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...     

Making environmental DNA count

The arc of reception for a new technology or method – like the reception of new information itself – can pass through predictable stages, with audiences’ responses evolving from ‘I don't believe it’, through ‘well, maybe’ to ‘yes, everyone knows that’ to, finally, ‘old news’. The idea that one can sample a volume of water, sequence DNA out of it, and report what species are living nearby has experienced roughly this series of responses among biologists, beginning with the microbial biologists who developed genetic techniques to reveal the unseen microbiome. ‘Macrobial’ biologists and ecologists – those accustomed to dealing with species they can see and count – have been slower to adopt such molecular survey techniques, in part because of the uncertain relationship between the number of recovered DNA sequences and the abundance of whole organisms in the sampled environment. In this issue of Molecular Ecology Resources, Evans et al. ( 2015 ) quantify this relationship for a suite of nine vertebrate species consisting of eight fish and one amphibian. Having detected all of the species present with a molecular toolbox of six primer sets, they consistently find DNA abundances are associated with species’ biomasses. The strength and slope of this association vary for each species and each primer set – further evidence that there is no universal parameter linking recovered DNA to species abundance – but Evans and colleagues take a significant step towards being able to answer the next question audiences tend to ask: ‘Yes, but how many are there?’     

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 analysis for macro-organisms: species distribution and more

In an era of severe biodiversity loss, biological monitoring is becoming increasingly essential. The analysis of environmental DNA (eDNA) has emerged as a new approach that could revolutionize the biological monitoring of aquatic ecosystems. Over the past decade, macro-organismal eDNA analysis has undergone significant developments and is rapidly becoming established as the golden standard for non-destructive and non-invasive biological monitoring. In this review, I summarize the development of macro-organismal eDNA analysis to date and the techniques used in this field. I also discuss the future perspective of these analytical methods in combination with sophisticated analytical techniques for DNA research developed in the fields of molecular biology and molecular genetics, including genomics, epigenomics, and single-cell technologies. eDNA analysis, which to date has been used primarily for determining the distribution of organisms, is expected to develop into a tool for elucidating the physiological state and behaviour of organisms. The fusion of microbiology and macrobiology through an amalgamation of these technologies is anticipated to lead to the future development of an integrated biology.     

基于环境DNA技术的赤水河秋季鱼类多样性分布特征

赤水河是长江上游少有的仍保持自然流态的大型一级支流,是长江鱼类重要的繁衍场和珍稀物种的保护地,摸清其鱼类多样性现状及鱼类群落结构特征对赤水河水生态恢复评估极为重要。于2021年9月对赤水河流域开展了鱼类多样性、分布及其特征调查,全流域共设置52个采样点,采用环境DNA技术采集并研究了赤水河鱼类的组成及其分布。结果显示通过环境DNA方法共调查到鱼类6目18科62属77种,包含16种长江特有鱼类。以鲤形目为主,占总数的87.87%。赤水河鱼类食性以杂食性和肉食性鱼类为主,群落结构上,处于下层水环境鱼类较多;赤水河鱼类优势种为宽鳍鱲(Y=0.205)、西昌华吸鳅(Y=0.085)、麦穗鱼(Y=0.068)、乌苏拟鲿(Y=0.033)、云南光唇鱼(Y=0.027);赤水河上游和下游鱼类群落(P<0.01)和Shannon-Wiener指数差异均显著(P<0.05)。海拔、流速、pH、电导率和温度是影响赤水河鱼类多样性的主要环境因素。为环境DNA技术在赤水河鱼类多样性调查中的应用提供了探索性研究,将有助于赤水河生物多样性的保护。     

Riverine distribution of mussel environmental DNA reflects a balance among density,transport, and removal processes

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Optimization and validation of a cost‐effective protocol for biosurveillance of invasive alien species

