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

Environmental DNA (eDNA) metabarcoding can rapidly characterise biodiversity, yet its accuracy and effectiveness are limited by incomplete DNA barcode reference databases. We evaluated how comprehensive reference databases that include sequence variation within genomes (intragenomic) and across individuals and species (intergenomic) improve eDNA-based biodiversity assessments. We collected coral tissue and water samples at deep sites offshore Puerto Rico for reference barcoding and eDNA metabarcoding. Genome skimming coral specimens yielded 28S barcodes for 314 of 346 samples (90.8%) and revealed divergent intragenomic 28S lineages in multiple octocoral families. Incorporating local reference barcodes substantially changed ASV taxonomic classifications: 22 ASVs (8.9%) gained genus-level resolution, 19 ASVs (7.7%) were reassigned to different genera, and 14 ASVs (5.7%) lost incorrect genus-level classifications. Thus, incomplete reference databases produce not only unclassified ASVs but also false positive detections and ecologically meaningful misclassifications. When intragenomic 28S lineages were excluded from the reference database, 18 ASVs (7.4%) could not be classified to family or genus, demonstrating that unrecognised intragenomic variation can be mistaken for unsampled taxa. Integrating reference genome skimming and eDNA metabarcoding expanded known coral family richness by 36% at depths shallower than 1000 m and by 181% at depths greater than 1000 m. eDNA also detected two coral families previously unknown off Puerto Rico and nearby islands, underscoring its potential for biodiversity discovery.

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.

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.

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.

Monitoring of marine protected areas (MPAs) is critical for marine ecosystem management, yet current protocols rely on SCUBA-based visual surveys that are costly and time consuming, limiting their scope and effectiveness. Environmental DNA (eDNA) metabarcoding is a promising alternative for marine ecosystem monitoring, but more direct comparisons to visual surveys are needed to understand the strengths and limitations of each approach. This study compares fish communities inside and outside the Scorpion State Marine Reserve off Santa Cruz Island, CA using eDNA metabarcoding and underwater visual census surveys. Results from eDNA captured 76% (19/25) of fish species and 95% (19/20) of fish genera observed during pairwise underwater visual census. Species missed by eDNA were due to the inability of MiFish 12S barcodes to differentiate species of rockfishes (Sebastes, n = 4) or low site occupancy rates of crevice-dwelling Lythrypnus gobies. However, eDNA detected an additional 23 fish species not recorded in paired visual surveys, but previously reported from prior visual surveys, highlighting the sensitivity of eDNA. Significant variation in eDNA signatures by location (50 m) and site (~1000 m) demonstrates the sensitivity of eDNA to address key questions such as community composition inside and outside MPAs. Results demonstrate the utility of eDNA metabarcoding for monitoring marine ecosystems, providing an important complementary tool to visual methods.

Monitoring arthropod pests and their natural enemies provides essential information for pest control decisions in agricultural production. Traditional monitoring methods, such as trapping and visual surveys, have practical limitations because of their time-consuming preprocessing steps and the need for knowledgeable taxonomists. We tested the environmental DNA (eDNA) metabarcoding approach for monitoring cowpea (Vigna unguiculata) arthropod pests and their natural enemies across seven climatic sub-regions of China, comparing it with traditional sample collection and morphological identification methods. We showed that eDNA metabarcoding reveals a higher species richness of cowpea arthropods than the traditional visual method. The eDNA method detected 163 species, of which 72, 33 and 57 were pests, natural enemies and other insects, respectively, whereas the traditional method only detected 70 species. There were significant differences in alpha- and beta-diversity for arthropods on cowpea in different climatic sub-regions. We also found that longitude, altitude and geographical distance are associated with species diversity, but latitude, mean air temperature and temperature range are not. This study represents the first use of eDNA metabarcoding to investigate cowpea arthropod communities, providing a novel method for pest monitoring. © 2025 Society of Chemical Industry.

