Expanding Monitoring Capacity for Potential Invasive Species in Arctic Canada With Environmental DNA Metabarcoding

Coral reefs are biodiversity hotspots, places of high endemicity and provide essential services to billions of people globally. With increasing threats to these reefs worldwide, there is a need to implement faster, more efficient ways to monitor spatial and temporal patterns of biodiversity. Environmental DNA (eDNA) metabarcoding offers a promising tool to address this issue, as it has revolutionized our ability to monitor biodiversity from complex environmental samples such as seawater. However, the capacity for eDNA to resolve fine scale shifts in community composition across habitats in seascapes is yet to be fully explored. Here, we applied eDNA metabarcoding using the rRNA 18S Universal eukaryote assay to explore differences in community profiles between samples collected from the lagoon and reef slope habitats across more than 170 km of the Ningaloo Coast World Heritage Area in Western Australia. We recovered 2061 amplicon sequence variants that comprised of 401 taxa spanning 14 different metazoan phyla such as cnidarians, poriferans, molluscs, algae, worms, and echinoderms. Our results revealed strong clustering of samples by habitat type across the length of the reef. Community dissimilarity (beta diversity) between samples collected from the reef slope and lagoon habitats was high and was driven largely by a strong rate of spatial turnover, indicating a distinct set of taxa representing each reef zone community. We also detected a strong pattern of isolation by distance within our slope samples, suggesting that communities are spatially stratified across the length of the reef. Despite high connectivity due to regular flushing of the lagoon environment, our results demonstrate that metabarcoding of seawater eDNA from different habitats can resolve fine scale community structure. By generating multi-trophic biodiversity data, our study also provided baseline data for Ningaloo from which future changes can be assessed.

The karst aquifer of the Yucatán Peninsula (YP) in southeastern Mexico is a unique ecosystem in which water-filled sinkholes, locally known as cenotes, connect subterranean waters with the surface. This system is home to around 20 species of freshwater fishes, including several that are endemic and/or threatened. Studies on this unique ichthyofauna have been partially hampered by the technical difficulties associated with sampling these habitats, particularly submerged caves. In this proof-of-concept study, we use environmental DNA (eDNA) metabarcoding to survey the diversity of freshwater fishes associated with the YP karst aquifer by sampling six cenotes from across the Ring of Cenotes region in northwestern Yucatán, a 180-km-diameter semicircular band of abundant karst sinkholes. Through a combination of conventional sampling (direct observation, fishing) and eDNA metabarcoding, we detected eight species of freshwater fishes across the six sampled cenotes. Overall, our eDNA metabarcoding approach was effective at detecting the presence of fishes from cenote water samples, including one of the two endemic cave-dwelling fish species restricted to the subterranean section of the aquifer. Although our study was focused on detecting fishes via eDNA, we also recovered DNA from several other vertebrate groups, particularly bats. These results suggest that the eDNA metabarcoding approach represents a promising and largely noninvasive method to assay aquatic biodiversity in these vulnerable habitats, allowing more effective, frequent, and wide-ranging surveys. Our detection of DNA from aerial and terrestrial vertebrate fauna implies that eDNA from cenotes, besides being a means to survey aquatic fauna, may also offer an effective way to quickly survey non-aquatic biodiversity associated with these persistent water bodies.

The environmental DNA (eDNA) metabarcoding is a methodology that, from environmental samples such as soil, water, air, and others, enables the simultaneous identification of multiple species, thus allowing for large-scale mapping of biological diversity in a specific study area. Due to its non-invasive sampling approach, where species are detected from the traces they leave in the environment, eliminating the need to isolate and capture organisms, eDNA metabarcoding emerges as a valuable tool in conservation strategies. This study aims to explore the use of eDNA methodology for biodiversity monitoring and environmental impact assessment caused by the 2020 megafire in the Pantanal of Brazil, focusing on vertebrates. Environmental samples were collected at two protected areas and their surrounding areas, Taiamã Ecological Station (TES) and Pantanal Matogrossense National Park (PMNP), Mato Grosso, Brazil. We identified in TES, 27 mammals, 56 fishes, 12 birds, 4 amphibians, and 4 reptiles, while in PMNP, 43 mammals, 45 fishes, 126 birds, 19 amphibians, and 11 reptiles. Soil sampling proved to be more efficient compared to water sampling: 26 species were exclusively identified in soil samples, while 9 were attributed to water samples. Here, we demonstrated that the primer 12SV5 only a superior efficacy in identifying mammal and herpetofauna species compared to the other markers used (16Smam and MiBird). Moreover, we confirmed the complementary role of eDNA alongside camera trapping, and its advantage to estimate species richness with a single field expedition. We stress the need to optimize sample collection methods for the target group and to reduce the influence of contamination and water flow. This study highlights the importance of eDNA methodology as a crucial tool for biodiversity monitoring and environmental impact assessment, enabling rapid access to biodiversity and long-term monitoring.

