Genetic Diversity and Population Structure of a Cyprinid Fish (Ancherythroculter Nigrocauda) in a Highly Fragmented River

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Institute of Hydrobiology, Chinese Academy Of Sciences Received: 9 October 2018 | Revised: 19 January 2019 | Accepted: 13 February 2019 DOI: 10.1111/jai.13897

ORIGINAL ARTICLE

Genetic diversity and population structure of a cyprinid fish (Ancherythroculter nigrocauda) in a highly fragmented river

1The Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Abstract Sciences, Institute of Hydrobiology, Chinese In this study, samples of Ancherythroculter nigrocauda, an endemic fish in the upper Academy of Sciences, Wuhan, China Yangtze River, were collected above and below dams in the Longxi River, a tributary 2University of Chinese Academy of Sciences, Beijing, China of the upper Yangtze River, China, to investigate the genetic impacts of dams. The mitochondrial cytochrome b gene (cyt b) and 13 microsatellite (SSR) loci were used to Correspondence Xin Gao, The Key Laboratory of Aquatic analyze whether dams have resulted in loss of genetic diversity of the two frag‐ Biodiversity and Conservation of mented populations or caused genetic differentiation between them. The results Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of showed that the haplotype diversity (0.488; 0.486), nucleotide diversity (0.084%; Sciences, Wuhan, China. 0.082%) and average expected heterozygosity (0.652; 0.676) of the two populations Email: [email protected] were all at a low level, and recent bottlenecks were detected. However, there was no Funding information genetic differentiation detected by the low genetic differentiation index (F , cyt b: Special Program for Basic of Science st and Technology Research, Grant/Award −0.1677, p = 0.99707; SSR: 0.00259, p = 0.81427). Besides, 11 pairs of half‐sibling Number: 2014FY120200; China Three relationship were found between the two populations indicating that there were in‐ Gorges Corporation, Grant/Award Number: 0799570; National Natural Science dividual movements and gene flow between them. This could be the larvae moving Foundation of China, Grant/Award Number: from upstream to downstream when water spilled over dams in flooding season. 31400359 and 51509239 Therefore, our analysis showed that the dams have caused a loss of genetic diversity of the populations of A. nigrocauda in the Longxi River, blocked the active upstream movement but allowing passive downstream drift of larvae.

KEYWORDS bottleneck effect, gene flow, genetic differentiation, genetic diversity, genetic drift, sibship

1 | INTRODUCTION structure of European grayling in the Skjern River. Faulks, Gilligan, and Beheregaray (2011) also demonstrated the appearance of ge‐ In riverine ecosystems, dams have been well known as one of the netic differentiation and loss of genetic diversity in the Macquarie most negative anthropogenic impacts on fish populations (Saunders, Perch after damming. However, the previous studies mainly focused Hobbs, & Margules, 1991). Dams reduce fish populations by blocking on the genetic impacts of dams on migratory fish, rather than resi‐ migration routes, and alter fish community structure upstream and dent fish. downstream through regulating flow (Agostinho, Pelicice, & Gomes, Ancherythroculter nigrocauda is an endemic cyprinid fish in the 2008; Gao, Zeng, Wang, & Liu, 2010; Neraas & Spruell, 2001; Quinn upper Yangtze River basin. It inhabits the main channel and trib‐ & Kwak, 2003). Moreover, dams decline genetic diversity of fish utaries of the upper Yangtze River, especially in tributaries. This populations and cause genetic differentiation among separated pop‐ species is a resident fish and reproduces adhesive eggs from April ulations. For instance, Meldgaard, Nielsen, and Loeschcke (2003) to August. Its absolute fecundity varied from 11,300 to 504,630 found the construction of dams apparently influenced the genetic eggs, with the mean of 162,377 eggs (Liu et al., 2013). It is also