Dragonflies (Odonata) are widely recognized as bioindicators of freshwater ecosystem quality, yet conventional morphological observations often face limitations in species identification. Environmental DNA (eDNA) metabarcoding offers a more sensitive and efficient approach to assess community responses to environmental changes. This study aimed to analyze the diversity of Odonata in an urban river corridor following riparian restoration using an eDNA metabarcoding approach. Water samples were collected from several restored riparian sites, followed by DNA extraction and amplification of the Cytochrome Oxidase I (COI) gene. Sequencing data were processed through bioinformatics pipelines for species identification and diversity analysis. The results showed that eDNA metabarcoding detected a greater number of Odonata species compared to conventional field observations. The Shannon-Wiener diversity index ranged from moderate to high (H’ = 2.1–2.8), with dominant species belonging to the families Libellulidae and Coenagrionidae. Moreover, sensitive indicator species were identified, indicating improved habitat quality after restoration. In conclusion, eDNA metabarcoding proved to be an effective tool for detecting Odonata diversity and can serve as a reliable biomonitoring method to evaluate the success of riparian restoration in urban river ecosystems.

Determining the timing and location of fish reproductive events is crucial for the implementation of correct management and conservation schemes. Conventional methods used to monitor these events are often unable to assess the spawning activity directly or can be invasive and therefore problematic. This is especially the case when threatened fish populations are the study subject, such as the Arctic charr ( Salvelinus alpinus L.) populations in Windermere (Cumbria, UK). Arctic charr populations have been studied in this lake since the 1940s, and the locations and characteristics of spawning grounds have been described in detail using techniques such as hydroacoustics, as well as physical and visual surveys of the lake bottom. Here, in conjunction with established netting surveys, we added an environmental DNA (eDNA) metabarcoding approach to assess the spatial distribution of Arctic charr in the lake throughout the year to test whether this tool could allow us to identify spawning locations and activity. Sampling was carried out between October 2017 and July 2018 at three locations in the lake, covering putative and known spawning sites. eDNA metabarcoding provided accurate spatial and temporal characterization of Arctic charr spawning events. Peaks of Arctic charr relative read counts from eDNA metabarcoding were observed during the spawning season and at specific locations of both putative and known spawning sites. Net catches of mature Arctic charr individuals confirmed the association between the Arctic charr spawning activity and the peaks of eDNA metabarcoding relative read counts. This study demonstrates the ability of eDNA metabarcoding to effectively and efficiently characterize the spatial and temporal nature of fish spawning in lentic systems.

Environmental DNA metabarcoding for benthic monitoring: A review of sediment sampling and DNA extraction methods

Understanding biodiversity in aquatic systems is critical to ecological research and conservation efforts, but accurately measuring species richness using traditional methods can be challenging. Environmental DNA (eDNA) metabarcoding, which uses high‐throughput sequencing and universal primers to amplify DNA from multiple species present in an environmental sample, has shown great promise for augmenting results from traditional sampling to characterize fish communities in aquatic systems. Few studies, however, have compared exhaustive traditional sampling with eDNA metabarcoding of corresponding water samples at a small spatial scale. We intensively sampled Boardman Lake (1.4 km2) in Michigan, USA, from May to June in 2019 using gill and fyke nets and paired each net set with lake water samples collected in triplicate. We analyzed water samples using eDNA metabarcoding with 12S and 16S fish‐specific primers and compared estimates of fish diversity among methods. In total, we set 60 nets and analyzed 180 1 L lake water samples. We captured a total of 12 fish species in our traditional gear and detected 40 taxa in the eDNA water samples, which included all the species observed in nets. The 12S and 16S assays detected a comparable number of taxa, but taxonomic resolution varied between the two genes. In our traditional gear, there was a clear difference in the species selectivity between the two net types, and there were several species commonly detected in the eDNA samples that were not captured in nets. Finally, we detected spatial heterogeneity in fish community composition across relatively small scales in Boardman Lake with eDNA metabarcoding, but not with traditional sampling. Our results demonstrated that eDNA metabarcoding was substantially more efficient than traditional gear for estimating community composition, highlighting the utility of eDNA metabarcoding for assessing species diversity and informing management and conservation.