Environmental DNA (eDNA) metabarcoding has been increasingly applied to biodiversity surveys in stream ecosystems. In stream networks, the accuracy of eDNA-based biodiversity assessment depends on whether the upstream eDNA influx affects downstream detection. Biodiversity assessment in low-discharge streams should be less influenced by eDNA transport than in high-discharge streams. We estimated α- and β-diversity of the fish community from eDNA samples collected in a small Michigan (USA) stream from its headwaters to its confluence with a larger river. We found that α-diversity increased from upstream to downstream and, as predicted, we found a significant positive correlation between β-diversity and physical distance (stream length) between locations indicating species turnover along the longitudinal stream gradient. Sample replicates and different genetic markers showed similar species composition, supporting the consistency of the eDNA metabarcoding approach to estimate α- and β-diversity of fishes in low-discharge streams.

The use of environmental DNA (eDNA) metabarcoding to analyze fish species diversity across different aquatic ecosystems is well documented. Nonetheless, there is a gap in validating eDNA metabarcoding studies on the diversity and structure of fish communities in coastal ecosystems, particularly in comparing these findings with bottom trawl catch data. In this study, we employed eDNA metabarcoding to explore species composition and relative abundance in fish communities, taxonomic-level diversity variations, and the interplay between community structures and environmental factors in the Yellow Sea and compared these results with those obtained from bottom trawl catches. In addition, we compared the various methods used to estimate the distributions of taxonomic, phylogenetic, and functional diversity factors. We found that eDNA metabarcoding detected a greater number of species (86 vs. 41), genera (73 vs. 37), and families (42 vs. 25) than bottom trawl results at each sampling station. eDNA metabarcoding provided higher Shannon, Simpson, and Chao1 alpha diversity indices than the bottom trawl results. The PCoA results showed that eDNA metabarcoding samples could be more clearly separated at the sampling sites in the Zhuanghe (ZH) and Lianyungang (LYG) areas than bottom trawling samples. The RDA analysis indicated that temperature, along with NO3- and NH4+ concentrations, were pivotal in shaping the geographical patterns of fish communities, as identified through eDNA metabarcoding, echoing findings from bottom trawling studies. Furthermore, our findings suggest that eDNA barcoding surpasses bottom trawling in detecting taxonomic and phylogenetic diversity, as well as in uncovering greater functional diversity at the local level. Conclusively, eDNA metabarcoding emerges as a valuable complement to bottom trawling, offering a multifaceted approach to biodiversity monitoring that not only boosts efficiency but also reduces environmental impact on coastal ecosystems.

Abstract. Roesma DI, Tjong DH, Syaifullah, Nofrita, Janra MN, Prawira FDL,Salis VM, Aidil DR. 2023.The importance of DNA barcode reference libraries and selection primer pair in monitoring fish diversity using environmental DNA metabarcoding. Biodiversitas 24: 2251-2260. Environmental DNA (eDNA) metabarcoding has become an alternative method used for biodiversity monitoring of an ecosystem. The eDNA metabarcoding has advantages compared to the conventional method because it is non-invasive, quick, and requires less cost. However, the effectiveness of the eDNA method is highly dependent on the coverage of the DNA barcode reference and primerpair. A study using the eDNA method was conducted for fish biodiversity monitoring in Singkarak Lake. Two-liter water samples were collected using sterile bottle samples at each sampling site (five sites). The universal primers (Fish FI and Fish R1) used for Next-generation sequencing (GRIDION, Nanopore, Oxford Technologies). The study detected 152 fish species using eDNA metabarcoding. Ten species out of the 30 originally reported in Singkarak Lake were detected using eDNA metabarcoding. The low percentage of fish detected is thought to be due to several factors; incomplete/unavailability of freshwater fish DNA barcodes in Indonesia registered in the database repository, inappropriate primer pair selection, low DNA quality, and the absence of target species DNA in collected water samples. The results demonstrated the significance of correctly registering DNA barcodes to the database and appropriate primer pair selection to identify eDNA metabarcoding. This study provides recommendations using eDNA metabarcoding for monitoring in future work.

