eDNA Samples Research Articles

Species-specific eDNA assay development for enhanced box jellyfish risk management in coastal environments

Human interaction with marine creatures holds both positive and negative dimensions. Coastal communities benefit from marine environments, relying on them for sustenance and livelihoods. Fishing activities support economies, and marine biodiversity contributes to overall ecosystem health. However, challenges like overfishing, habitat destruction, and pollution pose threats to both marine life and human communities. Recently, there has been widespread concern regarding the potential increase in jellyfish populations across global marine ecosystems, attributed mainly to environmental factors such as climate drivers and anthropogenic forces, or their complex interactions. Encounters with hazardous marine species, such as box jellyfish, exemplify the dangers associated with coastal activities. Unintended interactions may lead to stings, injuries, and even fatalities, necessitating proactive measures and advanced technologies. This study addresses the inadequacies of existing measures in preventing box jellyfish incidents by introducing environmental DNA (eDNA) assays for detecting the deadly Chiropsoides buitendijki and focuses on developing qPCR and dPCR-based eDNA assays. Emphasising prevention over treatment, the study establishes a proactive system to assess C. buitendijki distribution across 63 tourist beaches in the Gulf of Thailand. Comparative analysis highlights the superior performance of dPCR over qPCR and traditional surveys. The dPCR experiment yielded positive results for all eDNA samples collected at sites where C. buitendijki had previously been identified. Remarkably, the eDNA testing also detected positive results in 16 additional sample locations where no physical specimens were collected, despite reported jellyfish stings at some of these sites. These findings underscore the precision and efficacy of the proposed eDNA detection technology in the early detection and assessment of box jellyfish distribution. This advancement therefore not only aids ecological research but also serves as a valuable tool for safeguarding public health, providing an early warning system for potential jellyfish encounters. Balancing positive human-marine interactions with effective risk mitigation strategies is crucial for sustainable coexistence, the preservation of marine ecosystems, and human well-being.

Passive eDNA sampling facilitates biodiversity monitoring and rare species detection

Environmental DNA (eDNA) technology has revolutionized biomonitoring, but challenges remain regarding water sample processing. The passive eDNA sampler (PEDS) represents a viable alternative to active, water filtration-based eDNA enrichment methods, but the effectiveness of PEDS for surveying biodiverse and complex natural water bodies is unknown. Here, we collected eDNA using filtration and glass fiber filter-based PEDS (submerged in water for 1 d) from 27 sites along the final reach of the Yangtze River and the coast of the Yellow Sea, followed by eDNA metabarcoding analysis of fish biodiversity and quantitative PCR (qPCR) for a critically endangered aquatic mammal, the Yangtze finless porpoise. We ultimately detected 98 fish species via eDNA metabarcoding. Both eDNA sampling methods captured comparable local species richness and revealed largely similar spatial variation in fish assemblages and community partitions between the river and sea sites. Notably, the Yangtze finless porpoise was detected only in the metabarcoding of eDNA collected by PEDS at five sites. Also, species-specific qPCR revealed that the PEDS captured porpoise eDNA at more sites (7 vs. 2), in greater quantities, and with a higher detection probability (0.803 vs. 0.407) than did filtration. Our results demonstrate the capacity of PEDS for surveying fish biodiversity, and support that continuous eDNA collection by PEDS can be more effective than instantaneous water sampling at capturing low abundance and ephemeral species in natural waters. Thus, the PEDS approach can facilitate more efficient and convenient eDNA-based biodiversity surveillance and rare species detection.

