ABSTRACT Metabarcoding is a powerful tool for assessing microbial community composition in various ecosystems. It involves the amplification of a marker gene followed by high‐throughput sequencing and bioinformatic analysis to identify species. Second‐generation sequencing has democratized biodiversity studies by allowing high‐throughput sequencing of short DNA fragments. Long‐read sequencing now allows for the use of longer markers, potentially offering improved taxonomic resolution. Until recently, this advantage was partly offset by the higher error rate of long‐read sequencing. Here we demonstrate that with current Oxford Nanopore Technologies sequencing (Kit V14 chemistry and R10.4.1 flow cells) combined with an appropriate analysis pipeline, sequencing errors have a negligible impact on the accuracy of long‐read metabarcoding. Using DNA extracted from two protist cultures, we estimated taxonomic misassignment of individual long‐reads at genus level between 0% and 0.01% with long‐read metabarcoding using the 18S rRNA gene and between 0% and 0.01% with short‐read Illumina metabarcoding relying on the V4 region only. We also propose an optimized long‐read clustering procedure that incorporates pre‐sorting reads by quality. When applied to the same cultures, it produced fewer but larger sequence clusters and increased the average similarity to the reference sequences. Applied to environmental DNA extracted from 26 vineyard soil samples, this method identified stronger correlations between protist communities and environmental variables compared to short‐read metabarcoding. Notably, taxonomic assignment of individual long‐reads (without clustering) further increased sensitivity to environmental patterns. These results support the reliability of long‐read metabarcoding and highlight its strong potential for ecological research in general. Broader adoption of this approach may improve the accuracy of biodiversity assessments and in turn, future studies will benefit from the expanded representation of long sequences in public databases.
ABSTRACT Small subunit ribosomal RNA (SSU rRNA)—18S in eukaryotes—is a universally present gene that was central in resolving ancient relationships in early molecular phylogenies. Nowadays, despite multi‐locus phylogenomics dominating, SSU rRNA sequences still serve as a molecular identifier of biodiversity. Phylogenetic placement uses a reference phylogeny and a given evolutionary model to annotate taxonomically environmental DNA, enabling a more accurate identification of divergent sequences that may belong to unknown lineages. Here, we first created the largest dataset of 18S sequences belonging to non‐Bilateria metazoans by curating the sequences found in PR2 database. We then used it as a case study to test the performance of common 18S‐based barcodes (V4, V9, and full‐length 18S) within a phylogenetic placement framework. We found that the V9 region generally lacks sufficient phylogenetic signal for reliable placements. The V4 region is accurate when the environmental diversity is represented in the reference tree but struggles with divergent lineages. Full‐length 18S overcomes short‐read limitations and emerges as the most robust option to uncover major clades. Finally, we apply phylogenetic placement to empirical environmental 18S data. We observe geographical variation in non‐bilaterians communities and recover a putative clade sister to Ctenophora based on long‐sequence barcodes.
ABSTRACT Airborne environmental DNA (eDNA) is emerging as a powerful tool for noninvasive assessment of terrestrial biodiversity. We evaluated the effectiveness of three analytical pipelines for characterizing plant diversity in a botanical greenhouse, with a particular focus on how particle size and sampling height influence detection. Air was sampled for up to 4 days using active Total Suspended Particulate samplers, a 14‐stage impactor, and a vertical filter stack, all equipped with quartz filters. To assess methodological variability and identify optimal approach, three analytical pipelines were applied: (A) Illumina amplicon sequencing with a QIIME‐based workflow, and (B) Illumina and (C) Nanopore shotgun sequencing processed with Kraken2. Of ~2100 plant genera recorded in the greenhouse and adjacent garden, ~86% were considered detectable based on rbcL, trnL, ITS1, and ITS2 barcode coverage. Pipeline A (32 samples) detected 149 genera, 88% of which were known from the garden; 55% of species‐level assignments matched cataloged species, with ~11% attributable to misannotations or contamination. Pipelines B (6 samples) and C (2 samples) detected 76 and 88 genera, respectively, with 81%–82% corresponding to garden taxa and 50%–51% confirmed at species level. The two shotgun pipelines produced highly similar taxonomic profiles despite samples being collected 7 weeks apart, and both were strongly complementary to the amplicon results. Size‐dependent sampling yielded elevated detections in the 2.5 μm impactor fraction, indicate substantial contributions from sub‐pollen particles, while larger fractions (> 40 μm) also contained plant eDNA, suggesting multiple particle carriers or aggregates. Overall, airborne eDNA provided robust detection of local plant biodiversity and yielded new insights into particle‐size distributions relevant to eDNA transport and deposition.
