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。     

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。     

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.     

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。     

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.     

Identifying error and accurately interpreting environmental DNA metabarcoding results: A case study to detect vertebrates at arid zone waterholes

Environmental DNA (eDNA) metabarcoding surveys enable rapid, noninvasive identification of taxa from trace samples with wide‐ranging applications from characterizing local biodiversity to identifying food‐web interactions. However, the technique is prone to error from two major sources: (a) contamination through foreign DNA entering the workflow, and (b) misidentification of DNA within the workflow. Both types of error have the potential to obscure true taxon presence or to increase taxonomic richness by incorrectly identifying taxa as present at sample sites, but multiple error sources can remain unaccounted for in metabarcoding studies. Here, we use data from an eDNA metabarcoding study designed to detect vertebrate species at waterholes in Australia's arid zone to illustrate where and how in the workflow errors can arise, and how to mitigate those errors. We detected the DNA of 36 taxa spanning 34 families, 19 orders and five vertebrate classes in water samples from waterholes, demonstrating the potential for eDNA metabarcoding surveys to provide rapid, noninvasive detection in remote locations, and to widely sample taxonomic diversity from aquatic through to terrestrial taxa. However, we initially identified 152 taxa in the samples, meaning there were many false positive detections. We identified the sources of these errors, allowing us to design a stepwise process to detect and remove error, and provide a template to minimize similar errors that are likely to arise in other metabarcoding studies. Our findings suggest eDNA metabarcoding surveys need to be carefully conducted and screened for errors to ensure their accuracy.     

Seasonal and spatial variability of zooplankton diversity in the Poyang Lake Basin using DNA metabarcoding

Freshwater ecosystems face multiple threats to their stability globally. Poyang Lake is the largest lake in China, but its habitat has been seriously degraded because of human activities and natural factors (e.g. climate change), resulting in a decline in freshwater biodiversity. Zooplankton are useful indicators of environmental stressors because they are sensitive to external perturbations. DNA metabarcoding is an approach that has gained significant traction by aiding ecosystem conservation and management. Here, the seasonal and spatial variability in the zooplankton diversity were analyzed in the Poyang Lake Basin using DNA metabarcoding. The results showed that the community structure of zooplankton exhibited significant seasonal and spatial variability using DNA metabarcoding, where the community structure was correlated with turbidity, water temperature, pH, total phosphorus, and chlorophyll‐a. These results indicated habitat variations affected by human activities and seasonal change could be the main driving factors for the variations of zooplankton community. This study also provides an important reference for the management of aquatic ecosystem health and conservation of aquatic biodiversity.     

基于环境DNA-宏条形码技术的底栖动物监测及水质评价研究进展

底栖动物是淡水生态系统中物种多样性最高的类群,也是应用最广泛的水质监测指示生物之一。传统的底栖动物监测以形态学为基础,耗时费力,无法满足流域尺度大规模监测的需求。环境DNA-宏条形码技术是一种新兴的生物监测方法,其与传统方法相比优势在于采样方法简单、低成本、高灵敏度,不受生物样本和环境状况的影响,不依赖分类专家和鉴定资料,能够快速准确地对多个类群进行大规模、高通量的物种鉴定。然而,在实际应用中该方法的效果受诸多因素的影响,不同的方法、流程往往会产生差异较大的结果。鉴于此,着重分析总结了应用环境DNA-宏条形码技术监测底栖动物的关键影响因素,包括样品采集与处理流程、分子标记选择、引物设计、PCR偏好性、参考数据库的完整性及相应的优化。并基于此探讨了提高环境DNA-宏条形码技术在底栖动物监测效率和准确率的途径,以期为底栖动物环境DNA-宏条形码监测方案的制定提供可靠的参考。最后对该技术在底栖动物监测和水质评价中的最新发展方向进行了展望。     

Terrestrial mammal surveillance using hybridization capture of environmental DNA from African waterholes

Determining species distributions can be extremely challenging but is crucial to ecological and conservation research. Environmental DNA (eDNA) approaches have shown particular promise in aquatic systems for several vertebrate and invertebrate species. For terrestrial animals, however, eDNA‐based surveys are considerably more difficult due to the lack of or difficulty in obtaining appropriate sampling substrate. In water‐limited ecosystem where terrestrial mammals are often forced to congregate at waterholes, water and sediment from shared water sources may be a suitable substrate for noninvasive eDNA approaches. We characterized mitochondrial DNA sequences from a broad range of terrestrial mammal species in two different African ecosystems (in Namibia and Tanzania) using eDNA isolated from native water, sediment and water filtered through glass fibre filters. A hybridization capture enrichment with RNA probes targeting the mitochondrial genomes of 38 mammal species representing the genera/families expected at the respective ecosystems was employed, and 16 species were identified, with a maximum mitogenome coverage of 99.8%. Conventional genus‐specific PCRs were tested on environmental samples for two genera producing fewer positive results than hybridization capture enrichment. An experiment with mock samples using DNA from non‐African mammals showed that baits covering 30% of nontarget mitogenomes produced 91% mitogenome coverage after capture. In the mock samples, over‐representation of DNA of one species still allowed for the detection of DNA of other species that was at a 100‐fold lower concentration. Hybridization capture enrichment of eDNA is therefore an effective method for monitoring terrestrial mammal species from shared water sources.     