Development of a quantitative colorimetric LAMP assay for fast and targeted molecular detection of the invasive lionfish Pterois miles from environmental DNA

The Mediterranean basin has faced an increased influx of invasive species since the Suez Canal expansion in 2015. The invasive lionfish species, Pterois miles, has rapidly established new populations in the Eastern Mediterranean Sea, impacting local fish biodiversity. Here, we have developed a new, fast (< 35 min) molecular approach to detect and quantify P. miles environmental DNA (eDNA) in combination with a portable device for field-based analysis. Using a species-specific real-time colorimetric loop-mediated isothermal amplification (qcLAMP) for the cytochrome oxidase subunit 1 (COI) gene, we demonstrate a high sensitivity with a limit of detection of 0.002 ng DNA per reaction, equivalent to only 50 copies of the COI gene. The assay is specific to the target in the presence of closely related and co-occurring species, and it is quantitative over five orders of magnitude. We validated the assay using aquarium water samples and further demonstrated its utility on natural eDNA samples collected from locations around the island of Crete where P. miles had been sighted. P. miles was indeed detected in three out of nine locations, two nature reserves and a closed bay. Lack of detection in the remaining locations suggests that populations are still at a low density. We also demonstrate the feasibility of P. miles eDNA qualitative detection directly from the filter used to collect eDNA-containing particles, completely omitting DNA extraction. Overall, we present a new approach for fast and targeted eDNA quantification. The developed LAMP assay together, with the quantitative real-time colorimetric detection approach, open new possibilities for monitoring invasive P. miles in the field.

Advances in environmental DNA monitoring: standardization, automation, and emerging technologies in aquatic ecosystems.

Environmental DNA (eDNA) monitoring, a rapidly advancing technique for assessing biodiversity and ecosystem health, offers a noninvasive approach for detecting and quantifying species from various environmental samples. In this review, a comprehensive overview of current eDNA collection and detection technologies is provided, emphasizing the necessity for standardization and automation in aquatic ecological monitoring. Furthermore, the intricacies of water bodies, from streams to the deep sea, and the associated challenges they pose for eDNA capture and analysis are explored. The paper delineates three primary eDNA survey methods, namely, bringing back water, bringing back filters, and bringing back data, each with specific advantages and constraints in terms of labor, transport, and data acquisition. Additionally, innovations in eDNA sampling equipment, including autonomous drones, subsurface samplers, and in-situ filtration devices, and their applications in monitoring diverse taxa are discussed. Moreover, recent advancements in species-specific detection and eDNA metabarcoding are addressed, highlighting the integration of novel techniques such as CRISPR-Cas and nanopore sequencing that enable precise and rapid detection of biodiversity. The implications of environmental RNA and epigenetic modifications are considered for future applications in providing nuanced ecological data. Lastly, the review stresses the critical role of standardization and automation in enhancing data consistency and comparability for robust long-term biomonitoring. We propose that the amalgamation of these technologies represents a paradigm shift in ecological monitoring, aligning with the urgent call for biodiversity conservation and sustainable management of aquatic ecosystems.

EDNA for monitoring and conserving terrestrial arthropods: Insights from a systematic map and barcode repositories assessments

Abstract In the past decade, environmental DNA (eDNA) assays have become an essential tool to investigate species presence with samples from the environment instead of collected specimens. eDNA sampling techniques have proved their worth in freshwater and marine studies; now, some trends emerge for their use in terrestrial habitats and particularly to study arthropods. After a systematic review of the literature, we illustrate and analyse the diversity of such studies and discuss their benefits and drawbacks. We identified the most relevant research themes and focused on (i) the taxa and environmental sample types targeted and (ii) the details of the survey scheme. In parallel, we also assessed the available number of sequences from cytochrome c oxidase subunit I (COI), 16S and 18S barcode regions for four major taxa (spiders, centipedes, springtails and insects) in relation to their diversity. We found strong taxonomic and geographic biases regarding coverage per barcode. eDNA research on terrestrial arthropods mainly focuses on insect species that affect humanity in a positive or negative way, and the availability of sequences is much higher for species from temperate‐developed countries than from tropical ones. Moreover, although a high variety of environmental samples are being used, most studies do not assess the barcode completeness of the target taxa nor compare the efficacy of eDNA monitoring technique to other well established and known traditional techniques. Careful workflow designs and comparisons are needed before giving any management or conservation advice as eDNA monitoring does not come without error. Strengths and weaknesses of eDNA assays for conservation are discussed.

