基于环境DNA宏条形码技术的辽东湾典型围海养殖池塘内水母多样性研究

研究使用环境DNA宏条形码技术(eDNA metabarcoding)检测辽东湾东北部河口区围海养殖池塘水母种类多样性,探索适用于水母种类物种鉴定和监测的新方法。利用环境DNA宏条形码技术,分别基于18S rDNA和COI宏条形码检测了辽东湾东北部河口区围海养殖池塘水母种类多样性,通过水样采集、过滤、eDNA提取、遗传标记扩增、测序与生物信息分析的环境DNA宏条形码标准化分析流程,从围海养殖池塘7个采样点中获得可检测的采样点数据。结果显示,基于18S rDNA宏条形码检测出8种水母种类,其中钵水母纲大型水母2种、水螅水母总纲小型水母6种;基于COI宏条形码技术共检测出19种水母种类,其中钵水母纲大型水母5种、水螅水母总纲小型水母14种;两种DNA条形码标记都显示养殖种类海蜇(Rhopilema esculentum)为优势种。研究结果表明,环境DNA宏条形码技术作为一种新兴的生物多样性监测手段可用于快速检测水母种类多样性,在水母类物种鉴定、监测及早期预警中有较大的应用潜能。     

基于eDNA技术的长江上游珍稀特有鱼类国家级自然保护区重庆段鱼类多样性研究

利用环境DNA宏条形码(environmental DNA metabarcoding;eDNA metabarcoding)检测长江上游珍稀特有鱼类国家级自然保护区重庆段鱼类多样性,探索适用于长江鱼类多样性监测和保护的新方法,为后期长江"十年禁渔"效果评估提供一定的基础资料.研究于2021年3月在保护区重庆段共设置6...     

基于环境DNA宏条形码技术的秦淮河生物多样性研究

秦淮河是南京的母亲河,其生物多样性受城市化进程影响面临严重威胁,而物种资源调研是生物多样性保护的基础。环境DNA宏条形码技术较形态学监测是一种简单高效、灵敏度高的新型监测技术。为探究秦淮河浮游生物、底栖动物及鱼类的生物多样性,于2019年7月,采用环境DNA宏条形码技术对其进行了探究,并分析了秦淮河上下游间的差异及环境因子对其群落结构的影响。结果表明:秦淮河共监测到浮游动物13属22种407个操作分类单元(Operational Taxonomic Units, OTUs),浮游植物85属60种4445个OTUs,底栖动物16属17种212个OTUs,鱼类53属44种1663个OTUs。其中浮游动物以游泳轮虫目(Ploima)和双甲目(Diplostraca)为主,共占浮游动物63.37%,浮游植物以隐藻门(Cryptomonas)和褐藻门(Ochrophyta)为主,共占浮游植物88.11%,底栖动物中节肢动物门(Arthropoda)占比最高,达91.67%,鱼类中鲤形目(Cypriniformes)占比最高,达69.99%。与秦淮河历史形态学监测数据相比,环境DNA宏条形码技术在...     

基于eDNA metabarcoding探究北京市主要河流鱼类分布及影响因素

使用eDNA宏条形码(eDNA metabarcoding)和地笼法检测了北京市3条水系在夏季和秋季两个季节的鱼类多样性, 旨在研究北京市鱼类群落的空间格局特征, 探索适用于北京鱼类生物多样性监测及保护的新方法。结果表明: 在北京市的34个采样点中, 利用eDNA metabarcoding共检测出鱼类55种, 显著高于传统方法所捕获的鱼类种数(35种), 鱼类组成以鲤形目和鲈形目为主。山区河流清水鱼的多样性要显著高于城区河流, 城区河流(北运河水系)群落结构较为单一, 以鲫(Carassius auratus)、麦穗鱼(Pseudorasbora parva)、泥鳅(Misgurnus anguillicaudatus)等耐污种为优势种; 山区河流(潮白河水系及大清河水系)以宽鳍鱲(Zacco platypus)、拉氏鱥(Rhynchocypris lagowskii)、马口鱼(Opsariichthys uncirostris)等为优势种。不同季节影响清水鱼群落结构的环境因子不同, 夏季主要是总溶解固体和电导率, 秋季主要是海拔和温度。清水鱼丰富度与环境因子及人类活动的相关性表明, 清水鱼的丰富度随着总溶解固体及灯光指数增加而显著降低, 且均与海拔、温度等存在显著相关性。本研究证明了eDNA metabarcoding方法用于监测北京市鱼类多样性及其时空分布的可行性。     

