Environmental DNA

Airborne environmental DNA (eDNA) has shown promise as a terrestrial biomonitoring tool and its ecological applications are expanding. Despite its growing use, airborne eDNA does not yet have the extensive body of supporting research like its aquatic counterpart, with considerable uncertainty remaining concerning how airborne eDNA behaves, with regards to signal duration, and how much sampling effort is needed to capture DNA in a given airspace. By using airborne eDNA in a semi-controlled environment which acted as an artificial roost where bat species and their abundances were known, we estimated the sampling intensity (both the number of samples and number of sampling events) required to capture bat diversity of a given airspace, as well as signal persistence of airborne eDNA. Together these data provide a temporal scale for airborne eDNA measurements. The majority of species richness was detected using as little as 4 samplers in this enclosed space and the greater the number of sampling events, the fewer samplers were needed. Both air movement and the type of environment (i.e., enclosed space, open area etc.) are likely to impact detection and need to be considered during study design. eDNA also appeared to settle out of the air quickly, suggesting that detections likely reflect recent activity, which also has important implication for rare species which may only have a narrow window for detection. Our results add to the growing body of literature that indicate airborne eDNA can be a useful biosurvey method, especially for rapid surveys in communities with high turnover rates.

Freshwater ecosystems, such as streams, are facing increasing pressures from agricultural land use. Aquatic insects and other macroinvertebrates have historically been used as indicators of ecological condition and water quality in freshwater biomonitoring programs; however, many of these protocols use coarse taxonomic resolution (e.g., family) when identifying macroinvertebrates. The use of family-level identification can mask species-level diversity, as well as patterns in community composition in response to environmental variables. Recent literature stresses the importance of robust biomonitoring to detect trends in insect decline globally, though most of these studies are carried out in terrestrial habitats. Here, we incorporate molecular identification (DNA metabarcoding) into a stream biomonitoring sampling design to explore the diversity and variability of aquatic macroinvertebrate communities at small spatial scales. We sampled twenty southern Ontario streams in an agricultural landscape for aquatic macroinvertebrates and, using DNA metabarcoding, revealed incredibly rich benthic communities which were largely comprised of rare taxa detected only once per stream despite multiple biological replicates. In addition to numerous rare taxa, our species pool estimates indicated that after 240 samples from twenty streams, there was a large proportion of taxa present which remained undetected by our sampling regime. When comparing different levels of taxonomic resolution, we observed that using OTUs revealed over ten times more taxa than family-level identification. A single insect family, the Chironomidae, contained over one third of the total number of OTUs detected in our study. Within-stream dissimilarity estimates were consistently high for all taxonomic groups (invertebrate families, invertebrate OTUs, chironomid OTUs), indicating stream communities are very dissimilar at small spatial scales. While we predicted that increased land use would homogenize benthic communities, this was not supported as within-stream dissimilarity was unrelated to land use.

The study of environmental DNA (eDNA) is increasingly becoming a valuable tool to survey and monitor aquatic communities. However, there are important gaps in our understanding of the dynamics governing the distribution of eDNA under natural conditions. In this report we carry out controlled experiments to assess the extent and timing of eDNA distribution along the water column. A sample of known eDNA concentration was placed at the bottom of a 5-m high tube (20 cm in diameter and total volume of 160 L), and water samples were obtained at different depths over an 8 h-period. The presence of the target eDNA was assessed by qPCR analysis. This sampling protocol allowed for assessing the timescale for the diffusion of eDNA while minimizing the influence of turbulence. We demonstrate that, after a time-period of as little as 30 min, the eDNA had spread across the entire container. The implications of these results for eDNA sampling protocols in the field are discussed.