Exploring the potential of extreme citizen science with Danish high school students using environmental DNA for marine monitoring

Citizen science involves non-professionals assisting with scientific research, contributing data, and conducting experiments under professional guidance. In this paper, we describe the citizen science project DNA & life, based at the Natural History Museum of Denmark, which actively engages high school students in both fieldwork and advanced laboratory analyses by collecting and analyzing eDNA samples from marine environments across Denmark. The analysis is performed via species-specific real-time PCR and in a case study we present data gathered in the project from 2017-2023 with focus on the potential of eDNA detecting in use for species monitoring. The results include seasonal occurrence of Mnemiopsis leidyi and national distributions of selected species of particular ecological interest Perca fluviatilis, Anguilla anguilla, Neogobius melanostomus and Alexandrium ostenfeldii. In addition to the eDNA case study, we present a new way of conducting citizen science and eDNA analysis, as a development of the established DNA & life project. Local DNA laboratories are created to shorten the path from sampling site to laboratory facility, creating the possibility for Danish high schools to participate in a new version of the project. In close collaboration with the academic staff of the DNA & life project both teachers and students help develop facilities, protocols and laboratory analyses in an extreme citizen science and co-creation approach, where the participants are involved in a higher level of laboratory work and data analysis. Allowing high school students to perform intricate molecular lab analyzes through an extreme citizen science approach has demonstrated encouraging outcomes and potential for data of high quality in terms of sampling and analyzing eDNA with the purpose of species monitoring and conservation.

From DNA to diagnostics: A case study using macroinvertebrate metabarcoding to assess the effectiveness of restoration measures in a Dutch stream

Stream ecosystems are under pressure due to multiple stressors. Restoration measures can halt further degradation and improve their ecological status. However, assessment of the effectiveness of the implemented measures is often insufficient because of logistic and financial constraints. DNA-metabarcoding has been proposed to scale up sample processing, although its application as a diagnostic tool has received less attention. The aim of our study was to evaluate if DNA-metabarcoding of stream macroinvertebrates can be used to compute a stressor-specific index to assess the effectiveness of a stream restoration project. For this purpose, we sampled the upstream, restored, and downstream section of a recently restored lowland stream in the Netherlands. At each site, we applied three different methods of macroinvertebrate identification: morphological identification of bulk samples (morphology), DNA-metabarcoding of the same bulk samples (DNA) and metabarcoding of eDNA extracted from the water (eDNA). First, we compared the community composition identified by each method. The communities identified by morphology and DNA were highly similar, whereas the communities generated by the eDNA differed. Second, we analysed whether the identification methods could be used to assess the effectiveness of the restoration project, focussing on a stressor-specific index for flow as the restoration measures aimed at improving flow conditions. Both the morphology and bulk DNA samples indicated improved flow conditions in the restored section of the stream (i.e., less stress from the reduction or absence of flow than in the unrestored sections). Contrary, the eDNA-water samples did not differentiate the amount of stress throughout the catchment, although applying recent developments in eDNA sampling could lead to more robust results. In conclusion, this study forms proof of concept that DNA from bulk samples can be utilized to assess the effectiveness of restoration measures, showing the added value of this approach for water managers.