FIGURE 1 A map showing the sampling sites (star) of Ancherythroculter nigrocauda and the locations of cascade hydroelectric dams (black square) in the Longxi River, China an important economic fish and aquaculture species in China. b gene and SSR markers. Our objective was to understand the ge‐ In the past decades, the abundance and distribution area of this netic impacts of dams on resident fish populations. species were reducing. Yan (2002) found that population of this species still remained abundant in the Longxi River, a tributary of 2 | MATERIALS AND METHODS the upper Yangtze River, in Sichuan Province, China. This river is 97 km long with drainage area of 512 km2. Eight dams have been 2.1 | Samples collection and DNA extraction constructed on the Longxi river since 1920s (Figure 1; Table S1; Wang, 1994) and fragmented the populations of A. nigrocauda. Samples of A. nigrocauda were collected above and below the cas‐ There were some studies suggested that the growth and repro‐ cade dams in Longxi River in 2016. A total of 52 samples were col‐ duction of the populations of A. nigrocauda in Longxi River have lected from the Yulong Lake (YLL), which is above the cascade dams. been adversely influenced, it might be related to damming (Liu, Another 64 samples were collected from the reach between the sev‐ 2013; Liu et al., 2013). However, impacts of dams on the genetic enth dam (Kuifeng Dam) and eighth dam (Dongwo Dam) (DK). The diversity and population structure of A. nigrocauda in Longxi River two sampling sites are separated by 7 hydroelectric dams (Figure 1). have been little investigated. For DNA extraction, the tissue samples from the dorsal muscle of In this study, samples of A. nigrocauda were collected above and these samples were preserved in 95% ethanol and stored at −20°C. below the dams in the Longxi River. We analyzed genetic diversity Total DNA was extracted using standard proteinase K digestion fol‐ and population structure of the fragmented populations by using cyt lowed by phenol/chloroform extraction (Kocher et al., 1989). ZHAI et al. | 703

The number of alleles (A), observed (Ho) and expected (He) het‐ 2.2 | MtDNA amplification and sequencing erozygosity, and polymorphic information content (PIC) per locus The fragments containing the mtDNA cyt b gene were obtained were calculated by Cervus3.0 (Kalinowski, Taper, & Marshall, 2007). using polymerase chain reaction (PCR) amplification with the primer Standardized allelic richness (Ar) and allele frequency distribution of sets L14724 5′‐GAC TTG AAA AAC CAC CGT TG‐3′ and H15915 5′‐ per population at each locus were calculated using Fstat software