环境DNA技术在长江口鱼类多样性分析中的应用

研究采用高通量测序技术对长江口水域环境DNA(Environmental DNA, eDNA)样品进行分析,并与传统渔业资源调查结果对比,阐述长江口鱼类群落在其生境内的多样性特征,探讨eDNA技术在长江口水域鱼类多样性研究中的应用前景。结果显示, eDNA技术共检测到10目21科41属45种鱼类,各站点鱼类丰富度之间无显著差异,而多样性之间存在显著差异性。底拖网法共捕获11目16科29属33种鱼类。有18种鱼类在两种方法中均检测到,占鱼类总数的30%。两种方法检测到的鱼类中均以鲈形目(Perciformes)最多,其次是鲤形目(Cypriniformes),两种方法的结果均表明刀鲚(Coilia nasus)和凤鲚(Coilia mystus)为优势物种。研究表明环境DNA技术在长江口水域渔业资源监测中具有可行性,在禁捕环境下可根据实际情况采用不同方法对渔业资源进行监测。     

基于环境DNA宏条码和底拖网的珠江河口鱼类多样性

文章采用环境DNA宏条码和底拖网对珠江河口鱼类多样性进行了研究, 并对两种方法进行了比较。利用环境DNA宏条码检测到了175种鱼类, 而利用底拖网采集到了47种鱼类, 结合两种方法共检测出179种鱼类, 隶属于15 目63科128属。其中两种方法共同识别了鱼类43种, 占总检测物种的24.02%, 基于底拖网的调查未能收集到基于环境DNA宏条码检测到的大多数物种。根据Shannon指数和Simpson指数显示, DNA宏条码所检测珠江河口鱼类群落α多样性显著高于底拖网方法(P<0.05)。两种方法的PCoA结果均显示珠江河口鱼类群落存在空间结构, 基于环境DNA宏条码的分析显示空间重叠更多。两种方法基于冗余分析均显示溶解氧和盐度是影响鱼类群落结构的主要环境因子。研究表明, 环境DNA 宏条形码是一种环保且可靠的评估方法, 将其搭载到现有调查可以更好地了解河口鱼类多样性。     

环境DNA宏条形码在生物多样性研究与监测中的应用

环境DNA宏条形码(eDNA metabarcoding)技术通过提取水体、土壤、空气中的环境DNA,使用引物PCR扩增与高通量测序,进行物种鉴定与生物多样性评估.作为一种新的监测技术,相比于传统监测技术更加快捷、准确以及对自然环境的破坏小,因此在一定程度上改变了我们调查地球生物多样性的方式.本文综述了环境DNA宏条形...     

DNA宏条形码技术在海洋浮游动物多样性和生态学研究中的应用

浮游动物是海洋生态系统的关键类群,其覆盖门类广泛,多样性高。传统形态鉴定技术需要检测人员具备专业的形态鉴定知识,且费时费力。宏条形码技术无需分离生物个体,而是提取拖网采集到的浮游动物混合样本的总DNA,或者水体中的环境DNA （eDNA）,依托高通量测序平台测序,能够实现对大规模样本快速、准确、经济的分析,在海洋浮游动物生态学研究中得到越来越广泛的应用。分析了DNA宏条形码技术常用的核糖体和线粒体分子标记,在浮游动物多样性和数量研究中的可靠性和不足,并给出在海洋浮游动物群落监测,食物关系分析及生物入侵早期预警等研究中的应用。未来,开发多基因片段组合条形码,发展完备的参考数据库及实现准确的量化研究是DNA宏条形码技术发展的重要方向。     

AeDNA: 水生生物eDNA数据库

环境 DNA (eDNA) 技术是一种生态和生物多样性监测和评价的新手段, 完整和准确的参考序列库是eDNA技术应用于水生生物多样性调查的基础。当前, 不同水生生物eDNA参考序列还存在诸多问题, 如不同类群使用的标记基因不同且资源较为分散, 部分参考序列分类不准确, 以及针对我国各类水体中水生生物eDNA参考序列不多等。针对上述问题, 研究构建了水生生物eDNA数据库(AeDNA, http://aedna.ihb.ac.cn/)。 AeDNA整合了DNA条形码和基因组两种类型参考序列。其中18S、28S、ITS、COΙ、12S、rbcL 等各类DNA条形码60余万条, 涉及2万余种鱼类、1万余种水生植物、1万余种底栖动物、1万余种浮游动物和1万余种浮游植物; 基因组包含线粒体、叶绿体等细胞器基因组6199个及万种鱼类基因组计划和万种原生生物基因组计划所产生的物种基因组。涉及的生境有江、河、湖、海、冰川和温泉等各类水环境, 尤其数据库构建团队贡献的6万余条参考序列, 具有我国丰富的各类水体生境信息。总体来说, AeDNA是一个数据量大、类群覆盖全、准确性高且具有我国水生生物特色的综合性eDNA参考序列库, 是水生态和水生生物多样性监测的重要基础资源。     