Environmental DNA metabarcoding differentiates between micro‐habitats within the rocky intertidal

AbstractWhile the utility of environmental DNA (eDNA) metabarcoding surveys for biodiversity monitoring continues to be demonstrated, the spatial and temporal variability of eDNA, and thus the limits of the differentiability of an eDNA signal, remains under‐characterized. In this study, we collected eDNA samples from distinct micro‐habitats (~40 m apart) in a rocky intertidal ecosystem over their exposure period in a tidal cycle. During this period, the micro‐habitats transitioned from being interconnected, to physically isolated, to interconnected again. Using a well‐established eukaryotic (cytochrome oxidase subunit I) metabarcoding assay, we detected 415 species across 28 phyla. Across a variety of univariate and multivariate analyses, using exclusively taxonomically assigned data as well as all detected amplicon sequence variants (ASVs), we identified unique eDNA signals from the different micro‐habitats sampled. This differentiability paralleled expected ecological gradients and increased as the sites became more physically disconnected. Our results demonstrate that eDNA biomonitoring can differentiate micro‐habitats in the rocky intertidal only 40 m apart, that these differences reflect known ecology in the area, and that physical connectivity informs the degree of differentiation possible. These findings showcase the potential power of eDNA biomonitoring to increase the spatial and temporal resolution of marine biodiversity data, aiding research, conservation, and management efforts.

DNA from dives: Species detection of humpback whales (Megaptera novaeangliae) from flukeprint eDNA

AbstractNorthern British Columbia has been identified as an important habitat for several coastal cetacean species, including humpback whales (Megaptera novaeangliae). This species is listed as being of “Special Concern” under Canada's Species at Risk Act, partly due to data deficiencies concerning genetic population structure and demographics in British Columbia. Anthropogenic activities threaten North Coast humpback whale populations, with particular concern for the impact of vessel noise, entanglement, and ship strikes. Current methodology (i.e., biopsy sampling) for obtaining cetacean genetic data is invasive, challenging, and costly; therefore, there is an urgency to develop effective and minimally invasive methodologies for efficiently collecting this data. Environmental DNA (eDNA) has been identified as an ideal tool for monitoring the presence and distribution of numerous species within marine ecosystems; however, the feasibility for cetaceans is not yet well established. In this study, we opportunistically collected targeted 1 L seawater eDNA samples from flukeprints when individual humpback whales were observed diving between the years of 2020 and 2022. A total of 93 samples were collected from individual humpback whales identified using a photographic identification catalogue. We successfully detected humpback whale eDNA in 28 samples using novel species‐specific qPCR primers (~500 mL of sample), with relatively equal successful detection between immediate (0 days) and delayed (up to 10 days) sample filtration. Here, we have validated a qPCR assay for detecting humpback whale DNA from flukeprints and highlighted the future optimizations required to improve the potential application of flukeprint eDNA for conservation management.

Comparing cost, effort, and performance of environmental DNA sampling and trapping for detecting an elusive freshwater turtle

AbstractEnvironmental DNA (eDNA) analysis is an effective and non‐invasive technique for surveying and monitoring rare, threatened, or endangered (RTE) species. Compared to conventional capture‐based sampling, eDNA analysis may offer a more cost‐effective approach for surveying RTE species, yet few studies have compared their cost‐efficiency—a critical consideration for conservation planning. We compared the costs, effort, and relative performance of aquatic eDNA sampling and conventional trapping for detecting the Alligator Snapping Turtle, Macrochelys temminckii Troost, 1835, in southwest Louisiana, United States. Environmental DNA was sampled quarterly over 1 year (2018–2019) at 19 streams, including three streams where M. temminckii presence had been previously confirmed via conventional trapping efforts (2012–2013). Water samples from each stream were analyzed using quantitative polymerase chain reaction (qPCR) to assess M. temminckii eDNA presence/absence. Time and costs (i.e., labor, travel, wages, and supplies) per detection via eDNA analysis and trapping were calculated and compared. Environmental DNA analysis documented the presence of M. temminckii DNA at two of the three streams where individuals had previously been trapped and yielded detections (qPCR amplifications) at 16 additional streams not previously sampled, expanding M. temminckii's documented distribution at our study sites by 84%. Environmental DNA analysis returned a detection rate (per site) 5.55 times higher than conventional trapping and was 18.7% less expensive. Our results provide evidence that strategically deployed eDNA surveys may be an effective and cost‐efficient approach for detecting freshwater RTE species. With eDNA analysis, additional resources can be invested toward expanding survey coverage and increasing sampling frequency, allowing managers to more effectively target subsequent intensive monitoring efforts.

