Biochemical Systematics and Ecology 57 (2014) 141e151

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Biochemical Systematics and Ecology

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Genetic diversity and population structure of Schizopygopsisyounghusbandi Regan in the Yarlung Tsangpo River inferredfrom mitochondrial DNA sequence analysis

Shan-Shan Guo a, b, Gui-Rong Zhang a, b, Xiang-Zhao Guo a, b, Kai-Jian Wei a, b, *,Rui-Bin Yang a, b, Qi-Wei Wei c

a Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, College of Fisheries, Huazhong Agricultural University,Wuhan 430070, PR Chinab Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, PR Chinac Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture, Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences, Wuhan 430223, PR China

a r t i c l e i n f o

Article history:Received 28 January 2014Accepted 26 July 2014Available online

Keywords:Schizopygopsis younghusbandiGenetic diversityPopulation genetic structureCytochrome bControl regionTibet

* Corresponding author. Key Laboratory of FreshwWuhan 430070, PR China. Tel./fax: þ86 27 8728211

E-mail addresses: kjwei@mail.hzau.edu.cn, kaijia

http://dx.doi.org/10.1016/j.bse.2014.07.0260305-1978/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The genetic diversity and population structure of Schizopygopsis younghusbandi from sixsites in the middle reach of the Yarlung Tsangpo River were examined based on mito-chondrial DNA cytochrome b (Cyt b) gene and control region (D-loop) sequences. 1141 bpof complete Cyt b sequences and 737 bp of partial D-loop sequences for 153 individuals ofS. younghusbandi were obtained by using PCR amplification and sequencing. The resultsshowed that S. younghusbandi populations had high haplotype diversity and low nucleo-tide diversity, and the population genetic diversity of this species was at a low level.Analysis of molecular variance revealed that most genetic variation occurred withinpopulations, and that genetic differentiation was at low or moderate levels. The network ofhaplotypes based on Cyt b gene showed that there were two groups within the examinedpopulations. Neutrality tests and mismatch distribution analysis suggested that this spe-cies might have undergone a population expansion and the expansion time was estimatedto be 0.25e0.46 Ma BP. All these results would be crucial to establish scientific strategiesfor effective conservation and sustainable exploitation of wild resources of S.younghusbandi.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The Yarlung Tsangpo River is the largest river system (2057 km) in the Tibetan Plateau and the highest river in the worldwith an average elevation of over 4000 m above sea level (Li et al., 2013). It has five tributaries (the Dogxung Tsangpo, NyangChu River, Lhasa River, Nyang River and Parlung Tsangpo) in Tibet, China. The Yarlung Tsangpo River basin is notable for highspecies richness and an abundance of endemic fish species. However, many fish species in this area are now considered to beunder threat in terms of reductions in their natural distributional ranges and population numbers and sizes as a consequence

ater Animal Breeding, Ministry of Agriculture, College of Fisheries, Huazhong Agricultural University,3.n.wei@gmail.com (K.-J. Wei).

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of pollution, biological invasion of exotic species, overfishing, dam construction and habitat change (Zhou et al., 2013). As aconsequence of growing slowly and maturing late, these endemic fishes are very vulnerable to the threats listed above,suggesting that it is difficult to replenish their populations if they are depleted (Chen et al., 2009). The conservation of naturalpopulations and sustainable exploitation of germplasm genetic resources of these endemic fishes have become increasinglymatters of concerns in China.

Schizopygopsis younghusbandi Regan 1905 (Cyprinidae: Schizothoracinae) is an endemic tetraploid fish that is mainlydistributed in the middle reach of the Yarlung Tsangpo River in Tibet (Fisheries Bureau of Tibet Autonomous Region,1995; Wu et al., 1999). S. younghusbandi is a cold-adapted, long-lived and medium-sized species. It can reach nearly40 cm in body length, 750 g in body weight, and live longer than 15 years (Chen et al., 2009). Observations in the wildsuggest that the females reach sexual maturity at approximately seven years and males at four years of age. For the pastfew decades, S. younghusbandi has experienced a sharp decline in population size due to overfishing and environmentaldeterioration. Chen et al. (2009) found that standard lengths of 84.8% captures were less than those at growth inflexionpoints, indicating conservation and management schemes for S. younghusbandi should be considered urgently.

