Preliminary Study on Environmental DNA Metabarcoding for Detecting Biodiversity in the Middle and Lower Reaches of the Yangtze River
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摘要: 长江生物多样性在人为影响下面临严重威胁，物种监测是生物多样性保护的基础，为完善长江水生态监测体系，实现高效无损伤的物种监测，在长江中下游干流3个江段（新滩、安庆和芜湖）采集水样，建立长江水样环境DNA宏条形码物种检测体系并评估其有效性.结果表明：①长江中下游环境DNA宏条形码检测到32个物种，包括20种鱼类、1种水生哺乳动物（长江江豚）和11种陆生动物，其中鱼类物种包括鲤形目、鲇形目、鲈形目和鲱形目，其种数占鱼类总种数的比例分别为60%、25%、10%和5%.②长江中下游渔获物中资源量居首位的鲤形目在环境DNA调查中序列数最多，占鱼类总序列的96.2%，其次为鲱形目（占比为3.5%），鲇形目和鲈形目占比较低，分别为0.2%和0.1%，4个类目序列相对丰度与渔获物种资源量组成差异较大.③环境DNA调查次数约占传统渔获物调查次数的几十至几百分之一，采样时间不足努力量最少的渔获物调查的1%，检测到的鱼类种数为传统调查总数的31%~49%.④安庆采样点位于长江中下游长江江豚密度最高的江段，其环境DNA检出率和序列相对丰度在3个采样点中均最高.研究显示：长江水样环境DNA包含水陆复合生态系统的生物多样性信息，利用水样环境DNA宏条形码可检测不同类群的水生和陆生物种；对于鱼类物种检测，环境DNA宏条形码比传统调查方法效率更高，可对传统调查结果进行补充；环境DNA宏条形码生物多样性检测主要受分子标记体系和核酸序列数据库限制，获取全面的物种多样性和资源量信息需要对检测分析方法进行进一步完善.Abstract: Biodiversity of the Yangtze River is facing a serious threat under the influence of human activities. Species investigation is the basis of biodiversity protection, and an efficient monitoring system is urgently needed to fully understand the current status of biodiversity. In order to improve the water ecological monitoring system of the Yangtze River and achieve efficient and non-invasive species monitoring, water samples were collected from three sections (Xintan, Anqing and Wuhu) in the mainstream of the middle and lower reaches of the Yangtze River, and the species detection system of environmental DNA metabarcoding in water samples was established and its effectiveness was evaluated in this study. The results showed that: (1) Through water enviromental DNA metabarcoding in the middle and lower reaches of the Yangtze River, 32 species, including 20 fish, 1 aquatic mammal (Yangtze finless porpoise) and 11 terrestrial animals, were detected. The detected fish species belong to the order of Cypriniformes, Siluriformes, Perciformes and Clupeiformes. The proportions of species in each order are 60%, 25%, 10% and 5%. (2) The most abundant order in catches, Cypriniformes, has the largest number of sequences in the eDNA survey, accounting for 96.2% of the total fish sequences, followed by Clupeiformes, Siluriformes and Perciformes, accounting for 3.5%, 0.2% and 0.1%, respectively. (3) The number of environmental DNA sampling accounts for tens to hundreds of times of traditional catches, and the sampling duration is less than 1% of the fishing investigation with minimal effort, while the number of fish species detected is 31%-49% of the total number of traditional investigation. (4) The Anqing sampling site is located in the section with the highest density of Yangtze finless porpoise in the middle and lower reaches of the Yangtze River, and the detection rate of environmental DNA and the relative abundance of sequence of Anqing are the highest at the three sampling sites. The research indicated that the environmental DNA of the Yangtze River contains the biodiversity information of aquatic-terrestrial complex ecosystem. Environmental DNA metabarcoding of water samples can detect different groups of aquatic and terrestrial species. For detection of fish species, environmental DNA metabarcoding is more efficient than the traditional investigation method and can supplement the traditional investigation results. Biodiversity analysis using environmental DNA metabarcoding is mainly limited by molecular marker system and nucleic acid sequence database. To obtain comprehensive species diversity and resource information, the detection and analysis methods need to be further improved.
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图 1 长江中下游水样环境DNA宏条形码检测种类组成
Figure 1. The number of species detected from water samples in the middle and lower reaches of the Yangtze River using environmental DNA metabarcoding
图 2 基于OTU水平的样本层级聚类树
注：1、2、3表示采样点样本序号.下同.
Figure 2. Hierarchical clustering tree on OTU level
图 5 长江中下游干流3个采样点位置和长江江豚环境DNA序列相对丰度
注：传统调查分布密度来源于农业农村部发布的2017年长江江豚科学考察结果[43].
Figure 5. Location of the three sampling sites in the middle and lower reaches of the Yangtze River mainstream and relative abundance of environmental DNA sequences of Neophocaena asiaeorientalis asiaeorientalis
表 1 长江中下游3个江段水样环境DNA宏条形码注释物种
Table 1. Species annotation of environmental DNA metabarcoding in water samples from three sections of the middle and lower reaches of the Yangtze River
物种名称 序号 物种 新滩江段 安庆江段 芜湖江段 辐鳍鱼纲
(Actinopterygii)1 鲢(Hypophthalmichthys molitrix) + + + 2 鳙(Hypophthalmichthys nobilis) + + + 3 团头鲂(Megalobrama amblycephala) + + + 4 青鱼(Mylopharyngodon piceus) + + + 5 草鱼(Ctenopharyngodon idella) + + + 6 鲤(Cyprinus carpio) + + + 7 鲫(Carassius auratus) + + + 8 翘嘴鲌(Culter alburnus) + + 9 红鳍原鲌(Chanodichthys ilishaeformis) + + + 10 达氏鲌(Chanodichthys dabryi) + + + 11 (Hemiculter leucisculus) + + + 12 寡鳞飘鱼(Pseudolaubuca engraulis) + 13 黄颡鱼(Tachysurus fulvidraco) + + 14 光泽黄颡鱼(Tachysurus nitidus) + + 15 长吻鮠(Leiocassis longirostris) + 16 大口鲇(Silurus meridionalis) + + 17 鲇(Silurus asotus) + + 18 鳜(Siniperca chuatsi) + 19 乌鳢(Channa argus) + + 20 短颌鲚(Thryssa kammalensis) + + 哺乳纲(Mammalia) 21 长江江豚(Neophocaena asiaeorientalis asiaeorientalis) + + 22 猪(Sus scrofa) + + 23 水牛(Bubalus bubalis) + 24 牛(Bos taurus) + + 25 山羊(Capra hircus) + + 26 绵羊(Ovis aries) + + 27 褐家鼠(Rattus norvegicus) + + + 28 人(Homo sapiens) + + + 鸟纲(Aves) 29 鸿雁(Anser cygnoides) + + 30 番鸭(Cairina moschata) + + 31 鹌鹑(Coturnix japonica) + 32 鸡(Gallus gallus) + + + 注：+表示采样点水样中检测到物种环境DNA。 
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表 2 长江中下游水样环境DNA宏条形码检测物种注释序列数
Table 2. vThe number of sequences for each order identified by environmental DNA metabarcoding of water samples in the middle and lower reaches of the Yangtze River
序号 物种分类 序列数 1 鲤形目 122 632 2 鸡形目 33 226 3 鲱形目 4 446 4 灵长目 923 5 偶蹄目 374 6 雁形目 279 7 鲇形目 276 8 鲈形目 169 9 啮齿目 41 10 鲸目 32
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