Generic qPCR assays for quality control in environmental DNA research

AbstractEnvironmental DNA (eDNA) has been widely used for species surveillance. However, the lack of adequate quality control in many eDNA research projects and applications can lead to false‐negative results, greatly affecting biosecurity surveillance and conservation efforts. Exogenous DNA is routinely added to eDNA samples and used as a positive control, typically after DNA extraction. However, this type of positive control is only able to identify false negatives due to errors at the amplification stage. Therefore, errors in upstream processes, such as sample collection will not be identified by an exogenous control. We designed two independent sets of generic quality control qPCR assays (QCqPCR) targeting abundant endogenous DNA that is obtained during sample collection. Our QCqPCR assays target the chloroplast 16S and 23S ribosomal RNA sequences. In silico analyses indicated these regions were highly conserved among plants, algae and bacteria commonly found in freshwater, marine, or terrestrial environments. These QCqPCR assays were purposely mismatched against the human genome to avoid false positives resulting from human DNA contamination. Both assays remained highly efficient and sensitive under annealing temperatures between 58 and 62°C, allowing them to be multiplexed with most qPCR analyses. We validated our assays by multiplexing with a species‐specific Murray cod (Maccullochella peelii) assay on field‐collected environmental water samples. Potential false‐negative reactions can be identified by the failed or suppressed QCqPCR assay and the negative species‐specific assay. We recommend incorporating either one of the QCqPCR assays in qPCR‐based eDNA analysis to identify potential false negatives and improve the reliability of eDNA surveys.

Design and partial validation of novel eDNA qPCR assays for three common North American tick (Arachnida: Ixodida) species

AbstractThe range expansion of ticks to higher latitudes poses a severe threat to human health exposing human populations who had no prior contact with ticks to several harmful tick‐borne diseases. Early detection of ticks in new areas is critical to help inform the public and medical professionals of the dangers associated with tick encounters. Environmental DNA represents a novel survey method that could provide reliable records of tick occurrences and timely warnings of their range expansions. In this study, we designed novel eDNA qPCR assays for three common North American tick species (Dermacentor variabilis, Amblyomma americanum, and Ixodes scapularis) and tested them on 51 samples of grasses and leaf litter collected from 12 grassland and forest sites in central and southern Illinois. We provide in silico and in vitro validation of all three assays; however, we were unable to generate any positive detections from field samples. Our lack of eDNA detections likely stems from low eDNA deposition rates coupled with rapid degradation in grasslands and forests, a problem exacerbated by terrestrial eDNA sampling methods limited by volume of substrate. We provide recommendations for improving sample collection methods to increase detection probability in future efforts. Continued research should focus on the viability of eDNA to detect small terrestrial invertebrates, like ticks, and it potential as early warning indicator of the spread of vector‐borne diseases.

Towards eDNA informed biodiversity studies – Comparing water derived molecular taxa with traditional survey methods

Managing natural resources in a sustainable manner requires understanding the complexity of ecosystems and the species that are associated with the different parts of the ecosystem. Much of this knowledge is derived from traditional sampling methods (e.g., different types of trawls). The analysis of environmental DNA (eDNA) can provide increased knowledge, complementary to the traditional methods. In the present pilot study, we sampled eDNA from two geographical areas, north and west of Svalbard (NWS) and in the southwestern Barents Sea (SWBS). The combination of trawling, visual identification of mammals and eDNA collection facilitated a robust analysis of fish and marine mammal diversity and species composition. Through 12S MiFish metabarcoding of the eDNA samples, we found that incorporating eDNA data provided an additional level of information on both the diversity of fish and marine mammals in the study areas. By adding eDNA data to the trawl data, we found that richness increased from 32 to 49 fish taxa. Significant differences in diversity and composition of the fish communities were detected by eDNA between the two study areas. Considering degradation and dilution factors it is postulated that the results represent resident species to the Barents Sea and that long -transported DNA from other areas are less likely.

