DNA barcoding and evaluation of genetic diversity in Cyprinidae fish in the midstream of the Yangtze River Yanjun Shen1,2, Lihong Guan3, Dengqiang Wang4 & Xiaoni Gan1 1The Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, Hubei, China 2University of Chinese Academy of Sciences, Beijing 100039, China 3College of Life Science and Technology, Xinxiang Medical University, He’nan Xinxiang 453003, China 4The Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China Keywords Abstract Cyprinidae, cytochrome c oxidase subunit I, DNA barcoding, identification, Kimura’s The Yangtze River is the longest river in China and is divided into upstream two-parameter. and mid-downstream regions by the Three Gorges (the natural barriers of the Yangtze River), resulting in a complex distribution of fish. Dramatic changes to Correspondence habitat environments may ultimately threaten fish survival; thus, it is necessary Dengqiang Wang, The Key Laboratory of to evaluate the genetic diversity and propose protective measures. Species iden- Freshwater Biodiversity Conservation, Ministry tification is the most significant task in many fields of biological research and in of Agriculture, Yangtze River Fisheries Research Institute, Chinese Academy of conservation efforts. DNA barcoding, which constitutes the analysis of a short Fishery Sciences, Wuhan, China. fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence, Tel: 027-81780116; has been widely used for species identification. In this study, we collected 561 Fax: 027-81780088; COI barcode sequences from 35 fish from the midstream of the Yangtze River. E-mail: wdq@yfi.ac.cn The intraspecific distances of all species were below 2% (with the exception of and Acheilognathus macropterus and Hemibarbus maculatus). Nevertheless, all species Xiaoni Gan, The Key Laboratory of Aquatic could be unambiguously identified from the trees, barcoding gaps and taxo- Biodiversity and Conservation of Chinese nomic resolution ratio values. Furthermore, the COI barcode diversity was Academy of Sciences, Institute of ≤ Hydrobiology, Chinese Academy of Sciences, found to be low ( 0.5%), with the exception of H. maculatus (0.87%), Wuhan 430072, Hubei, China. A. macropterus (2.02%) and Saurogobio dabryi (0.82%). No or few shared hap- Tel: 027-68780089; lotypes were detected between the upstream and downstream populations for Fax: 027-68780071; ten species with overall nucleotide diversities greater than 0.00%, which indi- E-mail: [email protected] cated the likelihood of significant population genetic structuring. Our analyses indicated that DNA barcoding is an effective tool for the identification of cypri- Funding information nidae fish in the midstream of the Yangtze River. It is vital that some protective This work was funded by the Key Laboratory of Freshwater Biodiversity Conservation, measures be taken immediately because of the low COI barcode diversity. Ministry of Agriculture. Received: 14 October 2015; Revised: 31 January 2016; Accepted: 2 February 2016 doi: 10.1002/ece3.2060 Introduction et al. 2001). The Yangtze River is divided into upstream and mid-downstream regions by the Three Gorges (TG; The Yangtze River is the longest river in China. It origi- the natural barriers of the Yangtze River), resulting in a nates from the Tibetan Plateau at an elevation higher complex distribution of fish (Wang et al. 2004). Nearly than 5,000 m, flows first south, then north and northeast, 300 species are estimated to live in the tributaries and and finally east to reach the coast, 6,300 km away (Chen main stream. Cyprinidae are the predominant family of ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. DNA Barcoding for the Yangtze River Cyprinidae Y. Shen et al. fish in the Yangtze River, representing 54.02% of all spe- et al. 2013). K2P is computationally fast and yields consis- cies (Institute of Hydrobiology 1976). However, many tent results for many species that exhibit the necessary dis- cyprinidae fish populations have been disturbed by parity between intra- and interspecific variation. However, human activities. For example, the number of Coreius the use of the K2P distance in barcode analyses has been guichenoti in the Yangtze River has declined significantly challenged and the p-distance has been proposed to be a due to environmental pollution, overexploitation, and better model (Collins et al. 2012; Srivathsan and Meier construction of electrical projects (Duan et al. 2002). 