ZOOLOGICAL RESEARCH Genetic diversity and temporal changes of an endemic cyprinid fish species, Ancherythroculter nigrocauda, from the upper reaches of Yangtze River Dong-Dong Zhai1,2, Wen-Jing Li1,2, Huan-Zhang Liu1, Wen-Xuan Cao1, Xin Gao1,* 1 Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan Hubei 430072, China 2 University of Chinese Academy of Sciences, Beijing 100049, China ABSTRACT Therefore, loss of genetic diversity and increased genetic differentiation were found in the A. Small populations with low genetic diversity are nigrocauda populations, which could be attributed to prone to extinction. Knowledge on the genetic dam construction, overfishing, and water pollution in diversity and structure of small populations and their the upper Yangtze River. It is therefore genetic response to anthropogenic effects are of recommended that the government should ban critical importance for conservation management. In fishing, control water pollution, increase river this study, samples of Ancherythroculter nigrocauda, connectivity, and establish artificial breeding and an endemic cyprinid fish from the upper reaches of stocking. Yangtze River, were collected from five sites to analyze their genetic diversity and population Keywords: Genetic diversity; Population structure; structure using mitochondrial cytochrome b gene Temporal change; Conservation and 14 microsatellite loci. Haplotype diversity, nucleotide diversity, and expected heterozygosity INTRODUCTION indicated that the A. nigrocauda populations had low genetic diversity, and decreased heavily from 2001 Genetic diversity of a species determines its adaptive capacity to 2016. Significant genetic differentiation was found and evolutionary potential (Altizer et al., 2003; Pinsky & among different populations in the cyt b gene and Palumbi, 2014). Small populations of narrowly distributed SSR markers based on the genetic differentiation species often have low genetic variation within populations but index (F ), whereas no differentiation was found in ST high genetic differentiation among populations due to genetic 2001. Haplotype genealogy showed that eight out of drift and restricted gene flow (Gibson et al., 2008; Hamrick & 15 haplotypes were private to one population. The Godt, 1996; Young et al., 1996). Moreover, adverse SSR STRUCTURE analysis showed that there were anthropogenic influences can accelerate loss of genetic four genetic clusters in the A. nigrocauda samples, with each population forming a single cluster, except diversity within populations and differentiation among for the Chishui River (CSR) and Mudong River (MDR) populations, which formed a common cluster. Received: 01 December 2018; Accepted: 16 April 2019; Online: 16 May 2019 Open Access Foundation items: This work was supported by the Special Program This is an open-access article distributed under the terms of the for Basic Science and Technology Research (2014FY120200), Creative Commons Attribution Non-Commercial License (http:// Research Project of China Three Gorges Corporation (0799570), creativecommons. org/licenses/by-nc/4.0/), which permits unrestricted Research Project of Hubei Key Laboratory of Three Gorges Project for non-commercial use, distribution, and reproduction in any medium, Conservation of Fishes (SXSN / 4381), and Sino BON-Inland Water provided the original work is properly cited. Fish Diversity Observation Network Copyright ©2019 Editorial Office of Zoological Research, Kunming *Corresponding author, E-mail: [email protected] Institute of Zoology, Chinese Academy of Sciences DOI: 10.24272/j.issn.2095-8137.2019.027 Science Press Zoological Research 40(5): 427-438, 2019 427 populations (Frankham, 2002). Genetic impoverishment can analyzed the genetic diversity and population structure of fish accelerate the process of local extinction of small populations samples from the five different sites based on the cyt b gene (Hedrick & Kalinowski, 2000). An understanding of the genetic and simple sequence repeat (SSR) markers and compared variability and structure of small populations and their genetic the results with those of Liu et al. (2005). The cyt b gene was responses to anthropogenic effects is of critical importance for used in the current study for better comparison with Liu et al. conservation management (Keyghobadi, 2007; Zhang et al., (2005). The cyt b gene is part of the mitochondrial genome, 2007) and for formulating the appropriate scales and subunits whereas SSR loci are distributed on genomes and have (Moritz, 1999) for sustainable long-term conservation. advantages of high polymorphism and codominance, The Yangtze River is the largest river in China and the third therefore, the combination of cyt b gene and SSR markers is longest river in the world, with a total length of 6 300 km and a a powerful tool in studies on population genetics. This study 2 drainage area of 1 800 000 km . The Yangtze River supports aimed to determine the genetic diversity