环境DNA技术在淡水底栖大型无脊椎动物多样性监测中的应用

环境DNA (eDNA)是指生物有机体在环境中(例如土壤、沉积物或水体)遗留下的DNA片段。eDNA技术是指从环境中提取DNA片段进行测序以及数据分析来反映环境中的物种或群落信息。与传统方法相比, eDNA技术具有高灵敏度、省时省力、无损伤等优点。目前, eDNA技术已成为一种新的水生生物监测方法, 主要应用于水生生物的多样性研究、濒危和稀有动物的物种状态及外来入侵动物扩散动态的监测等。本文从eDNA技术在水生生物多样性监测应用领域的发展历程、eDNA技术的操作流程以及其在监测淡水底栖大型无脊椎动物方面的应用进展、技术优势和局限性五个方面进行了综述。最后, 本文对eDNA技术在淡水底栖大型无脊椎动物多样性监测应用的发展趋势和前景作出展望。     

Marine water environmental DNA metabarcoding provides a comprehensive fish diversity assessment and reveals spatial patterns in a large oceanic area

Current methods for monitoring marine fish (including bony fishes and elasmobranchs) diversity mostly rely on trawling surveys, which are invasive, costly, and time‐consuming. Moreover, these methods are selective, targeting a subset of species at the time, and can be inaccessible to certain areas. Here, we used environmental DNA (eDNA), the DNA present in the water column as part of shed cells, tissues, or mucus, to provide comprehensive information about fish diversity in a large marine area. Further, eDNA results were compared to the fish diversity obtained in pelagic trawls. A total of 44 5 L‐water samples were collected onboard a wide‐scale oceanographic survey covering about 120,000 square kilometers in Northeast Atlantic Ocean. A short region of the 12S rRNA gene was amplified and sequenced through metabarcoding generating almost 3.5 million quality‐filtered reads. Trawl and eDNA samples resulted in the same most abundant species (European anchovy, European pilchard, Atlantic mackerel, and blue whiting), but eDNA metabarcoding resulted in more detected bony fish and elasmobranch species (116) than trawling (16). Although an overall correlation between fishes biomass and number of reads was observed, some species deviated from the common trend, which could be explained by inherent biases of each of the methods. Species distribution patterns inferred from eDNA metabarcoding data coincided with current ecological knowledge of the species, suggesting that eDNA has the potential to draw sound ecological conclusions that can contribute to fish surveillance programs. Our results support eDNA metabarcoding for broad‐scale marine fish diversity monitoring in the context of Directives such as the Common Fisheries Policy or the Marine Strategy Framework Directive.     

Unlocking biodiversity and conservation studies in high‐diversity environments using environmental DNA (eDNA): A test with Guianese freshwater fishes

Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and nondestructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large‐scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity.     

Comparative analysis of fish environmental DNA reveals higher sensitivity achieved through targeted sequence-based metabarcoding

Environmental DNA (eDNA)-based methods of species detection are enabling various applications in ecology and conservation including large-scale biomonitoring efforts. qPCR is widely used as the standard approach for species-specific detection, often targeting a fish species of interest from aquatic eDNA. However, DNA metabarcoding has the potential to displace qPCR in certain eDNA applications. In this study, we compare the sensitivity of the latest Illumina NovaSeq 6000 NGS platform to qPCR TaqMan assays by measuring limits of detection and by analysing eDNA from water samples collected from Churchill River and Lake Melville, NL, Canada. Species-specific, targeted next generation sequencing (NGS) assays had significantly higher sensitivity than qPCR, with limits of detection 14- to 29-fold lower. For example, when analysing eDNA, qPCR detected Gadus ogac (Greenland cod) in 21% of samples, but targeted NGS detected this species in 29% of samples. General NGS assays were as sensitive as qPCR, while simultaneously detecting 15 fish species from eDNA samples. With over 34,000 fish species on the planet, parallel and sensitive methods such as NGS will be required to support effective biomonitoring at both regional and global scales.     