Optimizing waterborne eDNA capture from waterholes in savanna systems under remote field conditions.

Environmental DNA (eDNA) is used for biodiversity assessments in a variety of ecosystems across the globe, whereby different eDNA concentration, preservation and extraction methods can outperform others depending on the sampling conditions and environment. Tropical and subtropical ecosystems in Africa are among the less studied systems concerning eDNA-based monitoring. Waterholes in arid parts of southern Africa represent important agglomeration points for terrestrial mammals, and the eDNA shed into such waterbodies provides a powerful source of information for monitoring mammalian biodiversity in the surrounding area. However, the applied methods for eDNA sampling, preservation and filtering in different freshwater systems vary greatly, and rigorous protocol testing in African freshwater systems is still lacking. This study represents the first attempt to examine variations in eDNA concentration, preservation and extraction methods under remote field conditions using waterborne eDNA in a savanna system. Collected samples were heavily affected by microalgal and bacterial growth, impeding eDNA capture and PCR success. We demonstrate clear effects of the methodological choices, which also depend on the state of eDNA. A preliminary metabarcoding run showed little taxonomic overlap in mammal species detection between two metabarcoding primers tested. We recommend water filtering (using filters with pore sizes >1 μm) over centrifugation for eDNA concentration, Longmire's solution for ambient temperature sample preservation and Qiagen's DNeasy PowerSoil Pro Kit for DNA extraction of these inhibitor-prone samples. Furthermore, at least two independent metabarcoding markers should be utilized in order to maximize species detections in metabarcoding studies.

EDNA metabarcoding reveals a rich but threatened and declining elasmobranch community in West Africa’s largest marine protected area, the Banc d’Arguin

AbstractElasmobranchs (sharks and rays) are the most threatened marine vertebrates, particularly in tropical and subtropical areas. Their population status is often poorly understood due to insufficient information. Despite reportedly harbouring critical elasmobranch habitats, the Banc d’Arguin National Park (PNBA) in Mauritania lacks comprehensive and updated information on the diversity of elasmobranch species in the area. We developed a baseline inventory based on morphological and molecular identification and metabarcoding. DNA barcoding of tissue samples from elasmobranch processing sites and freshly sampled specimens was used to build a genetic reference database of local elasmobranch species. The richness and diversity of species in the PNBA were described via metabarcoding of seawater eDNA samples using an elasmobranch-specific assay and our reference database. We detected 27 species, including 12 new species records for the PNBA. We further uncover potentially undescribed species of Gymnura and Torpedo, while taxonomic corrections are noted for previously reported species. In particular, the reportedly abundant Mustelus mustelus was absent from tissue and eDNA samples, while M. punctulatus was detected instead. Taxa that have anecdotally become regionally extinct or rare (e.g., sawfishes, wedgefishes, lemon sharks) were not detected, highlighting local species diversity shifts within the last few decades. Results show that 67.9% of elasmobranch species in the PNBA are threatened with extinction according to the IUCN Red List of Threatened Species. This study emphasises the importance of taxonomic identification in support of species management and provides a baseline to inform future studies and conservation measures to avoid further species losses.