ABSTRACT High‐frequency environmental DNA (eDNA) sampling offers new opportunities to reveal fine‐scale biodiversity patterns and short‐term ecological dynamics, yet its spatial representativity and capacity to capture diel shifts in communities remain poorly understood. Semi‐autonomous sampling devices can aid high frequency and standardized collection for such assessments, particularly in remote and low‐light conditions, as well as dynamic coastal ecosystems such as tropical coral reefs. Coral reefs are ideal habitats to test such systems, as they support exceptional but rapidly declining biodiversity. We constructed Unmanned Surface Vehicles (USVs) for high‐frequency and cost‐efficient geolocalized sample retrieval around the break between two reef flats (Hermitage Pass) of the La Saline−Hermitage coral complex on Reunion Island. The USVs were deployed to navigate between eight stations, pumping seawater through filters to capture eDNA. Sampling occurred during nighttime and daytime (“sampling events”), across four days, to assess potential shifts in metazoan communities. Samples underwent DNA metabarcoding, applying a Cytochrome Oxidase I (COI) gene marker, to target faunal assemblages. Analyses revealed significant differences between communities surveyed during the nighttime and the daytime on each collection day, and across differing stations. Although eDNA dispersal was largely confined to each reef flat and limited among some stations, some communities towards Hermitage Pass were not statistically distinct in composition, suggesting that localized environmental conditions may influence eDNA distributions in this area. Altogether, these findings reveal a diverse and dynamic seascape within less than 1 km 2 , and point to key sites for long‐term monitoring using low‐cost mobile devices for high‐frequency eDNA sampling.
ABSTRACT Mangroves are a key ecosystem for coastal fishery resources, hence understanding the spatial distribution of fish communities is crucial for sustainable management, especially given that conventional net fishing can be ecologically destructive and taxonomically selective. The present research aims to (1) assess and compare the total fish diversity from both environmental DNA (eDNA) metabarcoding and fish catch, (2) analyze the influence of physicochemical parameters on the distribution of fishes and (3) evaluate the complementarity between these two methods. We conducted fish sampling (using drift and trammel nets) and eDNA metabarcoding (using the 12S rRNA MiFish primers) along a salinity gradient in Selangor River, a key mangrove estuary and major fishery ground in Peninsular Malaysia. We detected 89 taxa (68 species) from the eDNA dataset, and 16 taxa (10 species) from the conventional catch. The dominant mangrove fish families found in the eDNA dataset (Gobiidae, Leiognathidae and Lutjanidae) and fish catch (Ariidae, Ambassidae and Sciaenidae) could be confidently matched to known habitat preference in coastal areas. Our data highlighted that water temperature, salinity, pH and wave height is strongly associated with fish community patterns. We infer that eDNA accumulates at the river mouth, particularly under low‐tide conditions. Although eDNA metabarcoding detected thrice as many species as compared to conventional catch, the results from these methods are complementary, as each contributed unique detections with some overlap. Our combined approach contributed a preliminary inventory of fish species to support the biodiversity management of Kuala Selangor and highlighted the importance of integrating eDNA metabarcoding as a biomonitoring tool for ichthyofaunal surveys. Beyond this local context, our findings serve as a scalable framework, for integrating methods to improve regional assessments of fish diversity across Southeast Asia.
