The rapid advancement of environmental DNA (eDNA) science in the past two decades has inspired a concomitant growth in the development of eDNA sampling and analytical methods. However, these methods are often developed by individual laboratories or institutions, which can isolate protocols within programmes, agencies or regions and prevent the beneficial exchange of data and ideas. Recent efforts to advance national and international coordination have resulted in a groundswell of standardisation efforts, but there is still considerable confusion around the role of formal standards for regulatory or research applications. With this commentary, we hope to provide clarity on the terminology used in standardisation discussions, including the differences between formal standards and best practice guidelines. Additionally, we discuss how eDNA method choice may be informed by environmental management scenarios and review examples of formal eDNA method standards being used to inform management action. The eDNA community now has an opportunity to develop a roadmap for method development to help close standardisation gaps, advance eDNA method adoption and accelerate our ability to monitor biological life at the scales our current environmental challenges demand.

There are many decisions to be made when sampling for environmental DNA (eDNA) analysis, whether using a targeted, single-species assay or community-based metabarcoding. Of the entire workflow from sampling water to bioinformatic analyses, the first steps in the process of collecting water, filtering it, and preserving the filter membranes represent major decision points upon which the success of downstream processes depend. Though many previous studies have compared water volume filtered, filter pore size, and preservation and extraction methods, the conclusions are often that they produce different results, but it is unclear which is the optimal approach for a given purpose. Here, rather than provide yet another methods comparison paper, we provide a framework for how to make informed decisions from a methods comparison and, importantly, how to combine data collected via different methodological choices. We investigate (1) the volume of water filtered and the filter pore size and (2) the preservation method and extraction method of samples with a specific lens on how these choices impact the detection of a single targeted species (Atlantic bottlenose dolphin, Tursiops truncatus, via quantitative PCR (qPCR)), although in principle these findings apply to single-species assays more generally. We find that larger pore size filters (5 µm vs. 1 µm) and larger volumes of water (3 L vs. 1 L) maximize the ratio of amplifiable target DNA to total DNA without compromising the absolute detection of target. We also find that maximizing total DNA yield during extraction (phenol chloroform vs. two commercial kits) does not always increase target detection likely due to the concentration of inhibitors and co-extraction of off-target DNA. We also comment on variation including technical and biological variability between replicates, finding that by homogenizing source water before filtering removes much of the biological variation. Finally, we present a statistical model that allows for inclusion of data from samples collected and processed in different ways, enabling researchers to change protocols or include data from other field sampling efforts, thereby opening up more possibilities to extend datasets and analyses.

Water and air are generally treated as separate reservoirs of environmental DNA (eDNA) derived from the species resident in those respective environmental compartments. However, it is likely that eDNA routinely crosses the air–water boundary in both directions as a result of deposition, evaporation, or other processes. Here, we systematically tested methods of sampling eDNA at the air–water interface, showing for the first time that aquatic life can be consistently detected under standardized field conditions from passive air samples. We deployed four simple air samplers — three different kinds of filters and one open tray of deionized water — alongside paired water samples and visual counts over a six-week peak run of Coho salmon (Oncorhynchus kisutch) at a local spawning stream. We then quantified eDNA concentrations in both air and water (air: copies/cm²/day; water: copies/L) using quantitative PCR, to estimate (1) the concentration of target eDNA in air vs. water, and (2) the capture performance of each filter type. Passive air collectors captured quantitative airborne eDNA signals that covaried with salmon counts, despite air eDNA concentrations being approximately 25,000 times more dilute than water, although eDNA recovery varied with sampler design and orientation. We show the air–water interface can be a quantifiable source of aquatic genetic information in this system using simple, passive samplers that do not require electricity, making them appealing for biomonitoring in remote or resource-limited settings. This work points the way to using airborne eDNA as a promising pathway for biological information critical to conservation, resource management, and public-health protection.

Rare species are difficult and time-consuming to detect, but environmental DNA (eDNA) methods can be used to increase data availability for monitoring and management. Here, we use the diverse rockfish species flock (genus Sebastes) to demonstrate the utility of eDNA as a tool for detecting rare and difficult to observe species in the marine environment. We describe the identification of a phylogenetically informative gene region for eDNA metabarcoding which uniquely identifies 93 of the 109 Sebastes species currently described. We then use this assay to differentiate rockfish communities in field samples collected from two sub-basins within Puget Sound in Washington, USA. Across three field sampling platforms, we found that sample collection location (distance from seafloor) has substantial impacts on rates of detection and on the diversity of species detected, likely reflecting the habitat preferences of the target species. This metabarcoding region provides an important tool for rockfish monitoring, both within Puget Sound and across the North Pacific. More generally, this work speaks to the usefulness of eDNA data as a tool for the conservation and management of rare and difficult-to-distinguish species.