An understanding of the genetic background is of great significance and urgent need for resource conservation andmanagement of wild species. Molecular markers have been widely used in population genetic study for this purpose (Sunet al., 2012; Zhao et al., 2013; Wei et al., 2013). Mitochondrial DNA (mtDNA) is an extranuclear DNA that has proven to bea useful molecular marker in the study of fish population and evolutionary genetics because of its small molecularweight, maternal inheritance, relatively rapid base substitution rate, and lack of recombination (Guo et al., 2004).Moreover, the evolution speed varying in different regions makes it suitable for evolutionary research on different levels.Cytochrome b (Cyt b) is a protein-coding gene of the mtDNA genome and usually has a moderate level of intraspecificvariation. The Cyt b gene can be obtained by universal primers and has been widely used in genetic diversity andphylogenetic analysis (He and Chen, 2007; Qi et al., 2007; Sun et al., 2012). The control region (D-loop) is a noncodingregion and recognized as the most variable portion of the mtDNA genome. It is commonly variable at the intraspecificlevel, making it widely used in studies of genetic variability among populations and phylogenetic analysis (Donaldsonand Wilson, 1999; Liang et al., 2011; Zhao et al., 2013). To date, little genetic characteristic of S. younghusbandi isavailable except for limited research on its chromosome, phylogeny and biology (Wu et al., 1999; He and Chen, 2007;Chen et al., 2009). To conserve natural resources of this species, it is especially crucial to assess its genetic diversityand population structure.

In this study, we examined variation of the Cyt b and D-loop region sequences of mtDNA in S. younghusbandi populationsfrom the Yarlung Tsangpo River. The aim of the study is to determine the genetic diversity, population genetic structure anddemographic history of S. younghusbandi and gain some insights for conservation, management, and sustainable utilization ofthis species.

2. Material and methods

2.1. Sample collection and DNA extraction

Samples of S. younghusbandi were collected at six sites from the middle reach of the Yarlung Tsangpo River, from 2012 to2013 (Fig.1). Four populations (Shigatse, Quxu, Shannan andMainling) were from themain stream, the other two populations(Zhaxue and Nyingchi) were from two tributaries respectively (we use the term ‘population’ for ease of use, although eachgroup tested here may not be a biologically defined population). A small piece of pectoral fin from each sample was clippedand stored in 95% ethanol until DNA extraction. Total genomic DNAwas extracted from fin tissue using the traditional phenol-chloroform method (Sambrook and Russell, 2001). Concentration and quality of DNA were assessed by agarose gelelectrophoresis.

2.2. PCR amplification and sequencing

A total of 153 individuals (mean of 25 individuals per population) was analysed. Polymerase Chain Reaction (PCR) wasperformed to amplify a complete mitochondrial DNA (mtDNA) gene, cytochrome b (Cyt b) and a partial mtDNA control region(D-loop) sequence. PCR was carried out in a final volume of 20 ml including 1 � Taq polymerase buffer (Fermentas, EU),0.25 mM of each primer, 2.5 mM of MgCl2, 1 U of Taq DNA polymerase (Fermentas, EU), 250 mM of each dNTP, and 40 ng ofgenomic DNA. PCR thermal conditions were as follows: an initial denaturation at 95 �C for 4 min, followed by 35 cycles of95 �C for 30 s, annealing temperature (at 55 �C for Cyt b and at 51 �C for D-loop) for 30 s, 72 �C for 40 s, and a final extension at72 �C for 10 min. For Cyt b, we used primers L14724 (50-GACTTGAAAAACCACCGTTG-30) and H15915 (50-CTCCGATCTCCG-GATTACAAGAC-30) (He and Chen, 2007). For D-loop, we designed a primer pair 15848-F (50-CTTCGCATTTCACTTTCT-30) and790-R (50-AACTTGTTGGCTGATACG-30) for amplification based on the mitogenome sequence of S. younghusbandi (GenBankacc. # KC351895). PCR products were purified and sequenced at the Sangon Biotech Company (Shanghai, China). Cyt b se-quences were sequenced in both forward and reverse directions and D-loop sequences were sequenced in the forwarddirection.

Fig. 1. Collection sites of Schizopygopsis younghusbandi in the Yarlung Tsangpo River. Shigatse (SG); Quxu (QX); Zhaxue (ZX); Shannan (SN); Mainling (ML);Nyingchi (NC).

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2.3. Data analysis

Sequences were multiply aligned using CLUSTAL X 1.81 (Thompson et al., 1997) after manual editing and checking usingthe DNASTAR software package. Population genetic diversity indices (e.g., haplotype number (h); haplotype diversity (Hd);nucleotide diversity (p); and number of segregating sites (S)) were calculated using DNASP 5 (Librado and Rozas, 2009). Basecomposition and substitution of two mtDNA segments, and genetic distances within/between populations based on theKimura 2-parameter model (Kimura, 1980) were calculated by using MEGA 5 (Tamura et al., 2011).