Environmental DNA (eDNA) metabarcoding has revolutionized biodiversity monitoring and invasive pest biosurveillance programs. The introduction of insect pests considered invasive alien species (IAS) into a non‐native range poses a threat to native plant health. The early detection of IAS can allow for prompt actions by regulating authorities, thereby mitigating their impacts. In the present study, we optimized and validated a fast and cost‐effective eDNA metabarcoding protocol for biosurveillance of IAS and characterization of insect and microorganism diversity. Forty‐eight traps were placed, following the CFIA''s annual forest insect trapping survey, at four locations in southern Ontario that are high risk for forest IAS. We collected insects and eDNA samples using Lindgren funnel traps that contained a saturated salt (NaCl) solution in the collection jar. Using cytochrome c oxidase I (COI) as a molecular marker, a modified Illumina protocol effectively identified 2,535 Barcode Index Numbers (BINs). BINs were distributed among 57 Orders and 304 Families, with the vast majority being arthropods. Two IAS (Agrilus planipennis and Lymantria dispar) are regulated by the Canadian Food Inspection Agency (CFIA) as plant health pests, are known to occur in the study area, and were identified through eDNA in collected traps. Similarly, using 16S ribosomal RNA and nuclear ribosomal internal transcribed spacer (ITS), five bacterial and three fungal genera, which contain species of regulatory concern across several Canadian jurisdictions, were recovered from all sampling locations. Our study results reaffirm the effectiveness and importance of integrating eDNA metabarcoding as part of identification protocols in biosurveillance programs.     

环境DNA metabarcoding及其在生态学研究中的应用

环境DNA metabarcoding(eDNA metabarcoding)是指利用环境样本(如土壤、水、粪便等)中分离的DNA进行高通量的多个物种(或高级分类单元)鉴定的方法。近年来,该方法引起了学者的广泛关注,逐渐应用于生物多样性研究、水生生物监测、珍稀濒危物种和外来入侵物种检测等生态学领域。介绍环境DNA metabarcoding的含义和研究方法;重点介绍环境DNA metabarcoding在物种监测、生物多样性研究和食性分析等生态学领域中的应用;总结环境DNA metabarcoding应用于生态学研究领域面临的挑战并对该方法的发展进行展望。     

Targeted and passive environmental DNA approaches outperform established methods for detection of quagga mussels,Dreissena rostriformis bugensis in flowing water

1. The early detection of invasive non‐native species (INNS) is important for informing management actions. Established monitoring methods require the collection or observation of specimens, which is unlikely at the beginning of an invasion when densities are likely to be low. Environmental DNA (eDNA) analysis is a highly promising technique for the detection of INNS—particularly during the early stages of an invasion.
2. Here, we compared the use of traditional kick‐net sampling with two eDNA approaches (targeted detection using both conventional and quantitative PCR and passive detection via metabarcoding with conserved primers) for detection of quagga mussel, Dreissena rostriformis bugensis, a high priority INNS, along a density gradient on the River Wraysbury, UK.
3. All three molecular tools outperformed traditional sampling in terms of detection. Conventional PCR and qPCR both had 100% detection rate in all samples and outperformed metabarcoding when the target species was at low densities. Additionally, quagga mussel DNA copy number (qPCR) and relative read count (metabarcoding) were significantly influenced by both mussel density and distance from source population, with distance being the most significant predictor.
4. Synthesis and application. All three molecular approaches were more sensitive than traditional kick‐net sampling for the detection of the quagga mussel in flowing water, and both qPCR and metabarcoding enabled estimates of relative abundance. Targeted approaches were more sensitive than metabarcoding, but metabarcoding has the advantage of providing information on the wider community and consequently the impacts of INNS.

     

Environmental DNA methylation of Lymnaea stagnalis varies with age and is hypermethylated compared to tissue DNA

Environmental DNA (eDNA) approaches contributing to species identifications are quickly becoming the new norm in biomonitoring and ecosystem assessments. Yet, information such as age and health state of the population, which is vital to species biomonitoring, has not been accessible from eDNA. DNA methylation has the potential to provide such information on the state of a population. Here, we measured the methylation of eDNA along with tissue DNA (tDNA) of Lymnaea stagnalis at four life stages. We demonstrate that eDNA methylation varies with age and allows distinguishing among age classes. Moreover, eDNA was globally hypermethylated in comparison to tDNA. This difference was age-specific and connected to a limited number of eDNA sites. This differential methylation pattern suggests that eDNA release with age is partially regulated through DNA methylation. Our findings help to understand mechanisms involved in eDNA release and shows the potential of eDNA methylation analysis to assess age classes. Such age class assessments will encourage future eDNA studies to assess fundamental processes of population dynamics and functioning in ecology, biodiversity conservation and impact assessments.     