ABSTRACT Changes in natural and artificial light presumably have dramatic influences on ecological communities, yet measuring these effects can be challenging due to the difficulty in distinguishing ecological signals from background variability. Here, we used environmental DNA (eDNA) to test for light‐associated changes in a nearshore marine community. Over 8 days at Friday Harbor Laboratories, Washington, USA, we sampled seawater at midday and midnight with and without an artificial light treatment, analyzing a total of 84 samples (3 L triplicate samples) using COI and 12S markers on an Oxford Nanopore MinION platform. We detected 229 eukaryotic (COI) and 77 vertebrate (12S) taxa across 11 trophic groups, dominated by primary producers (89 species), benthic invertebrates, and fishes. Metabarcoding captured community restructuring within 30 min, such that we could observe meaningful effects of artificial light treatments at night (particularly among spawning polychaetes), as well as more fundamental community shifts in day vs. night (e.g., with primary producers, gelatinous zooplankton, and filter feeders more common during the day). These techniques offer a means of near‐real‐time assessment of wholescale changes in ecological communities, and our results here illustrate species‐ and group‐specific effects of light levels that are likely important in structuring nearshore marine communities worldwide.
ABSTRACT Invasive fish are a serious threat to indigenous biodiversity. However, methods and tools to detect and control invasive fish can be poorly developed, costly, and/or labour‐intensive. Environmental DNA (eDNA) methods have significant potential for use in pest fish management. However, different studies asking how eDNA compares with conventional sampling, whether a relationship exists between eDNA data and population density, and how differences in field and laboratory eDNA techniques affect detection rates can return species‐ and context‐specific results. We addressed these questions for lacustrine environments using the brown bullhead catfish ( Ameiurus nebulosus ), an introduced pest in Aotearoa New Zealand. We collected eDNA samples alongside catch and biomass data from fyke nets set in two lake systems in the Bay of Plenty and Waikato regions of New Zealand's North Island. We investigated the effects of filter pore size (1.2 μm, 5 μm, dacron) and laboratory assay type (catfish‐specific metabarcoding, multi‐species metabarcoding) on catfish detection and compared our eDNA data to contemporary and historic catfish abundance records. All three filter types were similarly successful at detecting catfish presence when a minimum of five replicates was taken from each sampling site, while the catfish‐specific metabarcoding assays returned higher catfish compositional data and fewer false negatives compared to the multi‐species assays. We identified mixed results when examining the correlation between eDNA catfish read composition and historic or contemporary netting data, with significant correlations corresponding to random combinations of both filter size and eDNA assay type. Our results highlight the value of eDNA as a tool for pest fish detection that can complement more conventional biomonitoring methods, while limited correlations between eDNA data and field‐based fish netting rates emphasize the need for optimisation before eDNA can be used quantitatively for biosecurity surveillance efforts.
ABSTRACT Assessing fish diversity and abundance is crucial for effective marine conservation and management strategies, particularly in ecologically sensitive areas such as the North Sea. Bottom trawling, one of the most commonly used fishing methods, is facing growing criticism, even for scientific purposes, and has already been banned in ecologically sensitive zones. Therefore, it is crucial to rapidly establish noninvasive monitoring methods for solid biodiversity and abundance or biomass assessments in marine habitats. In this study, the potential of environmental DNA (eDNA) multiplex qPCR analyses, combined with computational modeling methods, for the quantitative assessment of common dab ( Limanda limanda ) and European plaice ( Pleuronectes platessa ) was explored. The concordance between the detectability of qPCR eDNA results and bottom trawling as the reference method was around 90% with a specificity of qPCR of 100% and a sensitivity of 82% for both species. For biomass estimation the outputs of a newly developed nonlinear model, with depth as an additional variable, showed correlations between the number of eDNA copies and biomass CPUE estimates from bottom trawl catches of 80% and 60% for plaice and dab, respectively. The model performance was validated by using data from other sampling missions in the North Sea and Baltic Sea. A good validation was recorded for plaice (RMSE 26%) and to a lesser extent for dab (MAE 34%). These results demonstrate the potential of eDNA as a noninvasive tool, not only for biodiversity monitoring, but also as a proxy for biomass estimations for certain species, although further optimization steps are certainly required for a stand‐alone use.