Neighbour-joining (NJ) analysis (Saitou and Nei, 1987) was performed with MEGA 5 using the Kimura 2-parameter model.Schizopygopsis malacanthus baoxingensis was used as outgroup, its Cyt b and D-loop sequences having been obtained fromGenBank (Cyt b acc. # DQ533798, D-loop acc. # FJ422893). Robustness of NJ phylogenetic hypothesis was tested by 1000bootstrap replications (Felsenstein, 1985). A median-joining network was built to detect evolutionary relationships betweenhaplotypes using NETWORK 4.6 (Bandelt et al., 1999). Differences between populations were assessed with pairwise geneticdifferentiation values (FST) and hierarchical analysis of molecular variance (AMOVA) in ARLEQUIN 3.11 (Excoffier et al., 1992,2005). The significances of FST were tested using 10,000 permutations, and P values were adjusted according to the sequentialBonferroni correction (Rice, 1989).

Two different approaches implemented in ARLEQUIN 3.11 (Excoffier et al., 2005) were used to investigate the de-mographic history of each population. First, Tajima's D (Tajima, 1989) and Fu's Fs (Fu, 1997) were used to test whether thetwo mtDNA sequences conformed to the expectations of neutrality. Tajima's D test uses the frequency of segregatingnucleotide sites and shows sensitivity to large numbers of private mutations (singletons); Fu's Fs test uses the distributionof alleles or haplotypes and is particularly sensitive to past population expansions, which typically generate large,negative numbers due to the predominance of new, rare haplotypes in the sample. Second, we examined the observeddistribution of pairwise differences between sequences (mismatch distribution; Rogers and Harpending, 1992; Rogers,1995). Populations that have been stable over time are predicted to have a bimodal or multimodal mismatch distribu-tion, whereas a unimodal distribution is generally found in populations having passed through a bottleneck or recentdemographic expansion (Rogers and Harpending, 1992). If evidence of population expansion was found, the time ofpossible population expansion (t, number of generations) was calculated through the relationship t ¼ 2 ut, where t is themode of mismatch distribution, and u is the mutation rate of the sequence considering that u ¼ 2 mk (m is the mutationrate per nucleotide and k is the number of nucleotides analysed). Finally, the approximate time of expansion in yearscould be calculated by multiplying t by the generation time of S. younghusbandi. The generation time was taken as sevenyears, only considering the female age because the mitochondria of males were not transmitted to offspring. In this study,

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the mutation rate of Cyt b and D-loop were assumed as 2% (Brown et al., 1979) and 3.6% (Donaldson and Wilson, 1999) permillion years respectively.

3. Results

3.1. Sequence variation and genetic diversity

Complete mtDNA Cyt b sequences were obtained from 153 individuals, each being 1141 bp in length. Forty-three variablesites were identified (3.77%) among 153 Cyt b sequences, including 27 parsimony informative sites and 16 singleton variablesites. No indels were found and all variable sites were transitions. The overall base composition of Cyt b genes was A (26.0%), T(30.6%), C (26.6%) and G (16.8%), showing obvious anti-G bias. A þ T content (56.6%) was higher than C þ G content (43.4%),showing AT bias, which was consistent with the known pattern in fishes. Thirty-six haplotypes (Cyt b) were defined and theirDNA sequences have been submitted to GenBank (accession nos. KF745794eKF745829). All segregating sites and distribu-tions of 36 haplotype sequences showed that there were 17 haplotypes shared by two or more populations, and each pop-ulation had its own private haplotypes (Fig. 2). The most frequent haplotype, Hap1, was present in all six populations,followed by Hap4 and Hap13, suggesting that Hap1 might be an ancestral haplotype. Haplotype diversity (Hd) of Cyt b washigh for all populations, the Hd value for each population ranging from 0.814 ± 0.071 (NC) to 0.957 ± 0.020 (SG), with anoverall value of 0.947 ± 0.006 across all populations (Table 1). However, nucleotide diversity (p) was low and ranged from0.0029 ± 0.0004 (NC) to 0.0041 ± 0.0003 (ZX) for each population, with an overall value of 0.0035 ± 0.0001 across allpopulations. Average number of nucleotide differences (K) for each population ranged from 3.260 to 4.641, with a value of3.989 across all populations.

A total of 153 of partial mtDNA D-loop sequences was obtained from six populations, each being 737 bp in length. Twenty-two variable sites were identified (2.99%), including 16 parsimony informative sites and six singleton variable sites. No indels

Fig. 2. Segregating sites and frequency distributions of mtDNA Cyt b haplotypes in Schizopygopsis younghusbandi. A small dot indicates the same base as the firstline. Base position at the top of the haplotype sequences represents variable sites. SG, QX, SN, ML, ZX and NC represent six populations.