2012). The lack of overlap between intra- and interspecific Today the situation may be even worse, especially after variation (dubbed the “barcoding gap”) has been deemed the construction of the Three Gorges Dam (TGD) in the to be of paramount importance for the accuracy and relia- middle of the Yangtze River in 2009. The environments bility of barcode genes (Meyer and Paulay 2005) and can for habitats and spawning fields have dramatically chan- be influenced by distance models (Collins et al. 2012; Sri- ged, particularly for downstream fish; these changes may vathsan and Meier 2012). Hebert et al. (2004) have ultimately threaten the survival of fish because the TGD defined the “barcoding gap” as the existence of at least a blocks natural fish migration patterns. Therefore, it is 10 times greater average interspecific distance than average necessary to evaluate the genetic diversity and propose intraspecific genetic distance. Therefore, in the present protective measures for cyprinidae fish in the midstream study, both the K2P and p-distance models were used in of the Yangtze River. the barcoding gap analysis. Species identification is the most significant task in The present study explored the utility of the DNA bar- many fields of biological research and conservation coding approach as a molecular technique for the identifi- efforts. Traditional morphological identification is not cation of Cyprinidae fish in the midstream of the Yangtze fully effective for eggs, fry, and adults lacking distinctive River and evaluated the identification success rates based morphological characteristics. Moreover, the number of on the K2P and p-distance models. Furthermore, a pre- specialists in alpha taxonomy is insufficient for conve- liminary genetic diversity analysis of COI was performed nient and complex morphological identification (Carvalho for some fish species to tentatively provide important et al. 2011, 2015). Thus, rapid, reliable, and reproducible information for the conservation of the Cyprinidae fish molecular tests to identify fish species are needed in many resource in the midstream of the Yangtze River. areas (Rasmussen and Morrissey 2009; Steinke et al. 2009). One proposed method is DNA barcoding, which Materials and Methods uses the mtDNA gene cytochrome oxidase subunit I (COI) as a global DNA barcoding identification system The experiments were performed in accordance with the for animals (Hebert et al. 2003a,b). Sequences for the Ethics Committee of the Institute of Hydrobiology at the same species are generally considered to be correctly iden- Chinese Academy of Sciences. The policies were enacted tified when they form a monophyletic cluster on a neigh- according to the Chinese Association for Laboratory bor-joining (NJ) tree with intraspecific distances that are Animal Sciences and the Institutional Animal Care and below a given threshold (Srivathsan and Meier 2012). At Use Committee (IACUC) protocols. present, this approach has proven to be highly efficient and reliable in many fish groups (Ward et al. 2005; Sample collection and morphological Hubert et al. 2008, 2010; Rock et al. 2008; Keskin et al. identification 2013; Loh et al. 2014) and is regularly used for a variety of applications, such as fishery management, biodiversity In 2011, a total of 561 samples from 35 species, 25 genera assessments and conservation (Triantafyllidis et al. 2011; and eight subfamilies of cyprinidae (Table S1) were col- Weigt et al. 2012; Keskin et al. 2013; Loh et al. 2014; lected from ten different sites in the midstream of the Shen et al. 2016). Yangtze River (Fig. 1). In most cases, the specimens were Species identification with DNA barcodes is reliable only obtained from research vessel trawling surveys conducted if a significant difference between the average intraspecific in multiple zones of the Yangtze River to inform fishery and the average interspecific genetic distances can be con- management of the status of fish stocks. Morphological sistently detected (Hebert et al. 2003a,b, 2004; Ward et al. identification was performed in situ by visual inspection, 2005). The use of Kimura’s two-parameter (K2P) model and the fish were taxonomically classified by employing (Kimura 1980) in DNA barcoding studies began with standard guides referencing Fauna Sinica (Chen 1998) (Hebert et al. 2003a,b) and is now widely used to assign and the FishBase databases (Froese and Pauly 2015). As an unknown specimen to a known species, to detect novel many individuals per species as possible were obtained sequences, and to determine whether an unknown for this study. However, in some cases, only one or two specimen is a distinct new species (Pereira et al. 2011; Hsu individuals per region per species could be collected, 2 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Y. Shen et al. DNA Barcoding for the Yangtze River Cyprinidae which precluded accurate calculations of population 0.375 lL of Taq DNA polymerase (2.5 U/lL, TaKaRa Bio, parameters (e.g., genetic diversity). Therefore, population Shanghai, China), and 1.0 lL of the DNA template (50– estimates were not made for these species, but this short- 100 ng/lL). The PCR amplification conditions were as fol- coming is unlikely to have affected the main conclusions lows: 94°C for 5 min, 32 cycles at 94°C for 30 s, 53°C for of this study.