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.     

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.     

应用DNA复合条形码技术研究秦岭水生动物多样性

基于新一代测序技术的DNA复合条形码技术,以其简便快捷和省时省力的特点,已经成为监测生物多样性的主要方法。采用这一技术,选取COⅠ和18S rRNA两个条形码标记,对秦岭5个淡水水域内,不同生境下10个样品的水生动物多样性进行了初步调查。区系组成结果显示:18S rRNA基因鉴定了9门42纲52目,COⅠ基因鉴定了5门11纲36目,而两个条形码标记共鉴定了10门48纲89目。群落组成分析结果显示:COⅠ分析得到样本中含量相对较高的类群是双翅目、毛翅目、基眼目、鞘翅目、蜉蝣目等;而18S rRNA分析得到样本中主要有节肢动物门、软体动物门和扁形动物门三个大门。此外,两者的分析结果也表明所选样地下游的类群数要高于上游。α多样性分析结果显示:金龙峡、沣峪口和石砭峪3个受人类影响较大样地的群落丰富度和群落多样性相对较高,而五台山和子午峪两个自然样地的物种丰富度和群落多样性相对较低,表明一定程度的外来干扰会对一个地方的水生生物多样性有明显的提高。β多样性分析结果显示,不同环境因素下样品的群落结构差异性较大,而相似环境因素下样品的群落结构差异性则相对较小。此外,在聚类分析时,环境相似性较高的样品首先聚集在一起,而且群落相似性也相对较高。     

DNA复合条形码在太白山土壤动物多样性研究中的应用

DNA复合条形码技术(metabarcoding)将DNA条形码与高通量测序技术相结合,快速便捷地鉴定群落混合样本中的物种,成为监测群落中物种组成和丰富度的可靠方法。采用这一方法分析了秦岭太白山5种不同生境的中小型土壤动物多样性,共得到土壤动物3门9纲28目199科。群落组成分析显示生境的变化对土壤动物群落组成有一定的影响。α多样性分析显示土壤动物群落丰富度指数最高的生境为针叶林,最低的为农田;土壤动物群落多样性指数最高的生境为针叶林,最低的为落叶小叶林。群落相似性分析显示高山草甸、针叶林和农田3种生境的土壤动物群落组成相似性较高,落叶小叶林和落叶阔叶林的土壤动物群落组成与这3种生境的差异较大,落叶小叶林与落叶阔叶林的土壤动物群落组成差异也较大。     

Invasive lionfish detected in estuaries in the northern Gulf of Mexico using environmental DNA

Environmental Biology of Fishes - Invasive lionfish are considered to be one of the worst marine invaders primarily due to their threat as predators to native species. The distribution of lionfish...     

Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring

Aquatic environmental DNA (eDNA) surveys are transforming how marine ecosystems are monitored. The time-consuming preprocessing step of active filtration, however, remains a bottleneck. Hence, new approaches that eliminate the need for active filtration are required. Filter-feeding invertebrates have been proven to collect eDNA, but side-by-side comparative studies to investigate the similarity between aquatic and filter-feeder eDNA signals are essential. Here, we investigated the differences among four eDNA sources (water; bivalve gill-tissue; sponges; and ethanol in which filter-feeding organisms were stored) along a vertically stratified transect in Doubtful Sound, New Zealand using three metabarcoding primer sets targeting fish and vertebrates. Combined, eDNA sources detected 59 vertebrates, while concurrent diver surveys observed eight fish species. There were no significant differences in alpha and beta diversity between water and sponge eDNA and both sources were highly correlated. Vertebrate eDNA was successfully extracted from the ethanol in which sponges were stored, although a reduced number of species were detected. Bivalve gill-tissue dissections, on the other hand, failed to reliably detect eDNA. Overall, our results show that vertebrate eDNA signals obtained from water samples and marine sponges are highly concordant. The strong similarity in eDNA signals demonstrates the potential of marine sponges as an additional tool for eDNA-based marine biodiversity surveys, by enabling the incorporation of larger sample numbers in eDNA surveys, reducing plastic waste, simplifying sample collection, and as a cost-efficient alternative. However, we note the importance to not detrimentally impact marine communities by, for example, nonlethal subsampling, specimen cloning, or using bycatch specimens.     