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 is a relatively new monitoring tool featuring in an increasing number of applications such as the facilitation of the accurate and cost effective detection of species in environmental samples. eDNA monitoring is likely to have a major impact on the ability of salmonid aquaculture industry producers and their regulators to detect the presence and abundance of pathogens and other biological threats in the surrounding environment. However, for eDNA metabarcoding to develop into a useful bio-monitoring tool it is necessary to (a) validate that sequence datasets derived from amplification of metabarcoding markers reflect the true species’ identity, (b) test the sensitivity under different abundance levels and environmental noise and (c) establish a low-cost sequencing method to enable the bulk processing of field samples. In this study, we employed an elaborate experimental design whereby different combinations of five biological agents were crossed at three abundance levels and exposed to sterile pre-filtered and unfiltered seawater, prior to coarse filtering and then eDNA ultrafiltration of the resultant material. We then benchmarked the low-cost, scalable, Ion Torrent sequencing method against the current gold-standard Illumina platform for eDNA surveys in aquaculture. Based on amplicon-seq of the 18S SSU rDNA v9 region, we were able to identify two parasites (Lepeophtheirus salmonis and Paramoeba perurans) to species level, whereas the microalgae species Prymnesium parvum, Pseudo-nitzschia seriata, and P. delicatissima could be assigned correctly only to the genus level. Illumina and Ion Torrent provided near identical results in terms of community composition in our samples, whereas Ion Torrent was more sensitive in detecting species richness when the medium was unfiltered seawater. Both methods were able to reflect the difference in relative abundance between treatments in 4 out of 5 species when samples were exposed to the unfiltered seawater, despite the significant amount of background noise from both bacteria and eukaryotes. Our findings indicate that eDNA metabarcoding offers significant potential in the monitoring of species harmful to aquaculture and for this purpose, the low-cost Ion Torrent sequencing is as accurate as Illumina in determining differences in their relative abundance between samples.

Environmental DNA (eDNA) metabarcoding is a powerful tool that can enhance marine ecosystem/biodiversity monitoring programs. Here we outline five important steps managers and researchers should consider when developing eDNA monitoring program: (1) select genes and primers to target taxa; (2) assemble or develop comprehensive barcode reference databases; (3) apply rigorous site occupancy based decontamination pipelines; (4) conduct pilot studies to define spatial and temporal variance of eDNA; and (5) archive samples, extracts, and raw sequence data. We demonstrate the importance of each of these considerations using a case study of eDNA metabarcoding in the Ports of Los Angeles and Long Beach. eDNA metabarcoding approaches detected 94.1% (16/17) of species observed in paired trawl surveys while identifying an additional 55 native fishes, providing more comprehensive biodiversity inventories. Rigorous benchmarking of eDNA metabarcoding results improved ecological interpretation and confidence in species detections while providing archived genetic resources for future analyses. Well designed and validated eDNA metabarcoding approaches are ideally suited for biomonitoring applications that rely on the detection of species, including mapping invasive species fronts and endangered species habitats as well as tracking range shifts in response to climate change. Incorporating these considerations will enhance the utility and efficacy of eDNA metabarcoding for routine biomonitoring applications.

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.

Environmental DNA (eDNA) metabarcoding is a method to detect taxa from environmental samples. It is increasingly used for marine biodiversity surveys. As it only requires water collection, eDNA metabarcoding is less invasive than scientific trawling and might be more cost effective. Here, we analysed data from both sampling methods applied in the same scientific survey targeting Northeast Atlantic fish in the Bay of Biscay. We compared the methods regarding the distribution of taxonomic, phylogenetic, and functional diversity. We found that eDNA captured more taxonomic and phylogenetic richness than bottom trawling and more functional richness at the local scale. eDNA was less selective than trawling and detected species in local communities spanning larger phylogenetic and functional breadths, especially as it detected large pelagic species that escaped the trawl, even though trawling detected more flat fish. eDNA indicated differences in fish community composition that were comparable to those based on trawling. However, consistency between abundance estimates provided by eDNA metabarcoding and trawl catches was low, even after accounting for allometric scaling in eDNA production. We conclude that eDNA metabarcoding is a promising method that can complement scientific trawling for multi-component biodiversity monitoring based on presence/absence, but not yet for abundance.