Adjacent to the spectacular, rugged coastline bordering the Olympic Peninsula of Washington State, Olympic Coast National Marine Sanctuary (OCNMS) encompasses 8257 km2 of coastal waters that support one of North Americas most productive marine ecosystems. These rich waters support important recreational, commercial and subsistence fisheries for Washington state and four sovereign tribal governments: the Quinault Indian Nation and the Makah, Quileute and Hoh Tribes. Given its ecological, cultural and economic significance, managers from OCNMS are tasked with monitoring and management towards conserving the areas ecological integrity. The development of metabarcoding with environmental DNA (eDNA) as an effective freshwater monitoring tool has potential applications in marine systems, but the statistical analysis and the interpretation of those eDNA results are still under development. One promising strategy to analyze eDNA is through the use of occupancy models. Occupancy models enable us to calculate probabilities of detection from each taxon sequenced and are designed to work with presence-absence data. To inform our long term monitoring design of this coastal ecosystem, we collected 44 eDNA samples at nine of OCNMS long term mooring sites in 2019 and tested four different molecular markers for species reported and taxonomic richness. Additionally, we assessed occupancy models for use with eDNA to estimate the number of samples required to accurately predict the presence of a species. Occupancy models show great promise for use in eDNA studies provided there is sufficient replication; additionally, the choice of molecular marker strongly influences a taxas probability of detection. Author SummaryWe show the utility of using occupancy models with eDNA in Olympic Coast National Marine Sanctuary for monitoring near-shore marine biological communities. Based on probabilities of detection, long term coastal monitoring projects using eDNA would benefit from designs prioritizing sample number (10-20 samples per site) over sites sampled. With proper study design, occupancy models provide a statistical framework for comparisons between sites and over time. By accounting for simple non-detection vs true absence, occupancy models help to eliminate noise from eDNA studies, increasing detection of true shifts in community composition. We also demonstrate that marker choice is an important study design consideration not only for the types of taxa recovered, but also for consistency between samples and timepoints.

DNA-based approaches utilizing high-throughput sequencing (HTS) (e.g. DNA metabarcoding) have revolutionized ecological biomonitoring by providing higher sample throughput, greater reproducibility, and better cost-benefits compared to traditional morphology-based bioassessment studies. Here, we utilized DNA metabarcoding in a watershed in Ontario (Canada) dominated by agricultural land uses. Our aim is to understand patterns of biodiversity in benthic taxa from data generated and inferred at various taxonomic scales and to compare these findings with over a decade of traditional morphological data. We sampled 18 watercourses during summer and fall 2023, spanning a forested-to-agricultural land-use gradient. We found significant differences between metabarcoding and historical morphology data where DNA provided more richness values at both the species (p = 2x10-5) and order (p = 0.008) levels. Whereas the morphology dataset contained many unresolved taxa, DNA metabarcoding captured a broader taxonomic breadth with diverse genetic profiles among taxa. Non-metric multidimensional scaling (NMDS) analyses on DNA metabarcoding data produced tighter clusters, more precise separation by land use, and greater consistency across taxonomic scales. Both urban context and land use had significant associations with metabarcoding patterns observed, with differences being strongest between agriculturally-dominated and primarily forested sites (median R{superscript 2} {approx} 0.08-0.11). We also found strong, consistent environmental signals linked to agricultural settings, such as water conductivity and turbidity, and pH. Altogether, our DNA-based results demonstrate the differences in community composition among different land uses in this watershed. Importantly, our work highlights the need for more taxonomic resolution (obtained through DNA analysis) to decipher community changes linked to anthropogenic and environmental drivers, as morphological data alone may lack the precision needed to capture these patterns.

Large-scale visual surveys are an integral part of waterfowl conservation and management programs. In this paper we explore the possibility of using environmental DNA-based surveys as a cost-efficient, complementary tool to estimate populations of North American waterfowl species. To achieve this, we first evaluated the performance of all currently available avian metabarcoding primers and compared them to newly designed primers targeting the mitochondrial ND2 gene within the Anatidae tribes of North America. All the existing avian assays showed strong cross-priming amplification with other vertebrates. In contrast, in-silico analyses of our waterfowl targeted assays showed a high degree (>90%) of avian specificity, encompassing all the 132 Anatidae species sequenced thus far. We used this targeted metabarcoding approach to track the temporal variation in the relative abundance of waterfowl species during the fall migration at Montezuma National Wildlife Refuge, New York, a major resting area for waterfowl on their journey to and from North American nesting areas. We compared eDNA results with visual surveys conducted by us and from those reported on eBird. Our results showed that eDNA detected all waterfowl species (n= 25) during fall migration. Positive correlations existed between standardized eDNA read counts and the relative abundance of waterfowl species as reported in eBird on the day of sampling and up to five days prior. However, this approach did not provide a good metric for absolute abundance of waterfowl species: only 8 out of 25 waterfowl species showed significant correlations between the number of eDNA reads and the total abundance of birds. Overall, while eDNA-targeted metabarcoding has not yet been applied to study bird communities extensively, our results demonstrate that this technique can be used as an effective complementary tool for assessing species composition of waterfowl communities and estimating relative abundance of species within those communities.