Environmental DNA (eDNA) analysis enables biodiversity monitoring by detecting organisms from trace genetic material, but high reagent costs, cold-chain logistics and computational demands limit its broader use, particularly in resource-limited settings. To address these challenges and improve accessibility, we directly compared multiple workflow components, including four DNA extraction methods, two primer sets, three Nanopore basecalling models, and two demultiplexing pipelines. Across 48 workflow combinations tested in an aquarium with 15 fish species, we mapped trade-offs between cost, sensitivity, and processing speed to assess where time and resource savings are possible without compromising detection. Workflows using the Qiagen Blood and Tissue (BT) extraction kit and amplification using the MiFish-U primer set provided the highest sensitivity, detecting ≥ 12 of 15 species by ~3–5 h and reaching the 15-OTU plateau at ~8–10 h with Oxford Nanopore’s high accuracy (HAC) basecalling model. Chelex, an alternative lower-cost extraction method, showed partial recovery only (≤ 9 OTUs by 61 h) even with extended sequencing, and did not recover all 15 OTUs. DirectPCR and QuickExtract offered field-friendly extraction alternatives that achieved comparable recovery in ~10–12 h, though their cost-effectiveness varied. While the MarVer1 primer was designed to broaden vertebrate detection, it recovered the same fish species as MiFish-U, though with fewer total reads. Real-time sequencing trials (0–61 h) revealed that high-efficiency workflows (BT + HAC) reached detection plateaus rapidly, indicating sequencing time can be reduced without sacrificing accuracy. The OBITools4 bioinformatics pipeline enabled automated demultiplexing but discarded more reads than an alternative, ONTbarcoder2.3, which retained low-abundance taxa at the cost of manual curation. Rather than identifying a single ‘best’ workflow, this study provides a transparent decision framework for prioritising cost, speed, and sensitivity in eDNA applications, supporting scalable, cost-effective eDNA monitoring in resource-limited settings.

Aquatic, aerial, and terrestrial habitats exist along a continuum, with biomass and energy flows transporting genetic material across environmental boundaries. Here, we use environmental DNA (eDNA) metabarcoding to characterize genetic information exchange between water and air. From 27 paired samples collected at two urban-wildland interface sites using passive air sampling and active water filtering, we recovered 35 vertebrate taxa, with 40% detected in both media, ranging from aquatic salmon to terrestrial cottontail rabbit. Cross-medium detection probability scales with DNA abundance: logistic models identify ∼660 water reads and ∼14 air reads as 50% detection thresholds. Peaks in coho and Chinook salmon eDNA in water and air align within 24 h, demonstrating that passive air sampling reflects temporal abundance trends. Low-abundance taxa appear sporadically, reflecting stochastic behavior at low DNA concentrations, and reliable detection requires intensified sampling in the primary habitat. Together, these findings establish a unified framework for holistic vertebrate biodiversity monitoring at the land-water interface, with applications in conservation, invasive species early warning, and One Health surveillance.

Environmental DNA (eDNA) analysis is a technique for detecting organisms based on genetic material in environments such as air, water, or soil. Observed eDNA concentrations vary in space and time due to biological and environmental processes. Here, we investigate variability in eDNA production and loss by sampling water adjacent to a managed population of non-native cetaceans on a near-hourly timescale for 48 hr. We used diverse sampling approaches and modeling methods to describe time variability in observed eDNA concentrations and then compare the magnitude of production and loss mechanisms. We parsed production and loss in a conceptual box model and compared biological and physical loss rates using a decay experiment and a physical transport-and-diffusion tracer model. We then evaluated eDNA concentrations along a transect away from the animal enclosure in light of model parameter estimates. We conclude that eDNA production is best conceptualized using a time-varying mixed-state model, and biological losses are small relative to physical losses in the marine environment. Because physical loss is unsteady and nonlinear, tracer models are especially helpful tools to estimate it accurately.

Environmental DNA (eDNA) is increasingly used for species detection and biodiversity monitoring in estuary and marine environments. The dynamic nature of these environments affects eDNA distribution relative to its source organisms, complicating the interpretation of eDNA observations and challenging the field sampling design. Here, an eDNA fate and transport model, built on an ocean model with Lagrangian particle tracking, provided a spatiotemporal estimate of the rapidly diluted eDNA shed by rare targets in an estuary environment before sampling. Based on the predicted particle densities, over 70% of the preselected stations detected the target eDNA. Despite potential variations in source strength and patchy distributions, the model explained approximately 40% of the observed variation in eDNA abundance; by comparison, eDNA concentration was uncorrelated with straight-line distance from the source or with a simplified oceanographic model. Our study revealed the extent of advective transport in shaping eDNA distribution and abundance and demonstrated the utility of ocean models and particle tracking in integrating marine eDNA observations with degradation, transport, and dilution processes; thus, it suggests broader applications to enhance understanding of eDNA signals and dispersal and optimize sampling strategies in other estuarine or marine environments.