Meta-Fish-Lib: A generalised,dynamic DNA reference library pipeline for metabarcoding of fishes

The accuracy and reliability of DNA metabarcoding analyses depend on the breadth and quality of the reference libraries that underpin them. However, there are limited options available to obtain and curate the huge volumes of sequence data that are available on public repositories such as NCBI and BOLD. Here, we provide a pipeline to download, clean and annotate mitochondrial DNA sequence data for a given list of fish species. Features of this pipeline include (a) support for multiple metabarcode markers; (b) searches on species synonyms and taxonomic name validation; (c) phylogeny assisted quality control for identification and removal of misannotated sequences; (d) automatically generated coverage reports for each new GenBank release update; and (e) citable, versioned DOIs. As an example we provide a ready-to-use curated reference library for the marine and freshwater fishes of the U.K. To augment this reference library for environmental DNA metabarcoding specifically, we generated 241 new MiFish-12S sequences for 88 U.K. marine species, and make available new primer sets useful for sequencing these. This brings the coverage of common U.K. species for the MiFish-12S fragment to 93%, opening new avenues for scaling up fish metabarcoding across wide spatial gradients. The Meta-Fish-Lib reference library and pipeline is hosted at https://github.com/genner-lab/meta-fish-lib .     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.     

Detection of invasive freshwater fish species using environmental DNA survey

Invasive species are one of the most significant problem in freshwater ecosystems. Most common non-native freshwater species in Turkish freshwater fish fauna are Prussian Carp (Carassius gibelio), North African Catfish (Clarias gariepinus), Nile Tilapia (Oreochromis niloticus) and Topmouth Gudgeon (Pseudorasbora parva).Recent studies showed that environmental DNA could be used to detect target species inhabiting the ecosystem with higher precision and less effort compared to traditional field surveys. In this study, eDNA approach was used to investigate non-native freshwater fish species from fifteen different locations of Upper Sakarya Basin. eDNA was successfully extracted from the water samples of locations where the species were visually observed. Mean amplification rate of eDNA was calculated as 77.03%.This study is the first environmental DNA study used in detection of four of the most common invasive freshwater fish species. Results clearly indicating that eDNA surveys could be used as an important molecular tool to monitor invasive fish species in freshwater ecosystems.     

Assessment of fish communities using environmental DNA: Effect of spatial sampling design in lentic systems of different sizes

Freshwater fish biodiversity is quickly decreasing and requires effective monitoring and conservation. Environmental DNA (eDNA)‐based methods have been shown to be highly sensitive and cost‐efficient for aquatic biodiversity surveys, but few studies have systematically investigated how spatial sampling design affects eDNA‐detected fish communities across lentic systems of different sizes. We compared the spatial patterns of fish diversity determined using eDNA in three lakes of small (SL; 3 ha), medium (ML; 122 ha) and large (LL; 4,343 ha) size using a spatially explicit grid sampling method. A total of 100 water samples (including nine, 17 and 18 shoreline samples and six, 14 and 36 interior samples from SL, ML and LL, respectively) were collected, and fish communities were analysed using eDNA metabarcoding of the mitochondrial 12S region. Together, 30, 35 and 41 fish taxa were detected in samples from SL, ML, and LL, respectively. We observed that eDNA from shoreline samples effectively captured the majority of the fish diversity of entire waterbodies, and pooled samples recovered fewer species than individually processed samples. Significant spatial autocorrelations between fish communities within 250 m and 2 km of each other were detected in ML and LL, respectively. Additionally, the relative sequence abundances of many fish species exhibited spatial distribution patterns that correlated with their typical habitat occupation. Overall, our results support the validity of a shoreline sampling strategy for eDNA‐based fish community surveys in lentic systems but also suggest that a spatially comprehensive sampling design can reveal finer distribution patterns of individual species.     