Selecting primers for DNA amplification poses a challenge in environmental DNA (eDNA) studies, particularly in biodiverse tropical ecosystems. Researchers must balance taxonomic coverage, primer specificity, and study feasibility when choosing between a single primer pair and multiple primer pairs. Empirical tests comparing these strategies are essential for improving eDNA protocols, enhancing the detection of aquatic biodiversity, and supporting conservation and environmental monitoring efforts. This study aimed to evaluate how different approaches affect the specificity and efficiency of detecting fish species in eDNA samples from the Teles Pires River basin. We (1) synthesized modified versions of primers commonly used in ichthyofaunal surveys; (2) compared the results generated by these modified primers with the standard primer versions; and (3) evaluated the species detection using two reference sequence databases: a global taxonomic database (GenBank) and a locally constructed taxonomic database containing samples of fish species previously collected in situ. The 12S-V5_mod primers detected the most fish species across the databases (67, 104, and 130 in the Midori2, Local, and joint databases, respectively), followed by the MiFish_mod primers, which detected 47, 101, and 114 species. Finally, the MiFish primers detected 39, 73, and 78 species. The 12S-V5_mod primers also had lower taxonomic specificity of fish species and amplified a broader range of vertebrate taxa than the MiFish_mod primers. The modified primers outperformed the MiFish primers, and detectability nearly doubled when the Local reference database was used. These results speak to the critical need for constructing reference databases of regional biodiversity, and the need to incorporate multiple primer pairs in eDNA studies.

Environmental DNA (eDNA) analysis has gained traction as a precise and cost effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA pipeline. At the same time, these two parameters are essential for the experimental design of a project. Here we evaluate and rank different eDNA protocols in the context of Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an aquatic environment, the San Francisco Estuary. We present a comprehensive evaluation of multiple eDNA protocol parameters, balancing time, cost and DNA yield. For estuarine waters, which are challenging for eDNA studies due to high turbidity, variable salinity, and the presence of PCR inhibitors, we find that a protocol combining glass filters and magnetic beads, along with an extra step for PCR inhibitor removal, is the method that best balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the optimal eDNA protocol for other studies on aquatic systems. Our findings should be applicable to most aquatic environments and provide a clear guide for determining which eDNA pipeline should be used for a given environmental condition. Author SummaryThe use of environmental DNA (eDNA) analysis for monitoring wildlife has steadily grown in recent years. Though, due to differences in the ecology of the environment studied and the novelty of the technique, eDNA currently shows a lack of standards compared to other fields. Here we take a deep look into each step of an eDNA assay, looking at common protocols and comparing their efficiencies in terms of time to process the samples, cost and how much DNA is recovered. We then analyze the data to provide a concise interpretation of best practices given different project constraints. For the conditions of the San Francisco Estuary we suggest the use of glass fiber filtration, the use of paramagnetic beads for DNA extraction and the use of a secondary inhibitor removal. We expect our findings to provide better support for managers to decide their standards ahead of project submission not only for estuarine conditions but for other waterine conditions alike.

The study of insect decline remains a major frontier in insect biodiversity and conservation. Despite growing concern about accelerating rates of insect decline generally, relatively little data has been compiled about species of aquatic insects. Data is particularly lacking on the distribution of aquatic insects in urban ecosystems. Here, we compare environmental DNA (eDNA) metabarcoding and community science observation as means of monitoring Odonata within an urban system in Southwest Idaho. We show that the distribution of Odonata across this urban landscape is not uniform and that both monitoring methods have different strengths and weaknesses. We found that eDNA metabarcoding was very sensitive to the identification of genera from underrepresented families in the region, but was unable to distinguish between closely related genera, particularly from localities where eDNA could accumulate more damage. On the other hand, community science observations effectively identified the presence of genera from more speciose families but missed the presence of relatively rare species, and those that had a short flight season. These findings suggest that, in our study system, eDNA and community science are highly complementary of each other. In cases where only one method is employed for a monitoring or conservation project, care should be given to account for the biases of each approach.

