Environmental DNA metabarcoding studies are critically affected by substrate selection

Effective biomonitoring is critical for driving management outcomes that ensure long‐term sustainability of the marine environment. In recent years, environmental DNA (eDNA), coupled with metabarcoding methodologies, has emerged as a promising tool for generating biotic surveys of marine ecosystems, including those under anthropogenic pressure. However, more empirical data are needed on how to best implement eDNA field sampling approaches to maximize their utility for each specific application. The effect of the substrate chosen for eDNA sampling on the diversity of marine taxa detected by DNA metabarcoding has not yet been systematically analysed, despite aquatic systems being those most commonly targeted for eDNA studies. We investigated the effect of four commonly used eDNA substrates to explore taxonomic diversity: (a) surface water, (b) marine sediment, (c) settlement plates and (d) planktonic tows. With a focus on coastal ports, 332 eDNA samples from Australia (Indian and Southern oceans) and Kazakhstan (Caspian Sea) were collected and analysed by multi‐assay DNA metabarcoding. Across study locations, between 30% and 52% of eukaryotic families detected were unique to a particular substrate and <6% of families were found in all four substrates. Taxonomic composition varied significantly depending on the substrate sampled implying that the suitability (and bias) of an eDNA substrate will depend on the focal taxa. These findings demonstrate that single substrate eDNA metabarcoding likely underestimates the total eukaryotic diversity. Future eDNA experimental design should consider incorporating multiple substrates or select substrate(s) best suited to the specific detection of target taxa.     

Environmental DNA (eDNA) metabarcoding reveals strong discrimination among diverse marine habitats connected by water movement

While in recent years environmental DNA (eDNA) metabarcoding surveys have shown great promise as an alternative monitoring method, the integration into existing marine monitoring programs may be confounded by the dispersal of the eDNA signal. Currents and tidal influences could transport eDNA over great distances, inducing false‐positive species detection, leading to inaccurate biodiversity assessments and, ultimately, mismanagement of marine environments. In this study, we determined the ability of eDNA metabarcoding surveys to distinguish localized signals obtained from four marine habitats within a small spatial scale (<5 km) subject to significant tidal and along‐shore water flow. Our eDNA metabarcoding survey detected 86 genera, within 77 families and across 11 phyla using three established metabarcoding assays targeting fish (16S rRNA gene), crustacean (16S rRNA gene) and eukaryotic (cytochrome oxidase subunit 1) diversity. Ordination and cluster analyses for both taxonomic and OTU data sets show distinct eDNA signals between the sampled habitats, suggesting dispersal of eDNA among habitats was limited. Individual taxa with strong habitat preferences displayed localized eDNA signals in accordance with their respective habitat, whereas taxa known to be less habitat‐specific generated more ubiquitous signals. Our data add to evidence that eDNA metabarcoding surveys in marine environments detect a broad range of taxa that are spatially discrete. Our work also highlights that refinement of assay choice is essential to realize the full potential of eDNA metabarcoding surveys in marine biodiversity monitoring programs.     

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.     

eDNA metabarcoding as a new surveillance approach for coastal Arctic biodiversity

Because significant global changes are currently underway in the Arctic, creating a large‐scale standardized database for Arctic marine biodiversity is particularly pressing. This study evaluates the potential of aquatic environmental DNA (eDNA) metabarcoding to detect Arctic coastal biodiversity changes and characterizes the local spatio‐temporal distribution of eDNA in two locations. We extracted and amplified eDNA using two COI primer pairs from ~80 water samples that were collected across two Canadian Arctic ports, Churchill and Iqaluit, based on optimized sampling and preservation methods for remote regions surveys. Results demonstrate that aquatic eDNA surveys have the potential to document large‐scale Arctic biodiversity change by providing a rapid overview of coastal metazoan biodiversity, detecting nonindigenous species, and allowing sampling in both open water and under the ice cover by local northern‐based communities. We show that DNA sequences of ~50% of known Canadian Arctic species and potential invaders are currently present in public databases. A similar proportion of operational taxonomic units was identified at the species level with eDNA metabarcoding, for a total of 181 species identified at both sites. Despite the cold and well‐mixed coastal environment, species composition was vertically heterogeneous, in part due to river inflow in the estuarine ecosystem, and differed between the water column and tide pools. Thus, COI‐based eDNA metabarcoding may quickly improve large‐scale Arctic biomonitoring using eDNA, but we caution that aquatic eDNA sampling needs to be standardized over space and time to accurately evaluate community structure changes.     

Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods

Terrestrial arthropods comprise the most species‐rich communities on Earth, and grassland flowers provide resources for hundreds of thousands of arthropod species. Diverse grassland ecosystems worldwide are threatened by various types of environmental change, which has led to decline in arthropod diversity. At the same time, monitoring grassland arthropod diversity is time‐consuming and strictly dependent on declining taxonomic expertise. Environmental DNA (eDNA) metabarcoding of complex samples has demonstrated that information on species compositions can be efficiently and non‐invasively obtained. Here, we test the potential of wild flowers as a novel source of arthropod eDNA. We performed eDNA metabarcoding of flowers from several different plant species using two sets of generic primers, targeting the mitochondrial genes 16S rRNA and COI. Our results show that terrestrial arthropod species leave traces of DNA on the flowers that they interact with. We obtained eDNA from at least 135 arthropod species in 67 families and 14 orders, together representing diverse ecological groups including pollinators, parasitoids, gall inducers, predators, and phytophagous species. Arthropod communities clustered together according to plant species. Our data also indicate that this experiment was not exhaustive, and that an even higher arthropod richness could be obtained using this eDNA approach. Overall, our results demonstrate that it is possible to obtain information on diverse communities of insects and other terrestrial arthropods from eDNA metabarcoding of wild flowers. This novel source of eDNA represents a vast potential for addressing fundamental research questions in ecology, obtaining data on cryptic and unknown species of plant‐associated arthropods, as well as applied research on pest management or conservation of endangered species such as wild pollinators.     

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.     

A DNA mini‐barcode for marine macrophytes

Studies focusing on marine macrophyte metabarcoding from environmental samples are scarce, due to the lack of a universal barcode for these taxa, and to their poor representation in DNA databases. Here, we searched for a short barcode able to identify marine macrophytes from tissue samples; then, we created a DNA reference library which was used to identify macrophytes in eDNA from coastal sediments. Barcoding of seagrasses, mangroves and marine macroalgae (Chlorophyta, Rhodophyta and Phaeophyceae) was tested using 18 primer pairs from six barcoding genes: the plant barcodes rbcL, matK and trnL, plus the genes ITS2, COI and 18S. The 18S gene showed the highest universality among marine macrophytes, amplifying 95%–100% of samples; amplification performance of the other barcodes was limited. Taxonomy was assigned using a phylogeny‐based approach to create an 18S DNA reference library. Macrophyte tissue sequences were accurately identified within their phyla (88%), order (76%), genus (71%) and species (23%). Nevertheless, out of 86 macrophytes tested, only 48% and 15% had a reference sequence at genus and at species level, respectively. Identification at these levels can be improved by more inclusive reference libraries. Using the 18S mini‐barcode and the reference library, we recovered eDNA from 21 marine macrophytes in sediments, demonstrating the barcode's ability to trace primary producers that contribute to blue carbon. We expect this barcode to also be useful for other ecological questions, such as tracing macro primary producers in marine food webs.     

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.     

eDNA metabarcoding survey reveals fine‐scale coral reef community variation across a remote,tropical island ecosystem