Table 1Genetic diversity results for six populations of Schizopygopsis younghusbandi based on Cyt b sequences: number of haplotypes (h), number of segregatingsites (S), haplotype diversity (Hd), nucleotide diversity (p), average number of nucleotide differences (K).

Population Sample size h S Hd (mean ± SD) p (mean ± SD) K

SG 24 14 19 0.957 ± 0.020 0.0034 ± 0.0003 3.888ZX 22 13 21 0.931 ± 0.036 0.0041 ± 0.0003 4.641QX 22 12 16 0.935 ± 0.029 0.0031 ± 0.0003 3.498SN 33 12 19 0.911 ± 0.024 0.0030 ± 0.0004 3.473ML 30 11 15 0.855 ± 0.041 0.0030 ± 0.0002 3.411NC 22 9 13 0.814 ± 0.071 0.0029 ± 0.0004 3.260Overall 153 36 43 0.947 ± 0.006 0.0035 ± 0.0001 3.989

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were found and all variable sites were transitions, except twowhich were transversions, indicating that D-loop sequences didnot reach saturation and were suitable for genetic analysis. D-loop sequence composition was A (30.5%), T (31.8%), C (22.5%)and G (15.2%), showing obvious anti-G bias. A þ T content (62.3%) was notably higher than C þ G content (37.7%), showingobvious AT bias, which was consistent with the known pattern in fishes. Twenty-nine haplotypes were generated and theirDNA sequences have been submitted to GenBank (accession nos. KF745830eKF745858). All segregating sites and distribu-tions of 29 haplotype sequences showed that there were 14 haplotypes shared by two or more populations, and each pop-ulation had its own private haplotypes (Fig. 3). The most frequent haplotype, Hap10, was present in all six populations,followed by Hap9 and Hap21, indicating that Hap10might be an ancestral haplotype. Haplotype diversity (Hd) was high for allpopulations, with values from 0.824 ± 0.048 (SN) to 0.909 ± 0.034 (ZX) for each population, with an overall value of0.922 ± 0.010 across all populations (Table 2). However, nucleotide diversity (p) was low and ranged from 0.0023 ± 0.0003(SN) to 0.0044 ± 0.0004 (ML) for each population, with a value of 0.0036 ± 0.0002 across all populations. Average number ofnucleotide differences (K) for each population ranged from 1.678 to 3.246 with a total of 2.622.

3.2. Phylogenetic reconstruction and population structure

The topologies built from the haplotype sequences of mtDNA Cyt b gene and D-loop region were dissimilar. Two NJ treesshowed that most of haplotypes wereweakly associated (less than 50% bootstrap support) or unresolved, which was possibly

Fig. 3. Segregating sites and frequency distributions of mtDNA D-loop haplotypes in Schizopygopsis younghusbandi. A small dot indicates the same base as thefirst line. Base position at the top of the haplotype sequences represents variable sites. SG, QX, SN, ML, ZX and NC represent six populations.

Table 2Genetic diversity results for six populations of Schizopygopsis younghusbandi based on D-loop sequences: number of haplotypes (h), number of segregatingsites (S), haplotype diversity (Hd), nucleotide diversity (p), average number of nucleotide differences (K).

Population Sample size h S Hd (mean ± SD) p (mean ± SD) K

SG 24 11 10 0.906 ± 0.036 0.0032 ± 0.0004 2.337ZX 22 10 13 0.909 ± 0.034 0.0036 ± 0.0004 2.654QX 22 11 12 0.909 ± 0.037 0.0034 ± 0.0005 2.476SN 33 9 7 0.824 ± 0.048 0.0023 ± 0.0003 1.678ML 30 10 14 0.853 ± 0.040 0.0044 ± 0.0004 3.246NC 22 10 13 0.905 ± 0.033 0.0036 ± 0.0004 2.636Overall 153 29 22 0.922 ± 0.010 0.0036 ± 0.0002 2.622

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due to low nucleotide acid differences among them (Fig. 4). The haplotypes of Cyt b gene clustered into two branches, theupper branch contained 13 private haplotypes (marked with circles) and most private haplotypes (ten in total) were frompopulations ZX and SG, the lower branch contained six private haplotypes and two of them were from population SN. Theprivate haplotypes of Cyt b gene indicated weak geographic structure between two branches and no obvious structure withineither branch (Fig. 4A). For D-loop region, the haplotypes clustered into more branches. Of 15 private haplotypes of D-loop,more private haplotypes (eight in total) were from populations ZX and SG, whereas there was no obvious structure among 15

Fig. 4. NJ trees for mtDNA haplotypes of Schizopygopsis younghusbandi. A, Cyt b gene; B, D-loop region; numbers at the nodes indicate the bootstrap values basedon 1000 replications, and only values higher than 50% are shown; the private haplotypes of each population are marked with circles, and the other sharedhaplotypes among populations are not marked; different colours represent different geographic locations as showed in Figure, the number in each parenthesesindicates the frequency of a private haplotype whilst it is more than one. (For interpretation of the references to colour in this figure legend, the reader is referredto the web version of this article).