Environmental DNA (eDNA) allows for efficient and convenient data collection using DNA segments to confirm species presence. There is little literature using eDNA to specifically examine tiger salamander presence in North Dakota. I sampled 8 different cattle ponds in Western North Dakota twice each using a set eDNA collection method from the United States Department of Agriculture. After collecting water samples, the filters were sent to Jonah Ventures for DNA extraction and metabarcoding. Samples were incubated, extracted, ran through a first round of Polymerase Chain Reaction (PCR) inspected on agarose gel. Then the amplicons-product of an amplified or replicated piece of DNA, were cleaned, a second round of PCR followed, then the amplicons were cleaned again. Finally sequencing and bioinformatics took place. Salamanders (genus Ambystoma) were detected at 5 of the 8 sites. Average single season Bayesian occupancy point estimate was 0.655 {+/-} (.165), with a detection probability of 0.735{+/-} (.156) over the two site visits. Of the covariates including elevation, surface area, and air temperature, air temperature on visit 2 had the best model but still was not significant. There was no significant correlation between any of the covariates and the naive detections. This method was effective in detecting the genus Ambystoma, however more work and greater sample site variation could elucidate impacts of species level presence absence data.

Bird nests are fascinating microcosms harboring a wide range of arthropods parasitizing the nesting birds or feeding on prey remains, feces, and the nest material. Studies of these communities have been entirely based on emergence traps which collect live organisms out of the nests. The analysis of nest contents and environmental DNA (eDNA) via metabarcoding could expand our knowledge and identify prey, exuviae, and other animal remains in bird nests. Here, we investigated the potential of arthropod remains, nest dust, and feathers to better describe taxonomic diversity accumulated in 20 bird nests collected in Guelph (Canada). We used subsampling strategies and tested two extraction approaches to investigate the distribution of DNA in nests, account for low-quality DNA, and the presence of inhibitory substances. In total, 103 taxa were detected via metabarcoding. Arthropod remains delivered the highest number of taxa (n=67), followed by nest dust (n=29). Extractions with the PowerSoil kit outperformed DNeasy extractions coupled with PowerClean Pro inhibitor removal. The subsamples of the same nest showed 5.5% and 47.1% taxonomic overlap for arthropod remains and PowerSoil extracted nest dust, respectively, indicating a heterogeneous eDNA distribution in nests. Most detected species were either feeding in the nest, i.e., herbivorous / predatory, or bird food. We also detected molecular traces of 25 bird species, whose feathers were likely used as nest material. Consequently, the metabarcoding of bird nest materials provides a more complete picture of nest communities, which can enable future studies on functional diversity and better comparisons between nesting species.

Environmental (eDNA) metabarcoding is recently being considered for bioassessment under the Water Framework Directive (WFD). While tissue-based ("bulk") metabarcoding of macroinvertebrates produces comparable ecological status class values to morphology-based methods, eDNA-based metabarcoding often includes DNA signals from upstream sites, limiting its site-specificity. A promising alternative involves the incubation of locally collected invertebrates in water, followed by eDNA metabarcoding of the incubated water. In this study, we tested the suitability of two different approaches of the incubation strategy for ecological status assessment under the WFD. The first approach uses conventional multi-habitat sampling (MH) for the collection of the local macroinvertebrates and the second natural substrate exposures (NSEs) that are actively colonised. Using metabarcoding of the incubated water from both approaches (MH/NSE eDNA), we compared community composition, trait diversity and the derived ecological status classes (ESCs) to the local signal reference (MH/NSE bulk DNA) and the regional signal reference (stream water eDNA). For both approaches, community and trait composition were highly congruent between the incubated water and bulk samples. Trait composition did not differ between MH and NSE samples or among NSE types (wood-leaf vs. gravel). However, we detected small-scale spatial differences in the trait composition between NSEs placed at different flow regimes (pool vs. riffle). ESCs derived from all approaches (MH/NSE incubated water eDNA, MH/NSE bulk DNA and stream water eDNA) were highly similar and consistent with those from morpho-taxonomic assessments. Our findings support the incubation strategy using the conventional MH sampling as the most suitable approach for a minimally invasive, site-specific stream bioassessment within the context of the WFD.

