Accounting for Intra- and Intergenomic Sequence Variation in Reference Barcodes Improves eDNA Metabarcoding Biodiversity Assessment.

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

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.

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.

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.

Fishery resources in Nigeria are experiencing a rapid decline, underscoring the urgent need for effective biodiversity surveys and comprehensive species documentation to guide conservation and management efforts. However, traditional methods for fish surveys, such as gillnets and trawl surveys, are intrusive, time-intensive, and costly. Environmental DNA (eDNA) metabarcoding has emerged as a non-invasive method for surveying fish diversity, yet its application in Nigerian aquatic ecosystems remains limited. This study evaluated the potential and limitations of 12S rRNA-based eDNA metabarcoding to survey fish communities across three water bodies (Jebba Hydroelectric Power Dam, Asa River, and Lagos lagoon). We also compared fish community composition obtained from our eDNA samples with previously published inventories based on traditional sampling techniques. Our study revealed several advantages of using eDNA metabarcoding for fish biodiversity surveys in Nigeria, including the rapid and non-invasive identification of fish taxa, the detection of differences in community composition and biodiversity metrics, the identification of species overlooked by traditional methods, and the ability to detect both threatened and invasive species. However, we identified three major constraints that must be addressed for the successful integration of eDNA sampling techniques in Nigeria. First are the logistical challenges, including determining the optimal number of replicates, selecting the appropriate water volume per site, determining the required number of sampling sites per study area, and identifying the most suitable time within the season for sampling. Second is the lack of a comprehensive DNA reference database for accurate species identification. Third are the issues with primer and marker specificity. Until a comprehensive regional DNA reference library is developed, suitable molecular markers are optimized, and standardized field protocols for eDNA sampling are established, we recommend using eDNA metabarcoding as a complementary survey tool rather than a replacement for conventional fish survey methods in Nigeria.

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.

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

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.

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.

Fish are among the main target groups when conducting biomonitoring and ecological assessment of aquatic environments. Environmental DNA (eDNA) metabarcoding provides a unique opportunity to enhance biomonitoring throughput, precision, and standardization. However, few fish monitoring studies based on eDNA metabarcoding have been conducted at the basin scale. Taking Dianchi Lake and the three inflowing rivers (Panlong River, Baoxiang River, and Chai River) as research objects, this study monitored fish diversity via eDNA metabarcoding, and the Fish Index of Biological Integrity (F-IBI) was constructed and applied for the ecological assessment of the aquatic environment. Here, we detected 41 species belonging to 9 orders, 15 families, and 35 genera in the sampling areas, including 17 native fish species such as Hemiculter leucisculus, Cyprinus carpio, and Silurus soldatovi. Distinct diversity patterns were observed among Dianchi Lake and the three inflowing rivers. Particularly, the fish diversity of Panlong River was richer than that of Dianchi Lake, Baoxiang River, and Chai River. Water temperature (WT), chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) significantly influenced the distribution of fish communities in most sampling sites of the Dianchi Lake basin (p < 0.05). Moreover, according to the F-IBI assessment system, 25% of the sampling sites were defined as ‘fine’ or above, and 75% were ‘impaired’ or below. This study provided insights into the ecological restoration and precise management of the aquatic environment of the Dianchi Lake basin.

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.