ABSTRACT Airborne environmental DNA (eDNA) offers a scalable tool for biodiversity monitoring, yet our limited understanding of airborne eDNA transport makes it challenging to define sample origin. We address this by investigating local spatial dynamics of eDNA dispersal using passive samplers and wind data. Over 3 weeks, we deployed passive samplers at Rotterdam Zoo (the Netherlands) and collected airborne eDNA at 5 locations for multiple durations over 96 h, where all samplers were started simultaneously. Vertebrate diversity was sequenced at 12S and 16S and resident zoo species identified (N = 24). Wind‐directed catchment areas were estimated using two models (Circular Sector model, using simple wind‐directed data from a nearby weather station, and Footprint model using gridded meteorological data). Catchment areas were defined by wind characteristics, with size and orientation determined by speed and directional variability. Results showed 100% species detections fell within predicted catchment areas up to 100 m (not all species in zoo detected). At longer ranges up to 655 m, 62% of the total detected species fell within the wind‐directed catchment areas. Species detections outside of the catchment areas suggest additional mechanisms may influence longer range transport of airborne eDNA. Longer sampling durations significantly increased the probability of species detections inside catchment areas and both models performed comparably well. Together, our results suggest simple wind characteristic models accurately identify vertebrate airborne eDNA catchments at local scales, increasing confidence that species signals originate at < 200 m downwind from sampler placement. Our work thus helps to enable more spatially explicit biomonitoring inferences.
ABSTRACT Freshwater ecosystems provide critical services to human societies; however, the plant communities that sustain these ecosystems face increasing threats from human activities, highlighting the need for effective monitoring strategies. While environmental DNA (eDNA) metabarcoding has emerged as a powerful, non‐invasive tool for biodiversity monitoring, its application to plant community monitoring remains underutilized. This study evaluated the use of eDNA metabarcoding based on the orbcL2 marker to simultaneously quantify aquatic, terrestrial, and wetland plant community composition and diversity within and among 22 inland lakes in Michigan, USA. eDNA metabarcoding surveys identified 221 plant species, including 28 aquatic, 62 wetland, and 131 terrestrial species. Notably, the richness of terrestrial species exceeded those reported in previous studies, comprising 44%–81% of all taxa detected per lake, with wind‐pollinated trees constituting a substantial component of the dataset. Terrestrial species were more prevalent in lakes within landscapes characterized by greater forest cover and lower human development. Sampling timing affected species richness. Lakes sampled early in the season had greater species richness from both terrestrial and wetland communities. Wind‐pollinated species dominated early‐season detections, indicating their disproportionate influence on diversity measures. The diversity of aquatic plants showed minimal sensitivity to sampling date, and no significant seasonal variation in alpha diversity, contrasting with previous studies. However, the relatively short duration of our sampling may explain the absence of trends typically observed later in the season when plant decay increases. Beta diversity of aquatic plant communities was affected by land use, particularly agricultural activity in surrounding landscapes. Results highlight how seasonal and landscape characteristics can affect diversity estimates of freshwater plant communities, underscoring the need to account for temporal and spatial dynamics in eDNA‐based biodiversity monitoring. The study also emphasizes the limitations of relying on a single marker for accurate detection of aquatic invasive plant species.