Context for and purposeRetrieval of modern and ancient environmental DNA (eDNA) from sediments has revolutionized our ability to reconstruct present and past ecosystems. Little emphasis has been placed, however, on the fundamentals of the DNA-sediment associations and, consequently, our understanding of taphonomy and provenance of eDNA in sediments remains extremely limited. If we are to be able to accurately infer community dynamics across time and space from eDNA data, we need to understand how depositional processes and sedimentary associations of DNA molecules in different settings influence our interpretation. Approach and methodsHere, we introduce interfacial geochemical principles to the field of eDNA and discuss current interpretational biases. We outline a way to increase the scope and resolution of ecological interpretations from eDNA by combining mineralogic composition with experimental adsorption data. We apply distribution coefficients to assess the relationship between the DNA fraction in water columns and DNA fraction sequestered by suspended sediment particles. We further evaluate the challenges with drawing ecological inference using eDNA from sedimentary systems that receive input from different ecosystem types as a consequence of sedimentary processes. Main results: We show thatO_LIThe retention of DNA in aqueous environments depends on the mineralogy of sediment particles and on the number of particles loaded in the water column. C_LIO_LIDNA attached to sediment particles from distal systems can be deposited in proximal systems and skew the interpretation of the proximal sediment samples. C_LIO_LIHigh particle loading in the water column can deplete suspended DNA and cause inaccurate interpretation of aqueous DNA samples. C_LIO_LIHigh particle loading in surface sediment pore waters enhances sequestration of DNA from benthic communities relative to that of water column communities, resulting in skewed estimates of species richness and abundance from sedimentary DNA. C_LI We discuss how to integrate taphonomy and provenance knowledge into the reconstruction of modern and past ecosystems, and ecosystem monitoring from eDNA data. Conclusions and the wider implicationsOur findings demonstrate that integrating information about eDNA taphonomy and provenance into modern and past ecosystem reconstruction from eDNA data can enhance the scope, resolution and accuracy of our interpretations.

O_LIIncreasingly, molecular methods of species monitoring are integrated into freshwater biodiversity surveys and fisheries management. Inferring organism abundance or biomass from sequence counts derived from metabarcoding data has been an exciting but contentious concept in the biomonitoring community for some time. Although demonstrating a strong correlation with abundance has proven difficult, many researchers have assumed that quantitative metabarcoding data can at least provide broad-scale ranking of abundance. However, robust field validations of this widely-held assumption remain scarce. C_LIO_LIHere, we analyse metabarcoding read counts of fish eDNA data derived from 20 lakes and use betabinomial mixed effects models to compare this to rank abundance generated from long-term fish survey data. Rank abundance data for 18 species was generated within-species across-sites, meaning that ranks compare the abundance of the same species in different lakes. We also investigated a possible allometric effect on eDNA production by analysing a subset of data for effects of fish body mass on the amount of eDNA sequences. C_LIO_LIWe found a good relationship between species-specific eDNA sequences and within-species rank abundance categories for fishes, with rare fish producing 3% of sequences in a library, moderately abundant producing 7% and abundant fish producing 29%, according to model predictions. C_LIO_LIWe found a small negative effect of body mass on the amount of eDNA sequences, where the proportion of reads recovered significantly decreased with increased mean body mass of the population. C_LIO_LISynthesis and applications: The benefit of this approach is the potential for rapid assessment of rank abundance for multiple species, including smaller species which are often missed by conventional methods such as gillnetting, with relatively low amounts of additional effort. This approach will assist practitioners taking a species-based approach to freshwater habitat management in lakes worldwide. C_LI