CTC CGA TCT CCG GAT TAC AAG AC‐3′ (Xiao, Zhang, & Liu, 2001). v2.9.3.2 (Goudet, 2001). The effective population size (Ne) was esti‐ PCR was performed at an initial denaturation step at 94°C for 3 min, mated by the Colony Version 2.0.6.4 (Jones & Wang, 2010). Arlequin followed by 35 cycles at 94°C for 30 s, 54°C for 45 s, 72°C for 1 min, version 3.0 (Excoffier et al., 2005) was used to perform an analysis and a final extension at 72°C for 8 min. The amplified fragments of molecular variance (AMOVA) to examine the degree of differenti‐ were purified with a BioStar glass‐milk DNA purification kit follow‐ ation between the two populations (Excoffier et al., 1992). Wilcoxon ing the manufacture's instruction. The purified fragments were se‐ signed rank test was performed to detect the difference of allele quenced by Shanghai DNA Biotechnologies Company. frequency distribution between the two populations. We calculated the M values (Garza & Williamson, 2001) to detect whether populations have suffered from recent bottleneck effect. 2.3 | MtDNA sequence analysis The formula is given as M = k/r where k is the number of alleles at a Nucleotide sequences were aligned and refined manually with given loci in a population sample, and r is the difference between the MEGA7 (Kumar, Stecher, & Tamura, 2016). The number of variable maximum and the minimum number of repeats. Loci were excluded site, haplotype and nucleotide diversity, and haplotype frequency from analysis that were monomorphic or that contained different were calculated with DnaSP v5.10 (Librado & Rozas, 2009). Arlequin repeat unit sizes (Garza & Williamson, 2001; Hundertmark & Daele, ver 3.0 (Excoffier, Laval, & Schneider, 2005) was used to perform an 2010). Based on simulations and experimental data, populations analysis of molecular variance (AMOVA) to examine the degree of that have experienced recent bottlenecks with the mean value of differentiation between the two populations (Excoffier, Smouse, & M < 0.68 (Garza & Williamson, 2001; Reid, Wilson, Mandrak, & Carl, Quattro, 1992). 2007). The detection of sibship among individuals was inferred by Colony Version 2.0.6.4 (Jones & Wang, 2010). The parameters were 2.4 | SSR amplification and electrophoresis set according to actual situation and the number of threads for PCR was performed at an initial denaturation step at 94°C for 3 min, the run was set to 10. The sibships were only accepted for poste‐ followed by 28 cycles at 94°C for 30 s, annealing temperature for rior probability values of more than 0.75 (Schunter, Pascual, Garza, 40 s, 72°C for 1 min, and a final extension at 72°C for 8 min. The Raventos, & Macpherson, 2014). specific sequences, repeat motif, optimum annealing temperature and accession number of GenBank for each primer was listed in 3 | RESULTS Table S2. The first 12 loci were developed using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol, another 3.1 | Cyt b marker 3 loci were quoted from Sun et al. (2014). The amplified fragments were electrophoresed in 8% non‐denaturing polyacrylamide gels on 3.1.1 | Genetic characterization and diversity Sequi‐Gen GT system (Bio‐Rad). Then the gels would be dyed by the GelRed Nucleic Acid Gel Stain for 30 min before being photo‐ Following alignment, 1,140 bp of cyt b gene were obtained for 116 graphed. At last, the size of band was read by referring to the marker individuals. No deletions and insertions were observed. Among of pBR322 DNA/Msp I (Tiangen) manually. Only unambiguous and the 1,140 bp, five sites were variable, of which two were parsi‐ clear bands were counted. mony informative. Only 6 haplotypes (GenBank accession number: MG756603‐MG756608) were identified from the total 116 individ‐ uals. The haplotype and nucleotide diversity of the DK population 2.5 | SSR data analysis were 0.488% and 0.084% respectively, and for the YLL population, Possible large allele dropout, scoring errors and null alleles were they were 0.486% and 0.082% respectively (Table 1). assessed by Micro‐Checker version 2.2.1 (Van Oosterhout, Hutchinson, Wills, & Shipley, 2004). Tests of deviation from Hardy– 3.1.2 | Population differentiation Weinberg equilibrium (HWE) and Linkage disequilibrium (LD) were implemented by GENEPOP version 4.7.0 (Rousset, 2008) with an Hap1, Hap2 and Hap3 were shared by the two populations, and the exact test using a Markov chain algorithm (Guo & Thompson, 1992; frequency of each haplotype in each population was nearly equal. p‐values were estimated from 10,000 dememorization, 100 batches Hap4, Hap5 and Hap6 were only found within one population, but and 5,000 iterations per batch). Significance levels for multiple com‐ these three haplotypes only represented a single individual with parisons of loci across samples were adjusted using the sequential relatively low frequency of 0.0086, respectively (Table 1). So there Bonferroni correction (Rice, 1989). were only slight differences between the haplotype frequency 704 | ZHAI et al.

TABLE 1 Number of samples, haplotype frequencies and indices of genetic diversity with standard deviations (SE) for the two populations

Haplotype Hap1 Hap2 Hap3 Hap4 Hap5 Hap6 Haplotype diversity Nucleotide diversity

Population DK(64) 0.6563 0.2969 0.0156 0.0156 0.0156 0.488 ± 0.050 0.00084 ± 0.00009 YLL(52) 0.6538 0.3077 0.0192 0.0192 0.486 ± 0.053 0.00082 ± 0.00009 Total(116) 0.6552 0.3017 0.0172 0.0086 0.0086 0.0086 0.483 ± 0.036 0.00082 ± 0.00006

Note. Numbers in brackets indicate the number of individuals from each population, Hap1‐Hap6 represent the six haplotypes.