ABSTRACT Environmental DNA (eDNA) has transformed biodiversity monitoring by enabling sensitive, non‐invasive species detection. However, its potential to resolve taxonomic uncertainties and uncover biogeographic patterns in tropical freshwater ecosystems remains underused. This study addresses an apparent discrepancy: while the Guiana Shield is recognized as a hotspot of aquatic biodiversity, the recorded diversity of freshwater bivalves in Guiana has been notably low, and their distribution patterns remain poorly understood. To address this paradox, we conducted the first large‐scale eDNA survey of freshwater bivalves in South America. Between 2019 and 2023, we collected water samples from 115 sites across the Maroni and Oyapock river basins in French Guiana. Cryptic species initially detected through eDNA were subsequently confirmed through targeted malacological sampling and molecular barcoding (COI, 16S) of collected specimens. These data were used to build a regionally calibrated reference database for taxonomic assignment of eDNA sequences. This integrative approach led to substantial taxonomic revisions, including the description of two new genera and two cryptic species. In addition, eDNA analysis revealed six additional Molecular Operational Taxonomic Units (MOTUs), likely corresponding to undescribed species. Overall, this study increases the number of known Unionid species in French Guiana from five to eighteen, more than tripling current species richness estimates. Beyond taxonomic discoveries, eDNA further revealed consistent basin‐scale patterns, with both species richness and intraspecific genetic diversity (as measured by Amplicon Sequence Variants) peaking in upstream areas with limited human disturbance and declining downstream in regions affected by gold mining and deforestation. These findings demonstrate the value of eDNA as a tool not only for monitoring but also for advancing taxonomy and biogeography in understudied tropical ecosystems. By revealing hidden diversity and expanding spatial coverage at minimal cost, eDNA enables more accurate assessments of freshwater biodiversity and informs conservation strategies at landscape scales.
ABSTRACT Mangroves support high faunal diversity and provide essential ecological services. However, biodiversity assessments in these habitats remain constrained by their structural complexity, resulting in limited accessibility, low visibility, and persistent taxonomic gaps. Rapid advances in molecular techniques use environmental DNA (eDNA) metabarcoding as an emerging, powerful alternative to conventional surveys. However, its effectiveness in tropical systems, such as Philippine mangroves, is constrained by gaps in publicly available genetic reference data, particularly for invertebrates. To address this, we systematically reviewed 48 years (1977–2025) of literature records of Philippine mangrove‐associated fauna and assessed their representation in public genetic databases to address three questions: (a) What is the current state of faunal biodiversity in Philippine mangroves? (b) How complete are public genetic reference libraries for eDNA applications? and (c) How can integrating eDNA with conventional surveys enhance ecological monitoring? We outlined a workflow for literature screening, taxonomic standardization, and programmatic retrieval and quality‐filtering of GenBank accessions. Across 999 species spanning nine phyla, the ray‐finned fishes (40.4%) dominated the records, followed by gastropods (21.7%) and bivalves (16.5%). Only 70.27% of species had at least one mitochondrial marker, dominated by COI (89.03%), followed by 16S (70.23%), 12S (60.97%), and Cytb (2.45%). Coverage was particularly low for invertebrates and some commercially important conservation‐relevant fishes, limiting species‐level detection in eDNA metabarcoding. Nevertheless, integrating eDNA with traditional methods increases the detection of cryptic, rare, juvenile, and hard‐to‐sample taxa, enabling more comprehensive biodiversity assessments. This combined approach enhances the capacity for spatial and temporal monitoring and supports more informed conservation strategies. Additionally, these data have the potential to enhance faunal assessments and strengthen biodiversity management practices. Overall, this review underscores the need to expand locally curated, voucher‐linked reference databases through targeted sequencing and highlights how national and regional partnerships can improve the accuracy and conservation relevance of eDNA‐based biodiversity monitoring.
ABSTRACT Airborne environmental DNA (eDNA) offers a powerful, noninvasive approach to monitor terrestrial biodiversity. However, its broader implementation is constrained by limited understanding of DNA persistence across the eDNA lifecycle, including biological shedding, environmental transport, capture efficiency, and post‐capture decay. While aquatic and soil studies provide valuable insight into eDNA degradation processes, airborne eDNA presents distinct challenges, and isolating individual lifecycle stages remains necessary for accurate signal interpretation. Here, we focus on one critical and underexplored stage: post‐capture DNA persistence on collection substrates. We conducted a 180‐day controlled laboratory trial to quantify how environmental exposure and material choice influence DNA signal retention following capture. Using a standardized vertebrate DNA source proxy (pork meat meal), we evaluated seven candidate collection materials, including five dry substrates and two liquid media, exposed to two temperature regimes (23°C and 40°C) and three light exposure levels. By directly applying DNA to substrates, we decoupled post‐capture DNA decay from variability in airborne transport and capture efficiency. We modeled DNA signal loss using exponential decay functions, revealing temperature as the dominant driver of post‐capture degradation across materials. Light exposure had no measurable effect under the low UV irradiance tested. DNA declined most rapidly within the first 2 weeks following capture, and decay constants (k) varied widely among substrates. Dry substrates retained DNA comparably to positive controls over extended periods, whereas liquid media exhibited accelerated signal loss and higher PCR inhibition. Pairwise statistical comparisons confirmed significant differences in both persistence and inhibition among materials. Together, these results demonstrate that collection substrate choice strongly shapes post‐capture airborne eDNA signal stability and downstream analytical performance. By quantifying material‐ and temperature‐dependent decay trajectories, this study provides empirical guidance for selecting sampling substrates and defining deployment duration, supporting more reliable interpretation of airborne eDNA detections in terrestrial biodiversity monitoring.