Dietary characterization of the endangered salt marsh harvest mouse and sympatric rodents using DNA metabarcoding

The salt marsh harvest mouse (Reithrodontomys raviventris; RERA) is an endangered species endemic to the coastal wetlands of the San Francisco Estuary, California. RERA are specialized to saline coastal wetlands, and their historical range has been severely impacted by landscape conversion and the introduction of non‐native plant and rodent species. A better understanding of their diet is needed to assess habitat quality, particularly in relation to potential competitors. We investigated three questions using DNA metabarcoding with ITS2 and trnL markers: (1) Do RERA specialize on the native plant, pickleweed (Salicornia pacifica), (2) Do RERA consume non‐native plants, and (3) What is the dietary niche breadth and overlap with three sympatric native and non‐native rodents? RERA diet was dominated by two plants, native Salicornia and non‐native salt bush (Atriplex spp.), but included 48 plant genera. RERA diet breadth was narrowest in fall, when they consumed the highest frequencies of Salicornia and Atriplex, and broadest in spring, when the frequencies of these two plants were lowest. Diet breadth was slightly lower for RERA than for co‐occurring species in pairwise comparisons. All four species consumed similarly high frequencies of wetland plants, but RERA consumed fewer grasses and upland plants, suggesting that it may be less suited to fragmented habitat than sympatric rodents. Diet overlap was lowest between RERA and the native California vole (Microtis californicus). In contrast, RERA diet overlapped substantially with the native western harvest mouse (R. megalotis) and non‐native house mouse (Mus musculus), suggesting potential for competition if these species become sufficiently abundant.     

Diet of the critically endangered blackchin guitarfish Glaucostegus cemiculus revealed using DNA metabarcoding

We present the first assessment of the diet of the blackchin guitarfish Glaucostegus cemiculus (Geoffroy Saint-Hilaire, 1817) for West Africa using DNA metabarcoding on stomach contents of individuals captured in the Bijagós Archipelago, Guinea-Bissau. The diet was dominated by crustaceans, particularly caramote prawn Penaeus kerathurus (frequency of occurrence [FO] = 74%, numerical frequency [NF] = 54%) and fiddler crab Afruca tangeri (FO = 74%, NF = 12%). Bony fishes were present in 30% of the stomachs. We highlight the importance of conservation action for intertidal habitats and their associated benthic invertebrates for the survival of the critically endangered blackchin guitarfish.     

Bird diversity and environmental gradients in Britain: a test of the species–energy hypothesis

1.  We tested the species diversity–energy hypothesis using the British bird fauna. This predicts that temperature patterns should match diversity patterns. We also tested the hypothesis that the mechanism operates directly through effects of temperature on thermoregulatory loads; this further predicts that seasonal changes in temperature cause matching changes in patterns of diversity, and that species' body mass is influential.
2.  We defined four assemblages using migration status (residents or visitors) and season (summer or winter distribution). Records of species' presence/absence in a total of 2362, 10 × 10-km, quadrats covering most of Britain were used, together with a wide selection of habitat, topographic and seasonal climatic data.
3.  We fitted a logistic regression model to each species' distribution using the environmental data. We then combined these individual species models mathematically to form a diversity model. Analysis of this composite model revealed that summer temperature was the factor most strongly associated with diversity.
4.  Although the species–energy hypothesis was supported, the direct mechanism, predicting an important role for body mass and matching seasonal patterns of change between diversity and temperature, was not supported.
5.  However, summer temperature is the best overall explanation for bird diversity patterns in Britain. It is a better predictor of winter diversity than winter temperature. Winter diversity is predicted more precisely from environmental factors than summer diversity.
6.  Climate change is likely to influence the diversity of different areas to different extents; for resident species, low diversity areas may respond more strongly as climate change progresses. For winter visitors, higher diversity areas may respond more strongly, while summer visitors are approximately neutral.     

Conservation in a cup of water: estimating biodiversity and population abundance from environmental DNA

Three mantras often guide species and ecosystem management: (i) for preventing invasions by harmful species, ‘early detection and rapid response’; (ii) for conserving imperilled native species, ‘protection of biodiversity hotspots’; and (iii) for assessing biosecurity risk, ‘an ounce of prevention equals a pound of cure.’ However, these and other management goals are elusive when traditional sampling tools (e.g. netting, traps, electrofishing, visual surveys) have poor detection limits, are too slow or are not feasible. One visionary solution is to use an organism’s DNA in the environment (eDNA), rather than the organism itself, as the target of detection. In this issue of Molecular Ecology, Thomsen et al. (2012) provide new evidence demonstrating the feasibility of this approach, showing that eDNA is an accurate indicator of the presence of an impressively diverse set of six aquatic or amphibious taxa including invertebrates, amphibians, a fish and a mammal in a wide range of freshwater habitats. They are also the first to demonstrate that the abundance of eDNA, as measured by qPCR, correlates positively with population abundance estimated with traditional tools. Finally, Thomsen et al. (2012) demonstrate that next‐generation sequencing of eDNA can quantify species richness. Overall, Thomsen et al. (2012) provide a revolutionary roadmap for using eDNA for detection of species, estimates of relative abundance and quantification of biodiversity.     

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.