TABLE 2 Summary statistics of SSR analysis of the two Ancherythroculter nigrocauda populations

Population Locus N Ho He A Ar M PIC Size range (bp)

DK An1 64 0.672 0.715 6 5.757 0.375 0.660 146−210 An10 64 0.531 0.585 5 4.594 0.509 140−156 An21 63 0.619 0.649 6 5.810 0.857 0.608 176−204 An52 64 0.547 0.581 9 7.944 0.474 0.508 189−227 An63 64 0.344 0.625 6 5.789 0.573 172−190* An68 64 0.750 0.777 8 7.905 0.727 0.735 156−178 An70 64 0.594 0.495 8 7.147 0.381 0.439 141−183 An76 64 0.641 0.667 6 5.797 0.300 0.624 262−302 An86 64 0.656 0.676 8 7.391 0.615 0.639 220−272 An95 64 0.500 0.589 5 4.959 0.556 0.498 160−178 An98 64 0.578 0.672 5 4.757 0.333 0.598 166−196 An114 62 0.387 0.753 11 10.108 0.714 144−204* hwb03 62 0.629 0.695 9 8.610 0.818 0.655 180−202 hwb08 62 0.548 0.637 4 3.823 0.267 0.555 261−291 hwb16 64 0.672 0.740 8 7.677 0.216 0.689 230−304 Mean 0.611 0.652 6.692 6.321 0.493 YLL An1 51 0.608 0.686 6 6.000 0.462 0.623 146−198 An10 52 0.538 0.656 4 3.981 0.580 144−156 An21 52 0.788 0.763 10 9.923 1.111 0.730 176−212 An52 52 0.654 0.623 7 6.961 0.500 0.570 189−217 An63 52 0.596 0.563 5 5.000 0.516 172−184 An68 51 0.686 0.787 7 7.000 0.778 0.747 160−178* An70 52 0.519 0.531 4 4.000 0.211 0.475 141−179 An76 52 0.673 0.663 5 5.000 0.385 0.613 262−288 An86 52 0.712 0.748 9 8.923 0.750 0.709 232−280 An95 52 0.481 0.584 5 4.981 0.556 0.496 160−178 An98 52 0.577 0.675 5 5.000 0.294 0.606 166−200 An114 51 0.392 0.788 9 9.000 0.752 144−220* hwb03 52 0.731 0.635 7 6.942 0.467 0.560 172−202 hwb08 52 0.558 0.658 5 4.962 0.250 0.580 261−301 hwb16 52 0.692 0.777 8 8.000 0.267 0.733 236−296 Mean 0.632 0.676 6.308 6.283 0.502

Note. N: ample size; Ho: observed heterozygosity; He: expected heterozygosity; A: observed allele; Ar: allelic richness; PIC: polymorphism information content. The calculation of mean values excluded the loci An63 and An114. *Indicates significant deviations from HWE after standard Bonferroni correction (p < 0.05). An10 was excluded from the calculation of M value due to the presence of different repeat unit size. ZHAI et al. | 705