Fig. 5. Median-joining networks of mtDNA haplotypes from six populations of Schizopygopsis younghusbandi: A, Cyt b gene; B, D-loop region; each circle rep-resents a haplotype, its size is proportional to its total frequency. And different colours indicate different geographic locations as showed in Figure. (For inter-pretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

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private haplotypes (Fig. 4B). The median-joining networks were constructed from the haplotype sequences of Cyt b gene andD-loop region respectively (Fig. 5). In the Cyt b network, haplotypes were grouped into two groups that were linked via oneprivate haplotype Hap32 (purplee populationML) in the centre of the plot.Whilst therewas no obvious geographic structurewithin either grouping, there were more black (individuals of population SN) in the left group, and more green (populationZX) and yellow (population NC) in the right group, suggesting that there was obvious geographic structure between twogroupings (Fig. 5A). For D-loop region, however, the network of haplotypes was starlike and had no obvious structure,indicating a signature of population expansion in S. younghusbandi (Fig. 5B).

For Cyt b gene, genetic distances between six populations were from 0.0030 to 0.0045 and those within populations werefrom 0.0029 to 0.0041. Fixation indices (FST) between populations ranged from 0.0108 (SN/QX) to 0.2145 (SN/NC) (Table 3).Significant genetic differentiation was observed between SN population and the other four populations (SG, ZX, ML, and NC),and FST values ranged from 0.0935 to 0.2145, suggesting that the SN population was genetically distinct from the other fourpopulations. The analysis of molecular variance (AMOVA) indicated that a high proportion (90.44%) of genetic variation wasattributed to differences within populations, and only 9.56% of the variationwas attributed to differences among populations(FST ¼ 0.0956, P < 0.001).

For D-loop region, genetic distances between populations ranged from 0.0030 to 0.0042 and those within populationswere from 0.0023 to 0.0044. FST values between populations ranged from �0.0174 (ML/NC) to 0.1920 (SN/ZX) (Table 4).Significant genetic differentiation was observed between SN population and the other four populations (SG, ZX, ML, and NC)with FST values ranging from 0.1042 to 0.1920, suggesting that the SN population was genetically distinct from the other fourpopulations. The analysis of molecular variance (AMOVA) showed that most of genetic variationwas attributed to differenceswithin populations (93.63%), and only 6.37% of the variation was attributed to differences among populations (FST ¼ 0.0637,P < 0.001).

3.3. Population historic dynamics

Conformations to the expectations of neutrality were examined in six populations of S. younghusbandi using Tajima'sD andFu's Fs tests (Table 5). All Tajima's D and Fu's Fs values based on both Cyt b and D-loop sequences were negative, but Tajima's Dvalues were not significant (P > 0.1). However, Fu's Fs tests based on Cyt b and D-loop sequences showed that significantdeviations from neutrality occurred in the SG population, the QX population and the overall population, indicating that S.younghusbandi had experienced expansion along the Yarlung Tsangpo River. Mismatch distributions of both Cyt b and D-loop

Table 3Pairwise genetic distance within population (shown in bold along diagonal), genetic distance (below diagonal) and fixation index (FST) (above diagonal)between populations of Schizopygopsis younghusbandi based on Cyt b sequences.

Population SG ZX QX SN ML NC

SG 0.0034 0.0694 0.0326 0.1248* 0.0390 0.0249ZX 0.0040 0.0041 0.1080 0.2090* 0.1165 0.0619QX 0.0034 0.0040 0.0031 0.0108 0.0549 0.1236SN 0.0037 0.0045 0.0031 0.0031 0.0935* 0.2145*ML 0.0033 0.0040 0.0032 0.0033 0.0030 0.0366NC 0.0032 0.0037 0.0034 0.0038 0.0030 0.0029

* Significant at a ¼ 0.05 after Bonferroni correction.

Table 4Pairwise genetic distance within population (shown in bold along diagonal), genetic distance (below diagonal) and fixation index (FST) (above diagonal)between populations of Schizopygopsis younghusbandi based on D-loop sequences.

