.31

FST: 0.51687

0.01760

139

130.814

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Among groups

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Concatenated sequences

Journal Pre-proof Table 4. Pair wise FST for cytochrome B (below diagonal), ATPase 6/8 (above diagonal) regions and Concatenated sequences in T. tor. (*p<0.01)

Berach Populations

Narmada Tawa

Penganga

Khargone

Godavari

Kolar

0.18214*

0.28539*

0.12271

0.64731*

0.78377*

Tawa

0.21651*

-

0.0000

0.0000

0.25876*

0.39893*

Khargone

0.20244*

0.0000

-

0.00990

0.18878*

0.38032*

Kolar

0.21827*

0.04938

0.05918

-

0.39969*

0.61638*

Penganga

0.54421*

0.42541*

0.39188*

0.55113*

-

0.07140

Godavari

0.85631*

0.72032*

0.75800*

0.25579*

-

0.20713*

-

Khargone

0.23910*

0.0000

Kolar

0.16897

0.02039

Penganga

0.57686*

0.37504*

Godavari

0.83377*

0.63408*

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-

0.04414

-

0.34250*

0.51889*

-

0.67410*

0.85436*

0.23436

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Tawa

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Berach

0.92405*

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Concatenated sequences

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Berach

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Journal Pre-proof Table 5. Neutrality (Tajima's D and Fu's FS) tests for Tor tor from for natural population

Berach Tawa

Narmada Khargone

Kolar

Godavar i

17

45

24

15

18

21

Tajima's D

0.97880

-1.08869

-0.63496

-1.68500*

1.27889

0.00000

FS Raggedness index Pattern of mismatch distribution ATPase6/8

0.63824 0.18745

-1.84316 0.16869

-0.27379 0.16190

-2.36912*** 0.18122

3.52530 0.69179

0.00000 0.00000

Unimodal

Multimodal

Multimodal

Unimodal

Multimod al

-

1.04864* * -0.12611 0.37630

-0.03726

0.27046

-0.52899

0.00000

-0.20366 0.20139*

0.27741 0.21775

-0.03956 0.12631

-0.01058 0.38212

0.00000 0.00000

Unimodal

Unimodal

-

Multimod Pattern of al mismatch distribution Concatenated sequences 17

Tajima's D

-0.21079

FS

-1.59357

Raggedness index Pattern of mismatch distribution

e-

0.03649

Unimodal

45

24

15

18

21

-0.86549

-0.34412

-1.15871

0.82716

0.00000

-1.15831

-3.70710**

-3.00952**

-2.78160**

0.81219

0.00000

-13.47188***

0.07353

0.05039

0.04602

0.07864

0.30493**

0.00000

0.02967

Multimod al

Unimodal

Unimodal

Unimodal

Unimodal

-

Unimodal

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Sample size

Unimodal

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FS Raggedness index

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Tajima's D

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Sample size Cytochrome B

Overall

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Statistics

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Penganga

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*(p<0.05) **(p<0.01) ) ***(p<0.005)

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Berach Tawa 2.24508 inf 9.62634 0.67535 0.19414

Narmada Khargone 1.25199 inf 7.94920 0.77591 0.15963

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Population

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Table 6. Gene flow estimates (No. of Migration Nm*) for Cytochrome B (below diagonal), ATPase 6/8 (above diagonal) regions and concatenated sequences. Kolar 3.57457 inf 50.00705 0.40723 0.04110

Penganga Godavari

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0.27243 0.13794 Berach 1.80933 1.43229 0.75335 Tawa 1.96984 2.14854 0.81468 Khargone 1.79075 0.75098 0.31119 Kolar 0.41876 6.50323 Penganga 0.08390 1.45471 Godavari Concatenated sequences Berach 1.91400 Tawa 1.59115 Khargone inf 2.45915 Kolar 24.02027 10.82687 0.36676 0.83320 0.95984 0.46360 Penganga 0.09969 0.28854 0.24173 0.08524 1.63347 Godavari *Lynch and Crease 1990 (with Jukes and Cantor correction), obtained by the permutation test with 1000 replicates

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Journal Pre-proof Names of Authors: Priyanka Sah1,

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, Sangeeta Mandal1 , Rajeev K. Singh1 , Raj Kumar1 , Abhinav Pathak1 ,

Nimisha Dutta1 , J.K. Srivastava2 , Ved Prakash Saini3 , Kuldeep K Lal1 and Vindhya Mohindra1 Title of the manuscript: Genetic structure of natural populations of endangered Tor mahseer, Tor tor (Hamilton, 1822) inferred from two mitochondrial DNA markers Affiliations and addresses of the authors: 1 ICAR- National Bureau of Fish Genetic Resources (NBFGR), Lucknow, India. Amity Institute of Biotechnology, Amity University, Lucknow Campus, Lucknow, Uttar

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Pradesh, India.

Aquaculture Research and Seed Unit, Maharana Pratap University of Agriculture and

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Technology, Udaipur, Rajasthan, India

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Corresponding author:

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Conflict of interest:

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Vindhya Mohindra ICAR-National Bureau of Fish Genetic Resources, Canal Ring Road, PO Dilkusha, Lucknow, Uttar Pradesh, India [email protected], [email protected] Telephone number-+91 9415234371

The authors declare that there is no financial or non-financial conflict of interest.

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