ABSTRACT The New Zealand mudsnail (NZMS; Potamopyrgus antipodarum) is a widespread aquatic invasive species that is parthenogenic, requiring only a single individual to initiate an infestation. Fish hatcheries–which are critical infrastructure that raise fish to support conservation, recreation, and subsistence fisheries–frequently use local water sources to provide cool water and are especially vulnerable to NZMS invasion from the contamination of water supplies. If an invasion proceeds undetected, hatcheries pose a risk for compounding the spread of NZMS because their operations transfer live organisms and associated water between hatchery facilities and, when stocking, to rivers and lakes. The U.S. Fish and Wildlife Service's Alchesay National Fish Hatchery, located on the Fort Apache Indian Reservation in Whiteriver, Arizona, produces trout to stock in Tribal reservoirs, lakes, and rivers across the southwestern U.S. New Zealand mudsnails were first documented in Arizona in 1995, are now widespread in this region, and occur at the confluence of the hatchery's outflow with the North Fork White River. Contamination of water supplies is the principal pathway for NZMS invasion into the hatchery. Here, we describe early detection environmental DNA (eDNA) surveillance efforts for NZMS at Alchesay National Fish Hatchery. Positive eDNA detections initiated a chain of events that ultimately led to four NZMS individuals being discovered and a rapid response eradication effort. Follow‐up eDNA sampling and visual observation efforts after the eradication effort have yielded no detections of NZMS eDNA. We credit the success of this case with four key elements: rapid turnaround times, a robust quality assurance scheme, a proactive eDNA sampling design, and established partnerships. To our knowledge, this is the first published case of eDNA monitoring being used for early detection and successful rapid response for complete removal of an invasive species in a fish hatchery.
ABSTRACT Environmental DNA (eDNA) analysis has transformed biodiversity monitoring by enabling rapid, non‐invasive species detection. eDNA is derived from multiple species, cell types, and individuals. The non‐cellular fraction of eDNA may contain a mixture of genotypes from multiple individuals. Standard eDNA sampling and analysis does not allow recovery of individual multi‐locus genotypes from this mixture. Solving this requires two innovations: (1) separating eDNA particles (emCells) belonging to individual organisms from a mixture; (2) generating multilocus genotypes from individual emCells. Having addressed partitioning individual emCells previously, here we tested whether multi‐locus nuclear single nucleotide polymorphism (SNP) genotypes can be reliably generated from lab‐generated and isolated single fish cells as a model for wild emCells. Our workflow “emCell‐Seq,” combines fluorescence‐activated cell sorting (FACS) to isolate environmental metazoan cells (emCells) with multiplex PCR targeting discriminatory nuclear SNPs. Individual zebrafish (Danio rerio) cells were created from tissue and pooled into mock emCells samples of known single and mixed source. Pooled samples were sorted by FACS, which isolated individual cells that were genotyped with a panel of 35 SNPs. SNP genotypes from emCells were compared to known genotypes to assess assignment accuracy. For emCells in artificial pool from multiple individuals, 91% (n = 129) were correctly assigned. No mixed genotypes were obtained. This study provides the first evidence that individual‐level nuclear genotypes can be obtained from isolated fish emCells. Extending these findings to field‐collected emCells will significantly expand the scope ecological analyses enabled by eDNA.