Diversity and ecology of fungal assemblages present in lake sediments at Clearwater Mesa, James Ross Island, Antarctica, assessed using metabarcoding of environmental DNA.

Simple SummaryThe selection of fish community survey methods based on their efficiency and reliability is critical for ecosystem conservation, protection and management. In this study, we used environmental DNA (eDNA) metabarcoding and traditional (gillnet) fishing to assess the diversity of fishes along a mangrove–seagrass–coral reef continuum of marine habitats in Hainan, China. Higher fish taxonomic diversity was identified using eDNA; however, gillnet fishing was better at identifying fish communities from different habitats along the sampled continuum. Results from both survey methods indicate that some fish species use multiple habitats along mangrove–seagrass–coral reef continuums. Therefore, the concurrent use of eDNA and gillnet survey methods provides a more comprehensive approach to understanding the heterogeneity of fish taxonomic and functional diversity along mangrove–seagrass–coral reef continuums.The effective and reliable monitoring of fish communities is important for the management and protection of marine ecosystems. Environmental DNA (eDNA) metabarcoding is a relatively new method that has been widely used in recent years, while traditional sampling via fish catching (i.e., gillnets) is one of the most common and reliable fish monitoring methods used to date. We compared the taxonomic and functional diversity of fish detected within a mangrove–seagrass–coral reef continuum using both survey methods. One liter seawater and gillnet samples were collected in August 2021 from mangrove forests, seagrass meadows and coral reef habitats (n = 3 each) in Hainan, China. Surveys using eDNA and gillnets identified 139 genera belonging to 66 families and 58 genera belonging to 42 families, respectively. Regardless of the survey method, fish detected in mangrove, seagrass and coral reef habitats were heterogeneous in their communities; however, the shared species between habitats suggest some degree of connectivity. There were no significant differences between habitats in terms of taxonomic and functional diversity, but a higher taxonomic diversity was detected using eDNA. Both methods were able to distinguish fish assemblages between different habitats; however, gillnet surveys performed better than eDNA surveys for distinguishing mangrove from seagrass assemblages. Therefore, the concurrent use of eDNA and gillnet survey methods provides a more comprehensive approach to understanding the heterogeneity of fish taxonomic and functional diversity along mangrove–seagrass–coral reef continuums.

ABSTRACTUnderstanding the intricate dynamics of biodiversity within and across riverine ecosystems, influenced by geological history and environmental factors, is crucial for effective conservation and management strategies. Italy, particularly the Ligurian region, harbors diverse freshwater fish communities and populations shaped by unique geological and hydrological conditions. Here, we investigated the suitability of environmental DNA (eDNA) metabarcoding to identify inter‐ and intraspecific diversity patterns of riverine fish populations in drainage basins on both sides of the main drainage divide (MDD) between the Adriatic and Ligurian river basins in Northern Italy. We collected 96 aquatic eDNA samples across 48 riverine sites, amplified them using a cytochrome b primer pair, and denoised the sequences to retrieve amplicon sequence variants (ASVs). We calculated communities' phylogenetic distance with betaMPD based on genetic distances derived from the ASVs, combined them with conductance‐based landscape metrics, and applied generalized dissimilarity models to assess spatial genetic structure. Our results reveal genetic differentiation among populations of several fish species, with some displaying clustering patterns across the main drainage divide and isolation by distance patterns. Overall, taxon richness was higher on the Adriatic side (12.82 ± 3.57; 26 unique taxa) than on the Ligurian side of the MDD (8.35 ± 3.66 SD, 25 unique taxa), but the other way around for ASV richness across all species (Ligurian side: 51.94 ± 25.79, 308 unique ASV, Adriatic side: 68.00 ± 32.97, 274 unique ASV). Our findings highlight the effectiveness of eDNA metabarcoding in uncovering various facets of diversity, shedding light on hidden genetic diversity among ASVs, and revealing significant spatial genetic structuring in freshwater fish populations across multiple species.