distribution of the two populations. The value of Fst between the TABLE 3 Specific individuals that participated in the two populations was −0.01677 (p = 0.99707 > 0.05), suggesting no construction of half‐sibling relationships between the two populations genetic differentiation between them. DK YLL Probability 3.2 | SSR marker DK33 YLL36 0.923 DK118 YLL4 0.810 3.2.1 | Characteristics of SSR loci DK124 YLL15 0.793 DK126 YLL46 0.751 The same 116 samples and 15 loci were used for SSR analysis. All DK139 YLL19 0.790 the loci were polymorphic in the two populations and easily scored. After Bonferroni correction, significant deviations from HWE were DK159 YLL19 0.912 found at locus An63 and An114 in the DK population, An68 and DK41 YLL14 0.757 An114 in the YLL population. All significant deviations from HWE DK42 YLL44 0.832 resulted from a deficit of heterozygotes (Table 2). No LD was found DK45 YLL44 0.908 among SSR loci pairs after Bonferroni correction. Micro‐Checker DK56 YLL44 0.934 detected no large allele dropout across all loci in the two popula‐ DK56 YLL61 0.759 tions. However, locus An63 suffered from possible scoring errors due to stutters in the DK population. And locus An63, An114 in the DK population and locus An114 in the YLL population showed 4 | DISCUSSION an excess of homozygotes, indicating the possibility of null alleles. Therefore, due to the possible presence of null alleles and deviations 4.1 | Genetic diversity from HWE, loci An63 and An114 were excluded in the subsequent In the present study, cyt b gene and SSR markers were used to evalu‐ data analysis. ate the genetic diversity. For cyt b gene, the haplotype and nucleo‐ tide diversity of the two populations were lower compared to those 3.2.2 | Genetic diversity and bottleneck effect of Liu, Zhu, Wang, and Tan (2005), sampled of A. nigrocauda from the same locality with DK population in 2001, with the value of 0.812 The average number of alleles, allelic richness and observed and ex‐ and 0.44% respectively. Moreover, the haplotype frequency distri‐ pected heterozygosity of DK population were 6.692, 6.321, 0.611 bution of DK population compared with those of Liu et al. (2005) and 0.652 respectively, and for YLL population, they were 6.308, was displayed by Figure 2, it showed that nine haplotypes were 6.283, 0.632 and 0.676 respectively. The mean M values of the two lost but only increased one haplotype in the present study (The se‐ populations were 0.493 and 0.502 respectively (Table 2). The N of e quence of each haplotype of Liu et al. (2005) were downloaded from DK and YLL populations were estimated to be 66 and 58 respectively. NCBI (GenBank accession number: AY493869‐AY493886), then we recovered the data of Liu et al. (2005) by referring the haplotype 3.3 | Population differentiation frequency distribution. Because the obtained length of cyt b was only 546 bp in Liu et al. (2005), we aligned and edited our sequences The allele frequency distribution of the two populations at each to 546 bp to do a better comparsion). While using SSR as the ge‐ locus was showed in Table S3, and the value of p of Wilcoxon Signed netic marker, the average expected heterozygosities were 0.652 and Rank Test was 0.656 (p > 0.05), showing no significant difference be‐ 0.676 for the two populations respectively, it showed a lower level tween the two populations in allele frequency distribution. Similarly, of genetic diversity while compared with other freshwater fishes in for SSR analysis, the Fst value between the two populations was the upper Yangtze River (Liu et al., 2017; Zhang, Duan, Cao, Wang, 0.00259 (p = 0.81427 > 0.05), suggesting no genetic differentiation & Tan, 2011; Zhang & Tan, 2010). Three of the used loci (hwb03, between them. hwb08, hwb16) were already published in Sun et al. (2014), and we found that the average number of alleles, observed heterozygosity and expected heterozygosity of the three loci in the present study 3.4 | Sibship among the samples were smaller than those in Sun et al. (2014), which the samples were The results of sibship test of every two individuals, one from the even collected from cultured population in Wu Lake, Wuhan. DK population and the other from the YLL population, showed that For cyt b gene, the low haplotype diversity (h < 0.5) and nu‐ there was no full‐sibling relationship, but included 11 pairs of half‐ cleotide diversity (pi<0.5%) suggested that A. nigrocauda in the sibling relationships. There were 10 individuals from DK population two localities might have suffered from bottleneck effect recently and 8 individuals from YLL population participated in the construc‐ (Grant & Bowen, 1998). For SSR analysis, both the mean values of M tion of 11 pairs of half‐sibling relationships, and their matching rela‐ were lower than the threshold value (M = 0.68), suggesting the two tionships were listed in Table 3. populations had undergone a recent bottleneck. Therefore, loss of 706 | ZHAI et al.