ABSTRACT Environmental DNA is being increasingly used for research and regulatory purposes, but currently the lack of standardization is holding it back. There are gaps in our understanding of the eDNA analysis pipeline and the potential impacts of areas of variability within that. Standardization is necessary for all uses of eDNA to allow comparison between sample sets and ensure accuracy and reproducibility. Understanding sample stability is particularly important for planning fieldwork and writing practicable guidance for regulatory compliance monitoring. Samples collected for eDNA analysis are preserved as soon as possible for stability, but practicalities of sampling in the field can lead to delays where the sample temperature may be uncontrolled. We collected eDNA sediment samples along an organic enrichment impact gradient and incubated them at 10°C, 20°C, and 40°C for up to 48 h prior to preservation, then sequenced the bacterial 16S gene. We show that bacteria families responded differently to the incubation temperatures and times to an extent that affected ecological interpretation. Predictions of benthic health using a trained random forest machine learning model were tolerant of incubation up to 20°C, and showed sensitivity to temperature within 3 h of incubation at 40°C. We show that the influence of temperature can depend on the study aim, taxa involved, and analysis used, such that some situations may allow temporary storage up to 20°C but others will be affected by 10°C. We confirm that keeping sediment temperature low is critical for many applications, and that potential temperature deviations must be reported.
ABSTRACT Environmental DNA (eDNA) metabarcoding is a powerful tool for monitoring elusive marine species, but its effectiveness is constrained by incomplete reference databases. This limitation is especially evident for epaulette sharks (Hemiscyllium), a genus of small benthic sharks endemic to the Indo‐Australian Archipelago. Six of the nine species occur in eastern Indonesia and are fully protected under national law, yet monitoring their distribution remains challenging using traditional methods. To address this gap, we developed the first genus‐specific eDNA metabarcoding assay for Hemiscyllium, targeting the mitochondrial NADH4 gene. Laboratory validation confirmed that the new ES‐200ND4 primer amplified Hemiscyllium DNA in simulated eDNA experiments, even at low concentrations (< 11 ng/μL). Field application in Raja Ampat detected the endemic H. freycineti at six of the seven sampling locations. The ES‐200ND4 primer demonstrated taxonomic specificity, with all 55 ASVs generated from field samples assigned exclusively to H. freycineti (mean genetic distance: 0.78%), while the universal 12S marker (elas02) exhibited non‐specific amplification but detected a broader range of marine taxa, including H. freycineti (17 ASVs), Carcharhinus sp. (1 ASV), and numerous phytoplankton groups. Notably, eDNA detection at Dayan, where daytime visual surveys recorded no sharks, underscores the method's capacity to reveal species presence when traditional approaches fall short. Overall, this study presents the first genus‐specific eDNA metabarcoding assay for Hemiscyllium spp. and the first application of NADH4 as a metabarcoding marker in marine environments. Finally, this scalable assay supports Indonesia's protective legislation for Hemiscyllium and offers a transferable framework for monitoring other data‐limited, cryptic, or threatened marine taxa.
ABSTRACT The Vietnamese Pond Turtle, Mauremys annamensis, is a species endemic to Central Vietnam. Its population has experienced a significant decline over the past several decades primarily due to habitat loss and targeted overexploitation for food, the international pet trade, and use in traditional medicine. As a result, this species has been listed among the 25 most endangered turtles in the world and considered likely extinct in the wild. This poorly studied species has only been recorded four times in its natural habitat using traditional survey methods, and its distribution remains largely unknown. In this study, we utilize an environmental DNA method to assess the distribution of M. annamensis across various habitats, including ponds, lakes, and shallow streams, in four provinces (Quang Nam, Quang Ngai, Phu Yen, and Dak Lak) in Central Vietnam. Our findings demonstrate that it is possible to detect M. annamensis' DNA in the aquatic habitats, even where the species has not been observed in the areas. Furthermore, our results provide the first evidence of the species' presence in Ea So Nature Reserve, a protected area in Dak Lak Province, where this turtle had not previously been documented in the wild. This discovery is crucial for informing conservation strategies by identifying a new candidate site for reintroduction programs for one of the most endangered turtles in the world, which currently has no known viable wild populations. Our results can help guide future eDNA surveys of this and other aquatic turtle species by focusing on suitable habitats and breeding seasons and promote the broader application of eDNA to assess distribution and population status of other threatened species in Vietnam.