Environmental DNA (eDNA) metabarcoding is a powerful tool for monitoring aquatic biodiversity in extreme environments like the Qinghai–Tibet Plateau (QTP), where traditional methods face significant challenges. However, key knowledge gaps remain, particularly in selecting optimal sampling substrates for target taxonomic groups and understanding substrate‐specific detection differences. Here, using eDNA metabarcoding with five primer sets (targeting mitochondrial, nuclear, and nucleoid sequences), we systematically compared biodiversity detection between sediment and water samples collected from five sites in the Dongjicuona Lake across the tree of life (encompassing archaea, bacteria, algae, fungi, plants, protozoa, invertebrates, fishes, birds, and mammals). The results showed that sediment eDNA exhibited significantly higher amplicon sequence variant (ASV) richness compared to water eDNA for most taxonomic groups. In contrast, the opposite pattern was observed for fishes, while no significant differences were observed for fungi and birds. Community composition differs significantly between sediment and water samples for most taxonomic groups, except for mammals. Across all taxonomic groups, 18.0% of ASVs were shared between sediments and water, with fishes showing the highest overlap (42.6%) and archaea the lowest (4.5%). Additionally, the percentages of ASVs contributing to major community dissimilarity (cumulatively accounting for 80%) ranged from 6.4% (invertebrates) to 17.4% (protozoa), with most taxa exhibiting distinct habitat preferences. These findings support water sampling as an effective method for monitoring fish diversity, while emphasizing the need for a multi‐substrate approach to comprehensively monitor other taxonomic groups in plateau lakes.

The need for efficient and more accurate ways of monitoring threatened ecosystems is becoming increasingly urgent as climate change intensifies. Coral reefs are an example of an ecosystem in crisis, with widespread declines in coral cover and diversity documented over recent decades. Novel molecular approaches such as biomonitoring using environmental DNA (eDNA) from seawater samples show great potential to complement future coral reef monitoring programs, especially when used in combination with conventional methods. However, eDNA metabarcoding studies often rely on public databases (e.g., GenBank) for assigning taxonomy, which generally limits the number of sequences that can be taxonomically identified. The extent to which building reference tissue sequences improves taxonomic resolution has yet to be fully examined. Here, we combined traditional coral reef monitoring data with eDNA assessments derived from seawater collected at the highly diverse Rowley Shoals in Western Australia. Using two ITS2 assays developed to target basal metazoan DNA and a reference database spiked with 70 local coral specimens, we identified 37 genera and 40 species from 56 1 L seawater samples. We identified considerable overlap of taxa with visual survey data and showed that assignment of amplicon sequence variants was significantly improved when “spiking” the taxonomic classifier with curated sequences of locally collected species. Our findings showcase the potential of eDNA metabarcoding for monitoring the biodiversity of reef corals and highlight the importance of custom reference sequence databases for improving taxonomic resolution in metabarcoding studies.

Remote polar and deepwater fish faunas are under pressure from ongoing climate change and increasing fishing effort. However, these fish communities are difficult to monitor for logistic and financial reasons. Currently, monitoring of marine fishes largely relies on invasive techniques such as bottom trawling, and on official reporting of global catches, which can be unreliable. Thus, there is need for alternative and non-invasive techniques for qualitative and quantitative oceanic fish surveys. Here we report environmental DNA (eDNA) metabarcoding of seawater samples from continental slope depths in Southwest Greenland. We collected seawater samples at depths of 188–918 m and compared seawater eDNA to catch data from trawling. We used Illumina sequencing of PCR products to demonstrate that eDNA reads show equivalence to fishing catch data obtained from trawling. Twenty-six families were found with both trawling and eDNA, while three families were found only with eDNA and two families were found only with trawling. Key commercial fish species for Greenland were the most abundant species in both eDNA reads and biomass catch, and interpolation of eDNA abundances between sampling sites showed good correspondence with catch sizes. Environmental DNA sequence reads from the fish assemblages correlated with biomass and abundance data obtained from trawling. Interestingly, the Greenland shark (Somniosus microcephalus) showed high abundance of eDNA reads despite only a single specimen being caught, demonstrating the relevance of the eDNA approach for large species that can probably avoid bottom trawls in most cases. Quantitative detection of marine fish using eDNA remains to be tested further to ascertain whether this technique is able to yield credible results for routine application in fisheries. Nevertheless, our study demonstrates that eDNA reads can be used as a qualitative and quantitative proxy for marine fish assemblages in deepwater oceanic habitats. This relates directly to applied fisheries as well as to monitoring effects of ongoing climate change on marine biodiversity—especially in polar ecosystems.