Environmental DNA metabarcoding of lake fish communities reflects long‐term data from established survey methods

Organisms continuously release DNA into their environments via shed cells, excreta, gametes and decaying material. Analysis of this ‘environmental DNA’ (eDNA) is revolutionizing biodiversity monitoring. eDNA outperforms many established survey methods for targeted detection of single species, but few studies have investigated how well eDNA reflects whole communities of organisms in natural environments. We investigated whether eDNA can recover accurate qualitative and quantitative information about fish communities in large lakes, by comparison to the most comprehensive long‐term gill‐net data set available in the UK. Seventy‐eight 2L water samples were collected along depth profile transects, gill‐net sites and from the shoreline in three large, deep lakes (Windermere, Bassenthwaite Lake and Derwent Water) in the English Lake District. Water samples were assayed by eDNA metabarcoding of the mitochondrial 12S and cytochrome b regions. Fourteen of the 16 species historically recorded in Windermere were detected using eDNA, compared to four species in the most recent gill‐net survey, demonstrating eDNA is extremely sensitive for detecting species. A key question for biodiversity monitoring is whether eDNA can accurately estimate abundance. To test this, we used the number of sequence reads per species and the proportion of sampling sites in which a species was detected with eDNA (i.e. site occupancy) as proxies for abundance. eDNA abundance data consistently correlated with rank abundance estimates from established surveys. These results demonstrate that eDNA metabarcoding can describe fish communities in large lakes, both qualitatively and quantitatively, and has great potential as a complementary tool to established monitoring methods.     

Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding

Freshwater fauna are particularly sensitive to environmental change and disturbance. Management agencies frequently use fish and amphibian biodiversity as indicators of ecosystem health and a way to prioritize and assess management strategies. Traditional aquatic bioassessment that relies on capture of organisms via nets, traps and electrofishing gear typically has low detection probabilities for rare species and can injure individuals of protected species. Our objective was to determine whether environmental DNA (eDNA) sampling and metabarcoding analysis can be used to accurately measure species diversity in aquatic assemblages with differing structures. We manipulated the density and relative abundance of eight fish and one amphibian species in replicated 206‐L mesocosms. Environmental DNA was filtered from water samples, and six mitochondrial gene fragments were Illumina‐sequenced to measure species diversity in each mesocosm. Metabarcoding detected all nine species in all treatment replicates. Additionally, we found a modest, but positive relationship between species abundance and sequencing read abundance. Our results illustrate the potential for eDNA sampling and metabarcoding approaches to improve quantification of aquatic species diversity in natural environments and point the way towards using eDNA metabarcoding as an index of macrofaunal species abundance.     

Environmental DNA metabarcoding primers for freshwater fish detection and quantification: In silico and in tanks

Environmental DNA (eDNA) techniques refer to utilizing the organisms’ DNA extracted from environment samples to genetically identify target species without capturing actual organisms. eDNA metabarcoding via high‐throughput sequencing can simultaneously detect multiple fish species from a single water sample, which is a powerful tool for the qualitative detection and quantitative estimates of multiple fish species. However, sequence counts obtained from eDNA metabarcoding may be influenced by many factors, of which primer bias is one of the foremost causes of methodological error. The performance of 18 primer pairs for COI, cytb, 12S rRNA, and 16S rRNA mitochondrial genes, which are all frequently used in fish eDNA metabarcoding, were evaluated in the current study. The ribosomal gene markers performed better than the protein‐coding gene markers during in silico screening, resulting in higher taxonomic coverage and appropriate barcode lengths. Four primer pairs—AcMDB07, MiFish‐U, Ve16S1, and Ve16S3—designed for various regions of the 12S and 16S rRNA genes were screened for tank metabarcoding in a case study targeting six freshwater fish species. The four primer pairs were able to accurately detect all six species in different tanks, while only MiFish‐U, Ve16S1, and Ve16S3 revealed a significant positive relationship between species biomass and read count for the pooled tank data. The positive relationship could not be found in all species within the tanks. Additionally, primer efficiency differed depending on the species while primer preferential species varied in different fish assemblages. This case study supports the potential for eDNA metabarcoding to assess species diversity in natural ecosystems and provides an alternative strategy to evaluate the performance of candidate primers before application of eDNA metabarcoding in natural ecosystems.