Environmental DNA (eDNA) metabarcoding, a technique for retrieving multispecies DNA from environmental samples, can detect a diverse array of marine species from filtered seawater samples. There is a growing potential to integrate eDNA alongside existing monitoring methods in order to establish or improve the assessment of species diversity. Remote island reefs are increasingly vulnerable to climate‐related threats and as such there is a pressing need for cost‐effective whole‐ecosystem surveying to baseline biodiversity, study assemblage changes and ultimately develop sustainable management plans. We investigated the utility of eDNA metabarcoding as a high‐resolution, multitrophic biomonitoring tool at the Cocos (Keeling) Islands, Australia (CKI)—a remote tropical coral reef atoll situated within the eastern Indian Ocean. Metabarcoding assays targeting the mitochondrial 16S rRNA and CO1 genes, as well as the 18S rRNA nuclear gene, were applied to 252 surface seawater samples collected from 42 sites within a 140 km² area. Our assays successfully detected a wide range of bony fish and elasmobranchs (244 taxa), crustaceans (88), molluscs (37) and echinoderms (7). Assemblage composition varied significantly between sites, reflecting habitat partitioning across the island ecosystem and demonstrating the localisation of eDNA signals, despite extensive tidal and oceanic movements. In addition, we document putative new occurrence records for 46 taxa and compare the efficiency of our eDNA approach to visual survey techniques at CKI. Our study demonstrates the utility of a multimarker metabarcoding approach in capturing multitrophic biodiversity across an entire coral reef atoll and sets an important baseline for ongoing monitoring and management.     

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.     

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology‐ and DNA‐based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta‐diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer‐binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well‐developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.     

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.     

Assessing eukaryotic biodiversity in the Florida Keys National Marine Sanctuary through environmental DNA metabarcoding

Environmental DNA (eDNA) is the DNA suspended in the environment (e.g., water column), which includes cells, gametes, and other material derived from but not limited to shedding of tissue, scales, mucus, and fecal matter. Amplifying and sequencing marker genes (i.e., metabarcoding) from eDNA can reveal the wide range of taxa present in an ecosystem through analysis of a single water sample. Metabarcoding of eDNA provides higher resolution data than visual surveys, aiding in assessments of ecosystem health. This study conducted eDNA metabarcoding of two molecular markers (cytochrome c oxidase I (COI) and 18S ribosomal RNA (rRNA) genes) to survey eukaryotic diversity across multiple trophic levels in surface water samples collected at three sites along the coral reef tract within the Florida Keys National Marine Sanctuary (FKNMS) during four research cruises in 2015. The 18S rRNA gene sequences recovered 785 genera while the COI gene sequences recovered 115 genera, with only 33 genera shared between the two datasets, emphasizing the complementarity of these marker genes. Community composition for both genetic markers clustered by month of sample collection, suggesting that temporal variation has a larger effect on biodiversity than spatial variability in the FKNMS surface waters. Sequences from both marker genes were dominated by copepods, but each marker recovered distinct phytoplankton groups, with 18S rRNA gene sequences dominated by dinoflagellates and COI sequences dominated by coccolithophores. Although eDNA samples were collected from surface waters, many benthic species such as sponges, crustaceans, and corals were identified. These results show the utility of eDNA metabarcoding for cataloging biodiversity to establish an ecosystem baseline against which future samples can be compared in order to monitor community changes.     

Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys.     

Metabarcoding of shrimp stomach content: Harnessing a natural sampler for fish biodiversity monitoring

Given their positioning and biological productivity, estuaries have long represented key providers of ecosystem services and consequently remain under remarkable pressure from numerous forms of anthropogenic impact. The monitoring of fish communities in space and time is one of the most widespread and established approaches to assess the ecological status of estuaries and other coastal habitats, but traditional fish surveys are invasive, costly, labour intensive and highly selective. Recently, the application of metabarcoding techniques, on either sediment or aqueous environmental DNA, has rapidly gained popularity. Here, we evaluate the application of a novel, high‐throughput DNA‐based monitoring tool to assess fish diversity, based on the analysis of the gut contents of a generalist predator/scavenger, the European brown shrimp, Crangon crangon. Sediment and shrimp samples were collected from eight European estuaries, and DNA metabarcoding (using both 12S and COI markers) was carried out to infer fish assemblage composition. We detected 32 teleost species (16 and 20, for 12S and COI, respectively). Twice as many species were recovered using metabarcoding than by traditional net surveys. By comparing and interweaving trophic, environmental DNA and traditional survey‐based techniques, we show that the DNA‐assisted gut content analysis of a ubiquitous, easily accessible, generalist species may serve as a powerful, rapid and cost‐effective tool for large‐scale, routine estuarine biodiversity monitoring.     