Population SG ZX QX SN ML NC

SG 0.0032 �0.0009 0.0227 0.1705* 0.0207 0.0190ZX 0.0034 0.0036 0.0436 0.1920* 0.0392 0.0272QX 0.0034 0.0037 0.0034 0.0539 0.0486 0.0512SN 0.0033 0.0036 0.0030 0.0023 0.1042* 0.1182*ML 0.0039 0.0042 0.0041 0.0037 0.0044 �0.0174NC 0.0035 0.0037 0.0037 0.0033 0.0039 0.0036

* Significant at a ¼ 0.05 after Bonferroni correction.

Table 5Tajima's D and Fu's Fs values of neutrality tests for six populations of Schizopygopsis younghusbandi based on Cyt b and D-loop sequences.

Population Cyt b D-loop

Tajima's D Fu's Fs Tajima's D Fu's Fs

SG �0.856 �5.138* �0.425 �4.308*ZX �0.726 �3.525 �0.906 �2.904QX �0.738 �3.827* �0.868 �4.397*SN �0.878 �2.175 �0.078 �2.669ML �0.333 �1.746 �0.272 �1.158NC �0.304 �1.180 �0.923 �2.933Overall �1.445 �17.257* �0.928 �15.986*

* Significant deviation from the expectation of neutrality at a ¼ 0.05.

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sequences were tested and shown to follow a unimodal pattern (Fig. 6), suggesting a recent history of demographicexpansion. Mean t values based on Cyt b and D-loop sequences were 5.99 and 3.75, respectively. The estimated expansiontime of S. younghusbandi was 0.25e0.46 million years before present (Ma BP), that is, as part to the Middle Pleistocene.

4. Discussion

4.1. Genetic diversity

The genetic diversity of a species is closely linked to its adaptability, viability and evolutionary potential. Abundant geneticdiversity implies high potential of adaptability, evolution, breeding and genetic improvement for a species, whereas poorgenetic diversity has negative effects on protection and utilization of germplasm resources (Hughes et al., 2008). In general,high levels of genetic variability in a given population can bemaintained if the population is large enough and stable for a longperiod of time. It is often assumed that when a population goes through a severe bottleneck, random genetic drift will inducea massive loss of genetic variability (Qi et al., 2007). In this study, S. younghusbandi exhibited high Hd values and low p valuesfor each population and across all populations as revealed by bothmtDNA Cyt b and D-loop sequences. TheHd and p values ofS. younghusbandiwere almost identical to those of Schizopygopsis pylzovi (Hd¼ 0.83e0.98, p¼ 0.0018e0.0045) from the samegenus (Qi et al., 2007), but were lower than those of Gymnocypris przewalskii (Hd ¼ 0.992, p ¼ 0.0082) and Schizothoraxprenanti (Hd ¼ 0.96, p ¼ 0.016) (Chen et al., 2006; Liang et al., 2011) from the same subfamily. Therefore, the populationgenetic diversity of S. younghusbandiwas lowwhen compared to closely related fishes. The high Hd value together with a lowp value in S. younghusbandi was consistently observed in other cyprinid fish (Qi et al., 2007; Zhao et al., 2013). Grant and

Fig. 6. Observed and expected mismatch distributions for Schizopygopsis younghusbandi: A, Cyt b gene; B, D-loop region; dashed line, observed distribution; solidline, theoretical expected distribution under a population expansion model.

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Bowen (1998) summarized the values ofHd and p from the literature and concluded that the occurrences of highHd and low p

in a population could be attributed to rapid population expansion after a period of small effective population size. This in-dicates that S. younghusbandi might have experienced population expansion. Rapid population growth enhanced the accu-mulation of mutations, and the expansion time was enough to accumulate their haplotype diversity, but insufficient for anincrease in nucleotide diversity.

In six populations of S. younghusbandi, the overall genetic diversity indices inferred from Cyt b gene were a little greaterthan or the same as those of the D-loop region. This was not congruent with the fact that population genetic variation inferredby D-loop region is greater than by Cyt b gene in vertebrates (Guo et al., 2004). One possible explanationwas that the D-loopsequences we used were partial ones and the Cyt b sequences were complete ones. Additionally, there were similar reportsthat the genetic variation in the D-loop was lower compared with Cyt b (Pereira et al., 2004; Zhao et al., 2010). This suggestedthat Cyt b gene and D-loop region were effective markers for assessing the genetic diversity of S. younghusbandi.