Environmental DNA (eDNA) has become an established and efficient method for monitoring biodiversity in aquatic systems. However, there is a need to compare and standardise sampling methods across ecosystem types, particularly complex ecosystems such as estuaries where unique challenges for monitoring fish populations are present due to fluctuating environmental factors. Here, we compare fish biodiversity metrics obtained from eDNA metabarcoding data using four different eDNA filtering methods: three manual filtering methods with different pore sizes (0.45, 1.2 and 5 {micro}m) and a newly established passive method, the metaprobe. The study was applied across a salinity gradient in a hyper-tidal estuarine ecosystem. Overall, 44 fish species were detected across the four methods used. The 0.45 {micro}m filter recovered the highest richness (39 species), then the metaprobe method (35), followed by the 1.2 {micro}m (34) and 5 {micro}m (33) filters. Filter performance between salinity gradients revealed that the 0.45 {micro}m and the 1.2 {micro}m methods recovered the highest species richness across all sampled zones. The 0.45 {micro}m also had the most consistent detection probabilities using representative species from each zone. While the 0.45 {micro}m method appeared to be the optimal method, each of the methods can be considered as a viable and comparable option for biomonitoring in dynamic ecosystems such as estuaries and rivers. In particular, the passive metaprobe (used in a freshwater system for the first time here) performed well in comparison to the manual filtering methods despite a short deployment time. This study provides critical insights for optimising fish biodiversity assessments using eDNA metabarcoding in estuarine ecosystems, providing a valuable framework for future monitoring efforts in similar systems worldwide.

There is an increasing need to move beyond evaluating the effect of land use on stream communities by only studying local variables, and instead incorporate a metacommunity perspective which integrates environmental and spatial factors across larger spatial scales. The use of molecular tools (DNA metabarcoding) to identify bioindicator groups, such as aquatic macroinvertebrates, can provide greater taxonomic resolution to explore patterns in stream metacommunities. In this study, we collected aquatic macroinvertebrates from streams in southern Ontario which spanned a gradient of agricultural disturbance and used DNA metabarcoding to identify the species composition from these samples. We address a significant knowledge gap in previous stream aquatic macroinvertebrate metacommunity studies by incorporating molecular identification as well as a temporal component. We observed that a combination of local habitat conditions, regional agricultural land use, and spatial position influenced aquatic macroinvertebrate community composition, suggesting there is an interaction between environmental filtering and dispersal processes that structures these communities. However, aquatic macroinvertebrate communities were also highly dissimilar between streams and composed of many rare species, and a large percentage of unexplained variation suggests that there is a strong stochastic component to community assembly. We also observed that there is a seasonal component to metacommunity dynamics, with different water quality variables being significant to community composition in each sampling month. While we expected that an increased percentage of surrounding agricultural land use would result in more homogenous macroinvertebrate communities, we only detected this relationship in May and found evidence that a larger riparian buffer width can mitigate the effects of agricultural land use. We demonstrate the utility of DNA metabarcoding for revealing patterns in metacommunity dynamics that may not be detectable using coarse taxonomic identifications, and reveal the importance of incorporating a seasonal component when evaluating the influence of land use on community composition.

Quantifying in-situ environmental DNA (eDNA) concentrations is increasingly used to infer fish abundance and biomass in freshwater ecosystems. While various sampling strategies (i.e. the combination of sample collection, eDNA preservation and extraction protocols) have been proposed to collect eDNA, little efforts have been undertaken to evaluate how these different strategies affect taxon-specific eDNA recovery and the subsequent estimates of in-situ eDNA concentrations. In this study, we compared a point (i.e. eDNA collection from relatively small and spatially separated water samples filtered using low capacity filter units) and a spatially integrated sampling strategy (i.e. eDNA collected from spatially integrated larger water volumes filtered using high capacity filter units) in two natural lakes. Through quantitative analyses of total DNA, total fish eDNA and species-specific eDNA, we assessed the performance of both strategies to infer in-situ eDNA concentrations. Our results showed that the integrated strategy led to a reduced recovery of fish eDNA and a subsequent underestimation of in-situ eDNA concentrations compared to the point sampling strategy. While the exact mechanisms underlying this pattern require further investigation, our findings highlight the importance of carefully selecting sampling strategies according to study objectives.