Marine ecosystems nestle species or populations known to be threatened due to human
 overexploitation. Reliable detection and monitoring of threatened organisms is crucial for
 data-driven conservation actions. Furthermore, misidentification of species represents a major
 problem. Here, we investigate the potential of using metabarcoding of environmental DNA
 (eDNA) obtained directly from seawater samples to detect endangered grouper species
 (Epinephelus spp.). Cytochrome c oxidase subunit I (COI) fragment of mtDNA was used to
 detect groupers species in the Mediterranean Coasts. We conducted eDNA sampling at sites
 by underwater diving across the range of the Grouper species habitats in Northeastern
 Mediterranean (Antalya-Kas Region and Iskenderun Bay). eDNA was isolated from 2 liter
 seawater samples which were vacuum-filtered onto 0.45-mm membrane filters. Filters were
 then folded inwards, placed in 2 ml tubes and stored at -20 oC until DNA extraction, which
 took place within 24 hours. DNA was extracted from the water sample filters using the
 DNeasy Blood and Tissue Kit (Qiagen, USA). Manufacturer’s protocols were used during all
 steps. PCR amplification of eDNA samples were done using selective primers of COI region
 of mitochondrial DNA, and next-generation DNA sequencing of PCR application was
 conducted. For the successfully obtained COI sequences, maximum matching rates were
 revealed as 80% for Epinephelus marginatus, 78,95% for Epinephelus aeneus, 73,48% for
 Epinephelus costae, 63,45% for Epinephelus caninus, 60,12% for Mycteroperca rubra and
 57,12% for Hyporthodus haifensis. Despite the methodological challenges inherent in eDNA
 analysis, the results demonstrated that eDNA method may be proved to step towards a new
 beginning to detect and monitor endangered grouper species.

Multi-marker eDNA metabarcoding reveals more abundant coral biodiversity in a subtropical coastal reef ecosystem.

Fish aquaculture potentially affects the local ecosystem through feed (leftovers) and feces. However, evaluation of its impacts on biodiversity, especially those of mobile organisms such as fish, is not straightforward. Here, we report a case study in which environmental DNA (eDNA) metabarcoding with MiFish universal primers for the 12S rRNA region of fish is used to detect and assess the impact of aquaculture on local fish communities. In Shizugawa Bay, Minamisanriku Town, Miyagi Prefecture, Japan, Oncorhynchus kisutch (coho salmon) is aquacultured in sea cages from November to July, but is absent from August to October. We conducted eDNA surveys in June, August, October, and December 2021 of water at depths of 5 m and 1 m above the bottom obtained from two sites, one approximately 100 m and the other approximately 2500 m from the nearest aquaculture site. The total number of operational taxonomic units (OTUs) detected was 122, with a maximum of 50 and a minimum of 10. With distance‐based redundancy analysis (dbRDA) and PERMANOVA applied to the eDNA‐based community compositional data, we detected a significant effect of the presence of O. kisutch aquaculture on fish biodiversity. The generalized linear model (GLM) applied to each detected OTUs indicated that the aquaculture was associated positively with Leucopsarion petersii (ice goby), and Oncorhynchus spp. and negatively with Chaenogobius annularis (forktongue goby). Environmental DNA metabarcoding surveys, combined with multivariate statics, are a promising tool to identify the dimension of biodiversity that human activity affects and would serve as a basic survey for sustainable use of the sea.