ABSTRACT The increasing prevalence of emerging chemicals poses a significant global threat to ecosystems, emphasizing the urgent need for efficient methods to assess pollutant impacts. Environmental DNA (eDNA) analysis has been employed as a rapid, labor‐saving biomonitoring tool. However, its application in pollutant toxicity testing has not yet been established. This study aimed to develop a soil toxicity test protocol using species‐specific eDNA from Allonychiurus kimi (Collembola). Quantity of DNA in quantitative polymerase chain reaction (qPCR) was determined by the semi‐quantitative measurement of the threshold cycle (Ct). To validate the effectiveness of this method in reflecting ecotoxicological endpoints, eDNA concentrations were measured across various age structures of A. kimi populations. Ct‐values decreased with increasing age and adult proportion, demonstrating that this approach can accurately detect demographic variations. Conventional (28‐day reproduction toxicity test) and eDNA‐based toxicity tests were conducted to compare their sensitivity for four heavy metals: arsenic, lead, cadmium, and copper. Both methods exhibited dose‐dependent responses to the tested heavy metals and yielded identical no observed effect concentration (NOEC) values for each metal. The eDNA tests indicated median effect concentration (EC50) values of 45.73, 684.70, 35.04, and 147.53 mg kg−1 for As, Pb, Cd, and Cu, respectively. Except for Cu, these values showed no statistically significant differences compared with those of conventional tests. Furthermore, the eDNA method produced a consistent EC50 equivalent Ct‐value of 29.91 across all the tested metals, suggesting its potential use as a benchmark in ecotoxicological risk assessment. These findings indicate that the eDNA‐based toxicity test offers sensitivity comparable to that of the conventional method and holds promise as an effective screening tool for assessing pollutant impacts on ecosystems.
ABSTRACT Environmental DNA (eDNA) from seabird fecal samples can provide insights into diet and the broader biodiversity of the ecosystems the birds inhabit. Seabirds like the little blue penguin (Eudyptula minor; kororā) are sensitive to changes in prey availability and habitat condition, yet their diet and foraging plasticity remain poorly resolved in some regions. We used DNA metabarcoding of the eukaryotic mitochondrial cytochrome c oxidase subunit I (COI) gene to analyze fecal samples collected from nests across eight locations in an E. minor colony on Matiu/Somes Island (Wellington Harbor, Aotearoa New Zealand) to investigate diet and bycatch DNA, and how these varied with nest location and time. We detected six marine fish species known to be components of the E. minor diet. In addition, the fecal samples contained a diverse range of nondiet eukaryotic taxa, including soil invertebrates, plants, fungi, arachnids, and other local species that reflected the surrounding microhabitat. Within this nondiet/bycatch data, community composition showed subtle temporal variation, with October samples exhibiting the highest number of unique specialist taxa (171 species) and a predominance of terrestrial‐associated groups such as Ascomycota, Arthropoda, and Basidiomycota. November samples showed intermediate unique diversity (74 specialist species), while December communities showed the lowest number of specialist taxa but an increased proportion of aquatic‐associated specialists, including Bacillariophyta and Rotifera, consistent with warmer‐season activity. Spatial differences were nonsignificant, but differences in data dispersion among sites may reflect the fine‐scale influence of local microhabitat conditions in some datasets. Collectively, our results demonstrate that, even in the absence of strong dietary signals, eDNA samples from seabird colonies can act as a valuable lens into local biodiversity and ecological processes. Thus, our findings underscore the potential of feces as so‐called “biodiversity capsules” for broad environmental monitoring, habitat assessment, and the identification of ecosystem change in coastal environments.