Isogenus nubecula is a critically endangered Plecoptera species. Considered extinct in the UK, the species was recently rediscovered in one location of the river Dee in Wales after 22 years of absence. As many species belonging to the Perlodidae, this species can be a bio-indicator, utilised for assessing water quality and health status of a given freshwater system. However, conventional monitoring of invertebrates via kick-sampling for example, is an invasive and expensive (time consuming). Further, such methods require a high level of taxonomic expertise. Here, we compared the traditional kick-sampling method with the use of eDNA detection using qPCR and ddPCR-analyses. In spring 2018, we sampled eDNA from twelve locations on the river Dee. I. nubecula was detected using kick-sampling in five of these locations, three locations using both eDNA detection and kick-sampling and one location using eDNA detection alone - resulting in a total of six known and distinct populations of this critically endangered species. Interestingly, despite the eDNA assay being validated in vitro and in silico, and results indicating high sensitivity, qPCR analysis of the eDNA samples proved to be ineffective. In contrast, ddPCR analyses resulted in a clear detection of I. nubecula at four locations suggesting that inhibition most likely explains the big discrepancy between the obtained qPCR and ddPCR results. It is therefore important to explore inhibition effects on any new eDNA assay. We also highlight that ddPCR may well be the best option for the detection of aquatic organisms which are either rare or likely to shed low levels of eDNA into their environment.

DNA based methods offer a rapid and cost-effective way for detecting species occurrence and monitoring biodiversity; among them bulk DNA metabarcoding is well-established, and recently developed environmental DNA (eDNA)-based methods offer a non-destructive alternative. With a goal to develop suitable methods for assessing insect biodiversity in ecosystems for which DNA reference libraries are not well developed and incomplete, such as remote islands, we compared established bulk DNA metabarcoding methods with eDNA across three replicated terrestrial ecosystem types (limestone forest, degraded forest, and grassland) in Guam. Using two mitochondrial COI primer pairs, we performed bulk DNA metabarcoding of standard entomological collection methods (malaise traps, pan traps, vegetation beating), and compared the assessment of biodiversity with that from different eDNA sources (flowers, spider webs, leaves, tree trunks). In our samples, eDNA and bulk DNA metabarcoding both detected a large proportion of overall taxa (OTUs, 86.6% and 60.3%, respectively). Although DNA metabarcoding detected significantly more taxa, eDNA proved to be a reasonable non-destructive alternative. As expected, because of limitations in existing reference databases for remote habitats, species-level identification was achieved for only a few OTUs. Overall, the sampling approach was the dominant driver of arthropod diversity, explaining [~]17% of the observed variation, while habitat type accounted for [~]4%. Thus, each sampling approach captured some unique diversity signals and contributed to the complementary effect of maximizing detection. For rapid insect biodiversity surveys of terrestrial arthropods, we recommend an integrated metabarcoding approach, and in sensitive habitats where insect capture is undesirable, eDNA offers a powerful alternative to monitor diversity and community change.

Robust methods to monitor species distributions are vital to ensuring successful conservation strategies, particularly in the case of conservation translocations. Environmental DNA (eDNA) from water samples is a cost-effective method to monitor species distributions without physical capture or disturbance. However, eDNA is vulnerable to long-distance transport depending on the hydrological and environmental characteristics which can lead to spatially false positives and ultimately inaccurate species distributions. Recently, the development of particle transport models has allowed researchers to integrate hydrological and environmental variables to predict how far eDNA will transport from a source point. Here, eDNA samples (n=218) were collected and quantified using digital PCR (dPCR) to study monthly changes in Eurasian beaver (Castor fiber) eDNA concentrations downstream of an enclosure which contained 4 - 5 beavers located in Scotland. The shortest eDNA transport distances (< 2 km) were observed in the summer which correlated with the lowest flows and highest temperatures. In contrast, throughout the winter eDNA was consistently detected up to 5.8 km downstream correlating with the highest discharge and lowest temperature. The eDNA transport model reliably reproduced the decrease in eDNA concentrations downstream of the enclosure, however there were challenges surrounding stream-specific decay rates following a confluence. To study localised species distributions, samples should be collected during summer low flow conditions. Conversely, to maximise species detections sampling should be conducted in winter which had the longest eDNA transport and highest detectability.