4.2. Population structure

Phylogenetic analysis of D-loop region sequences revealed no obvious phylogeography pattern separating the six locationsof S. younghusbandi and haplotypes from all locations were interspersed throughout the tree. This shallow phylogeny isconsistent with a population after a bottleneck (Slatkin and Hudson, 1991). However, the median-joining network of Cyt bhaplotypes indicated evidence of obvious geographic structure. There was two groups that were linked via one privatehaplotype in the centre of the network, most individuals of the population SN were in one group, and most individuals of thepopulations ZX and NC were in the other group. The populations ZX and NC were from two tributaries, they showed cleardifferentiation from the population SN in the main river system. This structure existed for the Cyt b but not for the D-loopresults. This difference in information content between the two mtDNA sequences might simply be a sampling artefact or itmight reflect a genuine difference between a gene and a control region of the mtDNA molecule.

Genetic distances between six populations and those within populations were at the same level, indicating that geneticdifferentiation was low. Fixation index (FST) represents the level of genetic differentiation among populations: an FST of0e0.05 represents ‘‘little differentiation’’, 0.05e0.25 ‘‘moderate differentiation’’, and values greater than 0.25 ‘‘very greatdifferentiation’’ (Wright, 1965). In this study, AMOVA analysis of six populations showed that genetic variation mostlyoccurred within populations, whilst there was moderate differentiation among populations (P < 0.001). The level of geneticdifferentiation between populations was little or moderate differentiation determined by pairwise FST, which was perhapsattributable to high gene flow. Lack of barriers to dispersal and strong dispersal capacity could facilitate genetic exchangesamong groups across their distribution, being possible reasons for the low genetic differentiation. Except for the populationSN, there were no significant differentiations between other five populations (SG, ZX, QX, ML, and NC). Whereas significantdifferentiations were observed between the SN population and the other four populations (SG, ZX, ML, and NC) inferred fromboth Cyt b gene and D-loop region, suggesting that the SN populationwas genetically distinct from the other four populations.SN Prefecture is located in the south of Tibet and is referred to as a “valley in southern Tibet”. There was a subpopulation of S.younghusbandi that distributed in small lakes of SN Prefecture and was geographically isolated from the middle reach of theYarlung Tsangpo River. The shape and structure of pharyngeal teeth, the number of vertebrae and the thin spine of dorsal finof this subpopulation were exactly the same with S. younghusbandi, whereas the morphological characteristics of smallsawteeth in dorsal fin spine andmore gill rakers made it be different from S. younghusbandi, therefore this subpopulationwasnamed a subspecies Schizopygopsis younghusbandi shannaensis (Wu and Wu, 1992). Probably due to human activities (Zhouet al., 2013), the subspecies S. y. shannaensis might have been introduced into the Yarlung Tsangpo River where S. young-husbandi is distributed. It was possible that the samples of the SN population collected from this region contained someindividuals of this subspecies that were hard to distinguish. This might be a possible explanation for the significant differ-entiation between SN and the other four populations. Further investigation was needed to focus on the SN population of thisspecies.

4.3. Historical demography

Neutral tests and mismatch distribution were used to investigate the historical demography. Tajima's D test gives a lowsignificant value if populations deviate from the neutral expectations (Tajima, 1989). Fu (1997) found that large negative Fsvalues indicate deviation from neutrality caused by population expansion and/or selection. The Tajima's D test puts moreweight on ancient mutation, thus revealing ancient population events, while the Fu's Fs test is more sensitive to recentpopulation events (Tajima, 1989; Fu, 1997). The mismatch distribution is usually multimodal in samples drawn from pop-ulations at demographic equilibrium and unimodal in populations following a recent population demographic expansion andpopulation range expansion (Rogers and Harpending, 1992; Rogers, 1995). In our investigation of demographic history acrossall populations, the negative but nonsignificant Tajima's D values, the significant Fu's Fs values, combined with the unimodalmismatch distribution, suggest that populations of S. younghusbandi from the Yarlung Tsangpo River may have passedthrough a recent bottleneck and experienced expansion. This was also evidenced by the starlike network and the distributionmode of high haplotype diversity and low nucleotide diversity that characterized these demographic events (Slatkin andHudson, 1991; Grant and Bowen, 1998). Corresponding to tau (t ¼ 3.75 and 5.99) values, the estimated time for popula-tion expansion was 0.25e0.46 Ma BP, reflecting the recent founding of S. younghusbandi since Middle Pleistocene. Recent

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founding and insufficient time to attain the migrationedrift equilibrium among populations of S. younghusbandi could be thepossible reason for lack of genetic structure observed in the current study.