FIGURE 2 Haplotype frequency distribution of Ancherythroculter nigrocauda in Liu et al. (2005) and the present study based on 546bp of cyt b sequences

genetic diversity and recent bottleneck effect were obviously de‐ them in flooding season. Therefore, although the adhesive eggs of tected in the two populations of A. nigrocauda in the Longxi River, it A. nigrocauda can't drift downstream, the larvae can drift down‐ might be related to damming. stream passively in the flooding season. In addition, A. nigrocauda Dams can fragment the riverine fish population and lower its ge‐ reproduces large number of offspring (absolute fecundity var‐ netic diversity by reducing effective population size, increasing genetic ied between 11,300 and 504,630 eggs, with the mean value of drift and limiting gene flow between populations (Jager, Chandler, 162,377) from April to August during the flooding season (Liu et al., Lepla, & Winkle, 2001). Lower genetic diversity had been measured 2013), making its larvae drift to the downstream from the Yulong in populations of steelhead (Oncorhynchus mykiss) (Nielsen, Hansen, Lake largely and easily. Eleven pairs of half‐sibling relationships & Loeschcke, 1997) and Sinibrama macrops (Zhao, Chenoweth, Liu, & between the two populations confirmed the presence of individ‐ Liu, 2016) isolated by dams. Of course, some other influences could ual movement and gene flow between them, which could weaken not been excluded in this study. It had been found that the degree of the genetic differentiation caused by the blocking effect of dams. fishing on A. nigrocauda in the Longxi River was over the appropriate This one‐way individual movement were supported by many stud‐ limit from 2001 to 2002 analyzed by the Yield per recruit (Gao, 2007), ies, where low‐head dams act as barriers to fish move upstream so was the year from 2011 to 2012 (Liu, 2013). Xiao and Hu (2015) but allowing individuals to drift downstream (Junker et al., 2012; indicated that the Longxi River had suffered from quite heavy pollution Meldgaard et al., 2003; Yamamoto, Morita, Koizumi, & Maekawa, because of the discharge of waste water from industry, agricultural 2004). non‐point and residency in recent years. However, some studies found that small isolated populations were prone to take place genetic drift that could increase genetic differentiation (Hänfling & Weetman, 2006; Jager et al., 2001). In the 4.2 | Population differentiation present study, the two fragmented populations are smaller than be‐ Generally speaking, dams can cause genetic differentiation among fore, especially for DK population, and recent bottleneck effects and fragmented populations due to its blocking effect on gene flow small Ne were found in the two populations, so the genetic drift will and the reduction in effective population size and increase in ge‐ be more intensified in the future if the living condition doesn't get netic drift associated with small, isolated populations (Hanfling & improvement. Over time, when the genetic drift was more influen‐ Weetman, 2006; Jager et al., 2001; Meldgaard et al., 2003). While tial than one‐way gene flow, there might be genetic differentiation our results indicated that there was no genetic differentiation be‐ between the two populations. tween the two populations, it might be attributed to dams being per‐ meable to downstream migration in the Longxi River and the high ACKNOWLEDGEMENTS level of fecundity of A. nigrocauda. It is notable that all the dams in the Longxi River are low‐head We were grateful to Baoyu Yuan who improved the English writ‐ dams that look like a waterfall with water passing through above ing and Chunchi Liu who performed the priliminary work of SSR ZHAI et al. | 707 primers screening. This study was supported by the Special Program Ecology Resources, 10, 551–555. https://doi.org/10.1111/j.1755- for Basic of Science and Technology Research (2014FY120200), the 0998.2009.02787.x Junker, J., Peter, A., Wagner, C. E., Mwaiko, S., Germann, B., Seehausen, National Natural Science Foundation of China (NSFC 31400359 and O., & Keller, I. 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