Bridging the gap: The integration of eDNA techniques and traditional sampling in fish diversity analysis

IntroductionBiodiversity loss poses a significant environmental challenge, particularly in aquatic ecosystems. The advent of environmental DNA (eDNA) sampling technology offers a promising tool for monitoring biological communities with purported high efficiency. Yet, its efficacy compared to traditional sampling methods remains underexplored, especially in fish diversity research.MethodsThis study conducted a comparative analysis of fish diversity and distribution across 29 sampling points within the rivers of the Changqing Nature Reserve, Central China, employing both eDNA techniques and traditional sampling methods.ResultsA total of 46 unique fish species were identified through this comprehensive approach. eDNA sampling detected 34 species, surpassing the 22 species identified by traditional methods. Interestingly, 10 species were detected by both methods, while traditional methods exclusively identified 12 species not detected by eDNA, and eDNA uniquely identified an additional 24 species. Despite eDNA's broader species detection range, traditional sampling methods typically yielded higher Shannon diversity index values. Both β-diversity indices (Bray-Curtis and Jaccard) and multivariate analyses (NMDS and PCoA) were applied, revealing no significant statistical differences in biodiversity measurement between the two sampling methods.DiscussionThe findings suggest that while eDNA sampling excels in identifying a wider range of species, it does not significantly outperform traditional methods in overall biodiversity assessment. By integrating both methodologies, this study demonstrates a more comprehensive and precise assessment of riverine biodiversity, underscoring the benefits of a synergistic approach for enhancing species detection and understanding distribution patterns. The combined methodology notably improves alpha diversity evaluations, particularly regarding Shannon diversity and Berger-Parker dominance. This integrated approach advocates for the amalgamation of data from both eDNA and conventional methods, fostering a robust and accurate biodiversity appraisal.

Improved eDNA assay evidences further refugia for critically endangered smalltooth sawfish (Pristis pectinata) in Mexico

Sawfishes are considered one of the most threatened marine fish taxa globally, with major contractions in their geographic range documented over the last 50 years across all oceans. Two sawfish species used to be found in Mexico, but a historical lack of research and first-hand baseline information recently raised concerns about the fate of both species there. Recent Local Ecological Knowledge (LEK) and environmental DNA (eDNA) research has shown that: a) Pristis pectinata and P. pristis were formerly abundant and widely distributed in the Mexican Atlantic coast, and the latter also in the Mexican Pacific coast, and b) sawfishes (at least P. pectinata) are still found in some coastal refugia in central Veracruz state and Campeche state on the Atlantic coast. In the present research we developed improved primers that allow for better species ID resolution for Pristis pectinata and P. pristis through successful sequencing of eDNA samples by amplifying a 270 bp fragment of the CO1 gene. Our results based on the analysis of 305 eDNA samples showed an improved positive species identification rate through sequencing, demonstrate that P. pectinata occurs in other coastal refugia along the Yucatán Peninsula in the Mexican Caribbean, and suggest this species might be extirpated from southern Veracruz, while no P. pristis eDNA has been found along the Atlantic coast.

Predicting downstream transport distance of fish eDNA in loticenvironments.

Environmental DNA (eDNA) is an effective tool for describing fish biodiversity in lotic environments, but the downstream transport of eDNA released by organisms makes it difficult to interpret species detection at the local scale. In addition to biophysical degradation and exchanges at the water-sediment interface, hydrological conditions control the transport distance. A new eDNA transport model described in this paper considers downstream retention and degradation processes in combination with hydraulic conditions and assumes that the sedimentation rate of very fine particles is a correct estimate of the eDNA deposition rate. Based on meta-analyses of available studies, the particle size distribution of fish eDNA (PSD), the relationship between the sedimentation rate and the size of very fine particles in suspension, and the influence of temperature on the degradation rate of fish eDNA were successively modelled. After combining the results in a mechanistic-based model, the eDNA uptake distances (distance required to retain 63.21% of the eDNA particles in the riverbed) observed in a compilation of previous experimental studies were correctly simulated. eDNA degradation is negligible at low flow and temperature but has a comparable influence to background transfer when hydraulic conditions allow a long uptake distance. The wide prediction intervals associated with the simulations reflect the complexity of the processes acting on eDNA after shedding. This model can be useful for estimating eDNA detection distance downstream from a source point and discussing the possibility of false positive detection in eDNA samples, as shown in an example.

