Environmental DNA metabarcoding reveals local fish communities in a species-rich coastal sea.
ABSTRACT: Environmental DNA (eDNA) metabarcoding has emerged as a potentially powerful tool to assess aquatic community structures. However, the method has hitherto lacked field tests that evaluate its effectiveness and practical properties as a biodiversity monitoring tool. Here, we evaluated the ability of eDNA metabarcoding to reveal fish community structures in species-rich coastal waters. High-performance fish-universal primers and systematic spatial water sampling at 47 stations covering ~11?km2 revealed the fish community structure at a species resolution. The eDNA metabarcoding based on a 6-h collection of water samples detected 128 fish species, of which 62.5% (40 species) were also observed by underwater visual censuses conducted over a 14-year period. This method also detected other local fishes (?23 species) that were not observed by the visual censuses. These eDNA metabarcoding features will enhance marine ecosystem-related research, and the method will potentially become a standard tool for surveying fish communities.
Project description:Environmental DNA (eDNA) analysis has seen rapid development in the last decade, as a novel biodiversity monitoring method. Previous studies have evaluated optimal strategies, at several experimental steps of eDNA metabarcoding, for the simultaneous detection of fish species. However, optimal sampling strategies, especially the season and the location of water sampling, have not been evaluated thoroughly. To identify optimal sampling seasons and locations, we performed sampling monthly or at two-monthly intervals throughout the year in three dam reservoirs. Water samples were collected from 15 and nine locations in the Miharu and Okawa dam reservoirs in Fukushima Prefecture, respectively, and five locations in the Sugo dam reservoir in Hyogo Prefecture, Japan. One liter of water was filtered with glass-fiber filters, and eDNA was extracted. By performing MiFish metabarcoding, we successfully detected a total of 21, 24, and 22 fish species in Miharu, Okawa, and Sugo reservoirs, respectively. From these results, the eDNA metabarcoding method had a similar level of performance compared to conventional long-term data. Furthermore, it was found to be effective in evaluating entire fish communities. The number of species detected by eDNA survey peaked in May in Miharu and Okawa reservoirs, and in March and June in Sugo reservoir, which corresponds with the breeding seasons of many of fish species inhabiting the reservoirs. In addition, the number of detected species was significantly higher in shore, compared to offshore samples in the Miharu reservoir, and a similar tendency was found in the other two reservoirs. Based on these results, we can conclude that the efficiency of species detection by eDNA metabarcoding could be maximized by collecting water from shore locations during the breeding seasons of the inhabiting fish. These results will contribute in the determination of sampling seasons and locations for fish fauna survey via eDNA metabarcoding, in the future.
Project description: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.
Project description:High-throughput sequencing of environmental DNA (i.e., eDNA metabarcoding) has become an increasingly popular method for monitoring aquatic biodiversity. At present, such analyses require target-specific primers to amplify DNA barcodes from co-occurring species, and this initial amplification can introduce biases. Understanding the performance of different primers is thus recommended prior to undertaking any metabarcoding initiative. While multiple software programs are available to evaluate metabarcoding primers, all programs have their own strengths and weaknesses. Therefore, a robust in silico workflow for the evaluation of metabarcoding primers will benefit from the use of multiple programs. Furthermore, geographic differences in species biodiversity are likely to influence the performance of metabarcoding primers and further complicate the evaluation process. Here, an in silico workflow is presented that can be used to evaluate the performance of metabarcoding primers on an ecoregion scale. This workflow was used to evaluate the performance of published and newly developed eDNA metabarcoding primers for the freshwater fish biodiversity of the Murray-Darling Basin (Australia). To validate the in silico workflow, a subset of the primers, including one newly designed primer pair, were used in metabarcoding analyses of an artificial DNA community and eDNA samples. The results show that the in silico workflow allows for a robust evaluation of metabarcoding primers and can reveal important trade-offs that need to be considered when selecting the most suitable primer. Additionally, a new primer pair was described and validated that allows for more robust taxonomic assignments and is less influenced by primer biases compared to commonly used fish metabarcoding primers.