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.     

Assessment of littoral algal diversity from the northern Gulf of Mexico using environmental DNA metabarcoding

Traditional methods for algal biodiversity monitoring are costly and time inefficient because they rely on high‐level taxonomic expertise to address species identity problems involving phenotypic plasticity and morphological convergence. These problems are exacerbated in regions such as the Gulf of Mexico, that has a limited history of phycological exploration, but that are economically important or threatened by numerous anthropogenic stressors. Given the high pace of disturbance to natural systems, there is a critical need for expedient and cost‐effective tools for the study of benthic algal communities. Here we document the use of environmental DNA metabarcoding, using the partial LSU rDNA and 23S rDNA plastid molecular markers, to elucidate littoral algal diversity in the Northern Gulf of Mexico. We assigned 73.7% of algal OTUs to genus and 59.6% to species ranks. Our current study detected molecular signals for 35 algal/protist species with no previous reports in the Gulf of Mexico, thus providing an important, molecular‐validated, baseline of species richness for this region. We also make several bioinformatic recommendations for the efficient use of high‐throughput sequence data to assess biological communities.     

Monitoring endangered freshwater biodiversity using environmental DNA

Freshwater ecosystems are among the most endangered habitats on Earth, with thousands of animal species known to be threatened or already extinct. Reliable monitoring of threatened organisms is crucial for data‐driven conservation actions but remains a challenge owing to nonstandardized methods that depend on practical and taxonomic expertise, which is rapidly declining. Here, we show that a diversity of rare and threatened freshwater animals—representing amphibians, fish, mammals, insects and crustaceans—can be detected and quantified based on DNA obtained directly from small water samples of lakes, ponds and streams. We successfully validate our findings in a controlled mesocosm experiment and show that DNA becomes undetectable within 2 weeks after removal of animals, indicating that DNA traces are near contemporary with presence of the species. We further demonstrate that entire faunas of amphibians and fish can be detected by high‐throughput sequencing of DNA extracted from pond water. Our findings underpin the ubiquitous nature of DNA traces in the environment and establish environmental DNA as a tool for monitoring rare and threatened species across a wide range of taxonomic groups.     

Applications of mitochondrial DNA analysis in conservation: a critical review

Patterns of variation in mitochondrial DNA (mtDNA) increasingly are being investigated in threatened or managed species, but not always with clearly defined goals for conservation. In this review I identify uses of mtDNA analysis which fall into two different areas: (i) 'gene conservation' - the identification and management of genetic diversity, and (ii) 'molecular ecology' - the use of mtDNA variation to guide and assist demographic studies of populations. These two classes of application have different conceptual bases, conservation goals and time-frames. Gene conservation makes extensive use of phylogenetic information and is, in general, most relevant to long-term planning. Appropriate uses here include identification of Evolutionarily Significant Units and assessment of conservation priority of taxa or areas from an evolutionary perspective. Less appropriate are inferences about fitness from within-population diversity and about species boundaries. Molecular ecology makes more use of allele frequencies and provides information useful for short-term management of populations. Powerful applications are to identify Management Units and to define and use naturally occurring genetic tags. Estimating demographic parameters, e.g migration rate and population size, from patterns of mtDNA diversity is fraught with difficulty, particularly where populations are fluctuating, and is unlikely to produce quantitative estimates sufficiently accurate to be useful for practical management of contemporary populations. However, through comparative studies, mtDNA analysis can provide qualitative signals of population changes, allowing efficient targeting of resource-intensive ecological studies. Thus, there are some relatively straightforward uses of mtDNA, preferably in conjunction with assays of nuclear variation, that can make a significant contribution to the long-term planning and short-term execution of species recovery plans.     

Environmental DNA metabarcoding: Transforming how we survey animal and plant communities

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High‐throughput sequencing (“HTS”) platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed “environmental DNA” or “eDNA”). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called “eDNA metabarcoding” and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.