Past geological and climatic events haveundoubtedly played amajor role inpopulation expansionof S. younghusbandi in theYarlung Tsangpo River. The intensive uplift of the QinghaieTibetan Plateau and repeated glacialeinterglacial changes aregenerally accepted to have had significant impact on the environment and climate. The geological studies indicated that theTibetan Plateau has entered a rapid uplift period since 3.6Ma ago, including threemajor phases. The early stage, the ‘QingzangMovement’, occurred between 3.6 and 1.7Ma and included three phases commencing at 3.6, 2.5, and 1.7Ma, respectively. Themiddle stage is the ‘Kunlun-HuangheMovement’,which occurredbetween1.1 and0.6Maand included threephases starting at1.1, 0.8 and 0.6Ma, respectively. The late stage, the ‘GongheMovement’, took place 0.15Ma ago (Li and Fang,1998; He andChen,2007). The Kunlun-HuangheMovement made the QinghaieTibetan Plateau approach a critical elevation of 3000 m and enterthe cryosphere, and this was perhaps the driving force of the tremendous environmental change in tectonic-climatic circu-lation (Cui et al.,1998). In this study, the estimated recent expansionperiod of S. younghusbandi populationswas 0.25e0.46Main themiddle Pleistocene period, whichwas after the last phase (0.6Ma) of the Kunlun-HuangheMovement. Our data supportthe assumption that the expansion and evolution of S. younghusbandi are associatedwith the environmental changes causedbythe violent upheaval of the plateau. On the other hand, the Pleistocene (0.01e1.9 Ma) was characterized by a series of largeglacialeinterglacial changes, and the geographical distributions and abundance of organismsmay be acutely affected by cyclesof water level rise-and-fall (Dynesius and Jansson, 2000). During glacial periods, most aquatic organisms might die withoutpassing on their genes or migrating to more suitable environments for survival (Hewitt, 2000). After that the survivors mightre-colonize the original regions and expand during interglacial periods. Thus, the repeated glacialeinterglacial changes duringthe Pleistocene might also have influenced the expansion of S. younghusbandi.

4.4. Conservation implications

Genetics can provide helpful information for establishing conservation planning and management strategies of species.According to Moritz's (1994) strict criteria for evolutionarily significant units (ESU), it required both significant divergence ofallele frequencies at nuclear loci and reciprocal monophyly at mtDNA alleles. The concept of a management unit (MU; Moritz,1994) was established for those cases in which reciprocal monophyly was not reached among lineages. This concept wasoriginally defined for populations (or groups of populations) identified by a significant divergence in the allele frequencies ofneutral loci (nuclear or mitochondrial). Therefore, MU is a group of individuals with a sufficiently low degree of ecological andgenetic connectivity, which justifies a separate monitoring and management for each group.

Data reported here did not meet the criteria of ESU, for there were no reciprocal monophyly, so we proposed a single ESUfor the six populations of S. younghusbandi. Given that there is clear differentiation between the two tributary populations (ZXand NC) and the SN population in the main river system, therefore each of these three populations should be defined as anMU. A series of fishery management strategies should be carried out in the local environment in Tibet: (1) establish fishingmoratorium, and prohibit fishing in breeding season (generally from April to July); (2) advocate rational fishing, includingfishing methods and increasing mesh size; (3) control exotic fishes; (4) protect the habitats from pollution or other humanactivities; (5) enhance artificial propagation and releasing; (6) establish and improve fishery legislation, providing legal basisfor the conservation and utilization of S. younghusbandi.

It is important to be aware that genetic data are only one component of defining conservation units, other data such asecological data should ideally be incorporated in the assessment of conservation units. Nevertheless, genetic data should beregarded as aminimum prerequisite for the definition of these units, since genetic data provide a rather conservativemeasureof population differentiation. We leave it to those involved with local management of the species to combine the analysispresented here with local ecological data to make the determinations.

In conclusion, we used cytochrome b and D-loop sequences to assess the genetic diversity and genetic structure of S.younghusbandi from six populations in the middle reach of the Yarlung Tsangpo River. Based on low population geneticvariations and low or moderate genetic differentiations between populations as revealed by this study, we propose a singleevolutionarily significant units for the six populations of S. younghusbandi. This research has provided detailed information onpopulation genetics and demographic history of S. younghusbandi, and provides a scientific basis for the evaluation ofgermplasm resources, rational utilization and resource management of this species.

Acknowledgements

We thank Jian-Hui Qin, Bin Huo and Hui-Juan Zhang for help with sample collection. This research was supported by theFundamental Research Funds for the Central Universities (Program No. 2013SC12), the Special Fund for Agro-scientificResearch in the Public Interest (Grant No. 201203086-13) and the Major Science and Technology Program for Water Pollu-tion Control and Treatment (Grant No. 2012ZX07202-004-02).

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