Simultaneous species detection and discovery with environmental DNA metabarcoding: A freshwater mollusk case study.

Environmental DNA (eDNA) sampling is a powerful tool for rapidly characterizing biodiversity patterns for specious, cryptic taxa with incomplete taxonomies. One such group that are also of high conservation concern are North American freshwater gastropods. In particular, springsnails of the genus Pyrgulopsis (Family: Hydrobiidae) are prevalent throughout the western United States where >140 species have been described. Many of the described species are narrow endemics known from a single spring or locality, and it is believed that there are likely many additional species which have yet to be described. The distribution of these species across the landscape is of interest because habitat loss and degradation, climate change, groundwater mining, and pollution have resulted in springsnail imperilment rates as high as 92%. Determining distributions with conventional sampling methods is limited by the fact that these snails are often <5 mm in length with few distinguishing morphological characters, making them both difficult to detect and to identify. We developed an eDNA metabarcoding protocol that is both inexpensive and capable of rapid, accurate detection of all known Pyrgulopsis species. When compared with conventional collection techniques, our pipeline consistently resulted in detection at sites previously known to contain Pyrgulopsis springsnails and at a cost per site that is likely to be substantially less than the conventional sampling and individual barcoding that has been done historically. Additionally, because our method uses eDNA extracted from filtered water, it is non-destructive and suitable for the detection of endangered species where "no take" restrictions may be in effect. This effort represents both a tool which is immediately applicable to taxa of high conservation concern across western North America and a case study in the broader application of eDNA sampling for landscape assessments of cryptic taxa of conservation concern.

Integrating spatial stream network models and environmentalDNAto estimate current and future distributions of nonnative Smallmouth Bass

AbstractObjectiveClimate change is fueling the rapid range expansion of invasive species in freshwater ecosystems. This has led to mounting calls from natural resource managers for more robust predictions of invasive species distributions to anticipate threats to species of concern and implement proactive conservation and restoration actions. Here, we applied recent advances in fish sampling and statistical modeling in river networks to estimate the current and future watershed‐scale spatial distribution of nonnative Smallmouth BassMicropterus dolomieu.MethodsWe integrated a spatial stream network (SSN) model of stream temperature, landscape environmental covariates, and Smallmouth Bass occurrence data based on environmental DNA (eDNA) detections to develop an SSN species distribution model (SDM) representing current Smallmouth Bass distributions in the Chehalis River, Washington State, a large coastal river basin of ongoing watershed‐scale restoration. The SDM was informed by spatially intensive eDNA sampling from 135 locations in the main stem and major tributaries. We then applied downscaled climate change projections to the SSN SDM to predict Smallmouth Bass range expansion in the basin by late century.ResultWe identified high levels of spatial autocorrelation at hydrological distances of ≤10 km in our eDNA data set, underscoring the importance of applying an SSN modeling framework. Stream temperature was identified as the most important environmental covariate explaining variability in Smallmouth Bass occurrence. Model predictions estimated that current suitable summer habitat for Smallmouth Bass habitat spans 681 km and is projected to nearly double by late century (1333 km) under a moderate climate change scenario. Current and future suitable habitat for Smallmouth Bass is prevalent in important tributaries for spring Chinook SalmonOncorhynchus tshawytscha, a species of major conservation concern in the Chehalis River and more broadly along the Pacific coast. In both the main stem and tributaries, the SSN SDM predictions of the upstream leading edges of Smallmouth Bass closely align with (within 4.8 km) edges identified by spatially intensive eDNA sampling.ConclusionOur study highlights the value of integrating SSN models with rapidly growing eDNA data sets for accurate and precise riverine fish distribution estimation. Our application provides crucial insights for anticipating the impacts of shifting invasive species on Pacific salmon Oncorhynchusspp. in a warming world.