Project description:Using environmental DNA (eDNA) metabarcoding, we compared fish diversity in two distinct water bodies within the Yangtze River Basin with known populations of the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis; YFP): the Tian-e-Zhou Reserve and Poyang Lake. We aimed to create a fish surveying tool for use in the Yangtze River Basin, while also gaining a better understanding of the prey distribution and diversity within two of the remaining strongholds of YFP. 16S rRNA universal primers were developed to amplify fish eDNA. After high-throughput sequencing and stringent data filtering, we identified a total of 75 fish species (6 orders, 9 families, 57 genera) across seasons and regions. Nine of the 75 fish species were among the 28 known YFP prey species, three of which were detected in all water samples. Our eDNA metabarcoding identified many species that had been previously captured using traditional netting practices, but also numerous species not previously collected in these water bodies. Fish diversity was higher in Poyang Lake than in Tian-e-Zhou Reserve, as well as higher in the spring than in summer. These methods provide a broadly applicable tool to quantify fish diversity and distributions throughout the Yangtze River Basin, and to inform conservation strategies of YFP.
Project description:The use of environmental DNA (eDNA) for monitoring aquatic macrofauna allows the non-invasive species determination and measurement of their DNA abundance and typically involves the analysis of eDNA captured from water samples. In this proof-of-concept study, we focused on the novel use of eDNA extracted from archived suspended particulate matter (SPM) for identifying fish species using metabarcoding, which benefits from the prospect of retrospective monitoring and also analysis of fish communities through time. We used archived SPM samples of the German Environmental Specimen Bank (ESB), which were collected using sedimentation traps from different riverine points in Germany. Environmental DNA was extracted from nine SPM samples differing in location, organic content, and porosity (among other factors) using four different methods for the isolation of high-quality DNA. Application of the PowerSoil DNA Isolation Kit with an overnight incubation in lysis buffer, resulted in DNA extraction with the highest purity and eDNA metabarcoding of these eDNA fragments was used to detect a total of 29 fish taxa among the analyzed samples. Here we demonstrated for the first time that SPM is a promising source of eDNA for metabarcoding analysis, which could provide valuable retrospective information (when using archived SPM) for fish monitoring, complementing the currently used approaches.
Project description: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.
Project description: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.
Project description: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.
Project description:Environmental DNA (eDNA) metabarcoding has been used increasingly to assess biodiversity of aquatic vertebrates. However, there still remains to be developed a sampling design of eDNA metabarcoding that can ensure high detection rates of species with minimum total survey effort, especially for large-scale surveys of aquatic organisms. We here tested whether pooling of eDNA samples can be used to evaluate biodiversity of freshwater fishes in four satellite lakes of Lake Biwa, Japan. Fish communities detected by eDNA metabarcoding of the mitochondrial 12S region were compared between the individual and pooled samples. In the individual samples, 31, 22, 33, and 31 fish lineages (proxies for species) were observed at the respective sites, within which moderate spatial autocorrelation existed. In the pooled samples, 30, 20, 29, and 27, lineages were detected, respectively, even after 15 PCR replicates. Lineages accounting for?<?0.05% of the total read count of each site's individual samples were mostly undetectable in the pooled samples. Moreover, fish communities detected were similar among PCR replicates in the pooled samples. Because of the decreased detection rates, the pooling strategy is unsuitable for estimating fish species richness. However, this procedure is useful potentially for among-site comparison of representative fish communities.
Project description:Biodiversity has to be accurately evaluated to assess more precisely possible dam effects on fish populations, in particular on the most biodiverse rivers such as the Mekong River. To improve tools for fish biodiversity assessment, a methodological survey was performed in the surroundings of a recent hydropower dam in the Mekong basin, the Nam Theun 2 project. Results of two different approaches, experimental surface gillnets capture and environmental DNA metabarcoding assays based on 12S ribosomal RNA and cytochrome b, were compared during 3 years (2014-2016). Pitfalls and benefits were identified for each method but the combined use of both approaches indisputably allows describing more accurately fish diversity around the reservoir. Importantly, striking convergent results were observed for biodiversity reports. 75% of the fish species caught by gillnets (62/82) were shown by the metabarcoding study performed on DNA extracted from water samples. eDNA approach also revealed to be sensitive by detecting 30 supplementary species known as present before the dam construction but never caught by gillnets during 3 years. Furthermore, potential of the marker-genes study might be underestimated since it was not possible to assign some sequences at lower taxonomic levels. Although 121 sequences were generated for this study, a third of species in the area, that exhibits high endemism, are still unknown in DNA databases. Efforts to complete local reference libraries must continue to improve the taxonomic assignment quality when using the non-invasive and promising eDNA approach. These results are of broader interest because of increasing number of hydropower projects in the Mekong Basin. They reveal the crucial importance to sample tissues/DNA of species before dam projects, i.e. before the species could become endangered and difficult to catch, to obtain more precise biomonitoring in the future as we believe eDNA metabarcoding will rapidly be integrated as a standard tool in such studies.