and population 378 fish species, of which 162 are endemic (Yu et al., 2005), structure, as well as temporal changes, of A. nigrocauda in the representing the highest fish diversity in the Palearctic region upper Yangtze River, and provide important information for the (Matthews, 1998). The upper Yangtze River refers to the conservation of this species. reach above Yichang City and reportedly contains 124 endemic fish species (Cao, 2011). Therefore, the upper Yangtze River is a crucial area for the conservation of fish MATERIALS AND METHODS diversity and genetic resources. However, due to dam construction, overfishing, and water pollution, fish diversity in Samples collection and DNA extraction this region has decreased sharply and many species have From 2016 to 2017, a total of 239 A. nigrocauda samples become endangered (Fu et al., 2003; Park et al., 2003; Zhong & were collected from five localities (Longxi River (LXR), Chishui Power, 1996). River (CSR), Mudong River (MDR), Modao Stream (MDS), Ancherythroculter nigrocauda, belonging to Cyprinidae in and Daning River (DNR)) in the upper Yangtze River (Figure Cypriniformes, is an important commercial and aquaculture 1, Table 1). Dorsal muscle used for DNA extraction was species in China and an endemic fish from the upper Yangtze clipped from each of the fish, and then preserved in 95% River, where it predominantly inhabits the main river and its alcohol in 5 mL cryogenic vials and stored at 20 ° C. Total − tributaries (Ding, 1994). This species is sedentary and lays DNA was extracted from alcohol preserved muscle tissue adhesive eggs from April to August during the rainy season using proteinase K digestion at 55 °C for 3–5 h, followed by (Cao et al., 2007; Liu et al., 2013). The minimum age of phenol/chloroform extraction (Kocher et al., 1989). sexual maturity of A. nigrocauda is one year, and the body length at 50% sexual maturity estimated to be 106 and 125 mm Table 1 Sampling sites, sample site codes, GPS locations, and for males and females, respectively (Liu et al., 2013). Its samples sizes ofA. nigrocauda in the upper Yangtze River, China absolute fecundity varies from 11 300 to 504 630 eggs, with a Sample Sample site Code GPS location (NE) mean of 162 377 eggs (Liu et al., 2013). In recent years, size (n) however, the natural populations and distribution areas of A. Longxi River LXR 28°54′ 46″ 105°30′ 51″ 64 nigrocauda have declined significantly (Liu, 2013). While Chishui River CSR 28°48′ 03″ 105°50′ 22″ 38 previous studies have reported on the age and growth (Xue & He, 2001), reproductive biology (Liu et al., 2013), and artificial Mudong River MDR 29°33′ 56″ 106°50′ 16″ 36 propagation (Tan et al., 2004; Yin & Lv, 2010) of A. Modao Stream MDS 30°49′ 04″ 108°51′ 46″ 39 nigrocauda, very little is known about the genetic diversity and Daning River DNR 31°16′ 37″ 109°49′ 00″ 62 structure of populations in the upper Yangtze River. Liu et al. (2005) conducted a study on the genetic diversity and mtDNA amplification and sequencing population structure of A. nigrocauda with samples collected All 239 samples were used for mtDNA amplification. The from 2001 to 2002 in the upper Yangtze River, and found high mtDNA cyt b gene was amplified using polymerase chain genetic diversity and no genetic differentiations among reaction (PCR) in 30 µL reactions containing 3 µL of reaction different geographical populations. However, that study had a limited sample size (43 samples from three localities) and only buffer (200 mmol/L Tris-HCL pH 8.4, 500 mmol/L KCL, recovered a 546 bp fragment of the cytochrome b (cyt b) 50 mmol/L MgCL), 1.5 µL of dNTPs (1 mmol/L), 1 µL of each gene. Moreover, due to a sharp decline in the natural primer (10 µmol/L), 0.25 µL (2.5 U) of Taq DNA polymerase, populations of A. nigrocauda over the past few decades and 3 µL of template DNA, and 20.25 µL of H2O. Primer sets were substantial environmental changes in the upper Yangtze River L14724 5'- GACTTGAAAAACCACCGTTG-3' and H15915 5'- after the impoundment of the Three Gorges Dam (TGD), it is CTCCGATCT CCGGATTACAAGAC-3' (Xiao et al., 2001). The likely that both the genetic diversity and population structure PCR profile was initial denaturation at 94 ° C for 4 min; of A. nigrocauda in the upper Yangtze River have been followed by 35 cycles at 94 °C for 45 s, 54 °C for 45 s, and impacted. 72 °C for 1 min; then one cycle at 72 °C for 10 min. The PCR In the current study, samples of A. nigrocauda were products were purified and sequenced by Shanghai DNA collected from five sites in the upper Yangtze River. We Biotechnologies Company. 428 www.zoores.ac.cn Figure 1 Sampling sites (stars) of A. nigrocauda in upper Yangtze River LXR: Longxi River; CSR: Chishui River; MDR: Mudong River; MDS: Modao Stream; DNR: Daning River. SSR amplification and electrophoresis 2005) to test for departure from neutrality due to population A total of 161 samples from the five localities (sample size of expansion or selection.