Introduction Human activities and their effects on ecosystems are threatening biodiversity and causing a decline in species, population, and genetic diversity. Environmental DNA (eDNA) metabarcoding represents a powerful instrument to assess marine biodiversity, enabling an efficient, cost-effective, and non-invasive approach. eDNA metabarcoding technology has significantly progressed in recent years and has been widely employed to assess marine fish populations. Methods This study assessed its potential, detection sensitivity, and effectiveness for an initial evaluation of fish biodiversity in the Black Sea. The efficacy of eDNA was evaluated by contrasting metabarcoding results with trawling data obtained from two sampling campaigns (summer and autumn 2022) across 16 selected locations along the Bulgarian Black Sea coast. Mitochondrial genes 12S and 16S were utilized as standard markers for metabarcoding given their high specificity and sensitivity in amplifying fish DNA, and the availability of large reference databases. A multi-model analytical approach utilizing Bayesian regression and Generalized Additive Models (GAMs) was implemented to assess the relationships between environmental DNA (eDNA) 12S metabarcoding data and abundance indices obtained from catch-per-unit-effort (CPUE). The method addresses significant challenges in predicting abundance from eDNA, including scarce, noisy datasets, excessive zeros, and nonlinear predictor-response relationships. Both frameworks reliably detected biologically meaningful associations between eDNA signal strength, environmental gradients, and trawl-derived abundance. Results Metabarcoding, employing the 12S mitochondrial gene with MiFish-U primers, exhibited superior sensitivity in identifying a broader array of fish species, highlighting its efficacy in detecting eDNA traces of rare and migratory species. During the autumn survey, environmental DNA (eDNA) analysis identified 23 fish species, compared to 15 species recorded through trawl sampling. In the summer expedition, 12 species were detected using molecular methods, while trawl surveys recorded 9 species. The Bayesian framework provided robust uncertainty quantification and reliable inference with few samples, whereas the GAM framework effectively captured nonlinear environmental and spatial effects. The results indicate that eDNA data can replicate patterns observed in conventional trawl surveys. This supports the assumption that eDNA data may serve as a low-impact, scalable method for monitoring fish populations. Model uncertainty informs the need for more sampling or a redesign of covariates to improve the accuracy of eDNA-based abundance predictions. Discussion Our findings demonstrate that the eDNA 12S primer combination exhibited superior taxonomic diversity compared to trawling, while necessitating reduced sampling effort. Furthermore, the eDNA-based approach can identify species across diverse life stages and sizes, unlike traditional trawling, which predominantly captures adult specimens within particular size categories. Although eDNA metabarcoding has limits in providing estimates for absolute biomass and size distributions, it provides significant insights on the presence of numerous fish species within the studied ecosystem. The consistency of results from multiple statistical frameworks supports the validity of eDNA counts as an abundance measure, provided that analytical models effectively account for dispersion and environmental variability. The Bayesian regression approach enabled thorough uncertainty quantification and dependable parameter inference with constrained sample sizes, whereas Generalized Additive Models (GAMs) provided an advanced representation of potentially nonlinear relationships among environmental DNA, environmental variables, and abundance.

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.

ABSTRACTIntensive agriculture and farming activities, industry, as well as human settlement can seriously affect freshwater ecosystems and the species they host. These disturbances have been previously monitored using traditional catch surveys. New methods, such as environmental DNA (eDNA) metabarcoding, can significantly improve biodiversity mapping by accessing rarely visited areas and by enhancing detection of rare and invasive species. We conducted an eDNA survey in a complex river and tributary system at Tenagi, Philippi that hosts approximately 12.5% of Greek freshwater ichthyofauna. A primer pair targeting the 12S rRNA was used to analyse eDNA samples from 30 sites. A reference sequence database was generated to improve regional diversity monitoring of endemic, native and invasive fishes. Results were compared with data collected using electrofishing from stations that allowed access. Two endemic, three invasive, two translocated and 10 native species were detected in their current known distribution using both methods. Overall, eDNA metabarcoding being more sensitive, consistently detected more freshwater species than traditional methods on a single fieldwork expedition. Co‐occurrence of species was also investigated for the first time, revealing minor variations in species composition within fish communities. Stations in intensive agricultural zones with potentially high accumulation of organic and inorganic pollutants (Tributary D), exhibited lower number of reads and species richness than other tributaries at Tenagi. Our results demonstrate the need for complementary use of both eDNA metabarcoding and electrofishing to assess fish assemblages in freshwater habitats, as a combined approach can address reliability issues and biases associated with each method.

The Mediterranean region with its islands is among the top biodiversity hotspots. It houses numerous freshwater taxa with a high rate of endemism, but is heavily impacted by anthropogenic pressures and global climate change. To conserve biodiversity, reliable data on species and genetic diversity are needed especially for the scarcely known insular freshwater ecosystems. Environmental DNA (eDNA) metabarcoding provides a straight-forward opportunity to assess aquatic biodiversity. Therefore, we conducted the first eDNA metabarcoding study in one stream catchment on Sicily. Specifically, we aimed to (i) investigate spatial diversity patterns of macroinvertebrate communities, (ii) assess seasonal changes (autumn and winter), and (iii) check if dispersal barriers can be identified. Water samples were taken at 27 different sites in two seasons and eDNA metabarcoding was performed using a fragment of the mitochondrial cytochrome c oxidase subunit I gene as a marker. In total, we detected 98 macroinvertebrate species, including 28 taxa potentially new to Sicily. Exact sequence variant and species composition data showed that diversity differed between seasons with less taxa detected in winter. We also detected a dispersal barrier, which had a stronger effect in autumn. Our findings show that eDNA metabarcoding provides valuable information on Sicilian freshwater biodiversity. We therefore encourage its application for understudied regions to better understand the state and dynamics of freshwater biodiversity.