Detections of environmental nucleic acids (eNA), such as DNA and RNA, are powerful tools for monitoring biodiversity. Yet, precise interpretation of these indirect detections requires understanding of eNAs persistence. We conducted a decay experiment to track degradation of six eNA components derived from the bottlenose dolphin Tursiops truncatus: mitochondrial eDNA of varying lengths, ribosomal eRNA, and messenger eRNA. Target eNAs were quantified over seven days via digital droplet PCR (ddPCR). Decay followed a biphasic exponential model with rapid initial loss (~ 24 h at 15 °C), followed by slower degradation. Mitochondrial messenger eRNA was least stable, disappearing within four hours. Ribosomal eRNA persisted longer but degraded slightly faster than its eDNA counterpart (decay rate λ₁ = 0.236 vs. 0.165 h⁻¹). Longest eDNA fragments decayed more rapidly (λ₁ = 0.190 h⁻¹) than shorter ones (λ₁ = 0.114 h⁻¹). These findings support using eDNA fragment length as a proxy for degradation and reinforce that combining multiple eNA components with distinct stabilities can provide a molecular clock to infer eNA age. This approach improves the spatiotemporal resolution of eNA-based monitoring, particularly for rare cetaceans that act as point sources. We also emphasize the importance of explicitly distinguishing between RNA types (ribosomal vs. messenger) in environmental studies, given their divergent stability and interpretability.

DNA metabarcoding is subject to observation bias associated with PCR and sequencing, which can result in observed read proportions differing from actual species proportions in the DNA extract. Here, we amplify and sequence a mock community of known composition containing marine fishes and cetaceans using four different primer sets and a variety of PCR conditions. We first compare metabarcoding observations to two different sets of expected species proportions based on total genomic DNA and on target mitochondrial template DNA. We find that calibrating observed read proportions based on template DNA concentration is most appropriate as it isolates PCR amplification bias; calibration with total genomic DNA results in bias that can be attributed to both PCR amplification bias and differing ratios of template to total genomic DNA. We then model the remaining amplification bias and find that approximately 60% can be explained by inherent species-specific DNA characteristics. These include primer-template mismatches, amplicon fragment length, and GC content, which vary somewhat across Taq polymerases. Finally, we investigate how different PCR protocols influence community composition regardless of expected proportions and find that changing protocols most strongly influence the amplification of templates with primer mismatches. Our findings suggest that using primer-template pairs without mismatches and targeting a narrow taxonomic group can yield more repeatable and accurate estimates of species’ true, underlying DNA template proportions. These findings identify key factors that should be considered when designing studies that aim to apply metabarcoding data quantitatively.

Environmental DNA (eDNA) provides powerful insights into species presence and community composition but remains limited in its capacity to infer species abundance or population structure. Here, we show that the deviation between within-sample haplotype frequencies and the overall population-level haplotype frequencies can be used to estimate the number of individual contributors to a given sample. We first establish the theoretical framework for approximating population haplotype frequencies directly from eDNA data, enabling application even in the absence of tissue-derived references. Building on this foundation, we introduce a maximum likelihood estimator to infer the number of contributors and assess its performance through simulations spanning a range of haplotype frequency distributions and noise scenarios. These approaches assume that all samples are drawn from a single, panmictic population. We find that accurate estimates are attainable when haplotypes are sufficiently variable, population frequencies are well-characterised, and samples are large enough to capture frequency deviations. By bridging population genetic theory and eDNA, our method complements existing molecular approaches and offers a novel path towards quantifying abundance from eDNA metabarcoding data.

Rare species are difficult to observe in the wild, particularly in the ocean where large spatial scales and accessibility hinder effective sampling. Environmental DNA (eDNA) is a non-destructive, scalable sampling method with the potential to inform the distribution of rare species in marine ecosystems. We sample eDNA within the California Current ecosystem to estimate the distribution of eulachon (Thaleichthys pacificus), a threatened anadromous smelt ranging along the coastal Northeast Pacific. We amplify eulachon DNA from thousands of water samples collected at night across two years and more than 200,000 square kilometers along the U.S. west coast. We then use spatiotemporal models to derive quantitative estimates of eulachon DNA across space, depth, and time relative to environmental covariates. We find that eulachon DNA has a distribution weighted towards the ocean surface, spatially associated with major river mouths and productive offshore banks. Temperature and prey density are key covariates, with eulachon more likely to be found in warmer waters with higher prey concentrations. We discuss how our results can augment the information currently used in eulachon recovery planning, and describe the wide applicability of our statistical models for estimating distribution and abundance for other species of conservation concern.

Environmental DNA (eDNA) is a rapidly emerging data source with the potential to support environmental monitoring and biodiversity conservation around the world. Current efforts to standardize eDNA methods and reporting are aimed at strengthening credibility and supporting adoption. In doing this, however, researchers must be mindful of diverse capacities and ecological contexts both regionally and around the world. The objective of our research is to understand how international standards for eDNA may support or hinder the uptake of eDNA methods and tools for conservation and biodiversity work. This was accomplished through two interactive workshops that brought together eDNA researchers and practitioners from around the world to surface broad and specific barriers to uptake of eDNA methods and tools.

The Maldives reopened a collapsed gulper shark fishery in November 2025 without stock assessment or recovery evidence. Applying the precautionary approach requires three minimum standards for managing reopened or proposed fisheries: transparent recovery assessment, independent population monitoring and funded operational plans with decision rules. I propose that environmental DNA (eDNA) offers a non-lethal, scalable monitoring tool to support evidence-based fishery decisions for threatened deep-sea sharks.