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

Preserving biodiversity is a global challenge requiring data on species’ distribution and abundance over large geographic and temporal scales. However, traditional methods to survey mobile species’ distribution and abundance in marine environments are often inefficient, environmentally destructive, or resource‐intensive. Metabarcoding of environmental DNA (eDNA) offers a new means to assess biodiversity and on much larger scales, but adoption of this approach for surveying whole animal communities in large, dynamic aquatic systems has been slowed by significant unknowns surrounding error rates of detection and relevant spatial resolution of eDNA surveys. Here, we report the results of a 2.5 km eDNA transect surveying the vertebrate fauna present along a gradation of diverse marine habitats associated with a kelp forest ecosystem. Using PCR primers that target the mitochondrial 12S rRNA gene of marine fishes and mammals, we generated eDNA sequence data and compared it to simultaneous visual dive surveys. We find spatial concordance between individual species’ eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 m. eDNA reliably detected vertebrates with low false‐negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.     

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。     

Taking eDNA underground: Factors affecting eDNA detection of subterranean fauna in groundwater

Stygofauna are aquatic fauna that have evolved to live underground. The impacts of anthropogenic climate change, extraction and pollution on groundwater pose major threats to groundwater health, prompting the need for efficient and reliable means to detect and monitor stygofaunal communities. Conventional survey techniques for these species rely on morphological identification and can be biased, labour-intensive and often indeterminate to lower taxonomic levels. By contrast, environmental DNA (eDNA)-based methods have the potential to dramatically improve on existing stygofaunal survey methods in a large range of habitats and for all life stages, reducing the need for the destructive manual collection of often critically endangered species or for specialized taxonomic expertise. We compared eDNA and haul-net samples collected in 2020 and 2021 from 19 groundwater bores and a cave on Barrow Island, northwest Western Australia, and assessed how sampling factors influenced the quality of eDNA detection of stygofauna. The two detection methods were complementary; eDNA metabarcoding was able to detect soft-bodied taxa and fish often missed by nets, but only detected seven of the nine stygofaunal crustacean orders identified from haul-net specimens. Our results also indicated that eDNA metabarcoding could detect 54%–100% of stygofauna from shallow-water samples and 82%–90% from sediment samples. However, there was significant variation in stygofaunal diversity between sample years and sampling types. The findings of this study demonstrate that haul-net sampling has a tendency to underestimate stygofaunal diversity and that eDNA metabarcoding of groundwater can substantially improve the efficiency of stygofaunal surveys.     

eDNA metabarcoding of avocado flowers: ‘Hass’ it got potential to survey arthropods in food production systems?

In the face of global biodiversity declines, surveys of beneficial and antagonistic arthropod diversity as well as the ecological services that they provide are increasingly important in both natural and agro-ecosystems. Conventional survey methods used to monitor these communities often require extensive taxonomic expertise and are time-intensive, potentially limiting their application in industries such as agriculture, where arthropods often play a critical role in productivity (e.g. pollinators, pests and predators). Environmental DNA (eDNA) metabarcoding of a novel substrate, crop flowers, may offer an accurate and high throughput alternative to aid in the detection of these managed and unmanaged taxa. Here, we compared the arthropod communities detected with eDNA metabarcoding of flowers, from an agricultural species (Persea americana—‘Hass’ avocado), with two conventional survey techniques: digital video recording (DVR) devices and pan traps. In total, 80 eDNA flower samples, 96 h of DVRs and 48 pan trap samples were collected. Across the three methods, 49 arthropod families were identified, of which 12 were unique to the eDNA dataset. Environmental DNA metabarcoding from flowers revealed potential arthropod pollinators, as well as plant pests and parasites. Alpha diversity levels did not differ across the three survey methods although taxonomic composition varied significantly, with only 12% of arthropod families found to be common across all three methods. eDNA metabarcoding of flowers has the potential to revolutionize the way arthropod communities are monitored in natural and agro-ecosystems, potentially detecting the response of pollinators and pests to climate change, diseases, habitat loss and other disturbances.     

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。