Combining environmental DNA and visual surveys can inform conservation planning for coral reefs.

Environmental DNA (eDNA) metabarcoding has the potential to revolutionize conservation planning by providing spatially and taxonomically comprehensive data on biodiversity and ecosystem conditions, but its utility to inform the design of protected areas remains untested. Here, we quantify whether and how identifying conservation priority areas within coral reef ecosystems differs when biodiversity information is collected via eDNA analyses or traditional visual census records. We focus on 147 coral reefs in Indonesia's hyper-diverse Wallacea region and show large discrepancies in the allocation and spatial design of conservation priority areas when coral reef species were surveyed with underwater visual techniques (fishes, corals, and algae) or eDNA metabarcoding (eukaryotes and metazoans). Specifically, incidental protection occurred for 55% of eDNA species when targets were set for species detected by visual surveys and 71% vice versa. This finding is supported by generally low overlap in detection between visual census and eDNA methods at species level, with more overlap at higher taxonomic ranks. Incomplete taxonomic reference databases for the highly diverse Wallacea reefs, and the complementary detection of species by the two methods, underscore the current need to combine different biodiversity data sources to maximize species representation in conservation planning.

A comparative study on eDNA-based detection of Siamese bat catfish (Oreoglanis siamensis) in wet and dry conditions.

The use of environmental DNA (eDNA) analysis has demonstrated notable efficacy in detecting the existence of freshwater species, including those that are endangered or uncommon. This application holds significant potential for enhancing environmental monitoring and management efforts. However, the efficacy of eDNA-based detection relies on several factors. In this study, we assessed the impact of rainfall on the detection of eDNA for the Siamese bat catfish (Oreoglanis siamensis). Quantitative polymerase chain reaction (qPCR) analysis indicated that samples from days with average rainfall exceeding 35 mm (classified as heavy and very heavy rain) yielded negative results. While eDNA detection remains feasible on light or moderate rainy days, a noteworthy reduction in eDNA concentration and qPCR-positive likelihood was observed. Analysis across 12 sampling sites established a statistically significant negative relationship (p < 0.001) between eDNA detection and rainfall. Specifically, for each 1 mm increase in rainfall, there was an observed drop in eDNA concentration of 0.19 copies/mL (±0.14). The findings of this study provide definitive evidence that precipitation has a significant impact on the detection of eDNA in Siamese bat catfish. However, in the case of adverse weather conditions occurring on the day of sampling, our research indicates that it is acceptable to continue with the task, as long as the rainfall is not heavy or very heavy. To enhance the effectiveness of an eDNA survey, it is crucial to consider many factors related to climatic conditions. The aforementioned factor holds significant importance not only for the specific species under scrutiny but also for the broader dynamics of the climate.

Environmental DNA unveils deep phylogeographic structure of a freshwater fish.

Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using an environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach Barbatula toni, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from B. toni in 189 rivers. A total of 51 samples, representing the entire island, were then selected from the B. toni eDNA-positive sample set for the subsequent analyses. To elucidate the phylogeographic structure of B. toni, newly developed eDNA metabarcoding primers (Barba-cytb-F/R) were applied to these samples, specifically targeting their haplotypic variation in cytochrome b. After a bioinformatic processing to mitigate haplotypic false positives, a total of 50 eDNA haplotypes were identified. Two regionally restricted, genetically distinct lineages of the species were revealed as a result of phylogeographic analyses on the haplotypes and tissue-derived DNA from B. toni. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonisation of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography.

TICI: a taxon-independent community index for eDNA-based ecological health assessment.

Global biodiversity is declining at an ever-increasing rate. Yet effective policies to mitigate or reverse these declines require ecosystem condition data that are rarely available. Morphology-based bioassessment methods are difficult to scale, limited in scope, suffer prohibitive costs, require skilled taxonomists, and can be applied inconsistently between practitioners. Environmental DNA (eDNA) metabarcoding offers a powerful, reproducible and scalable solution that can survey across the tree-of-life with relatively low cost and minimal expertise for sample collection. However, there remains a need to condense the complex, multidimensional community information into simple, interpretable metrics of ecological health for environmental management purposes. We developed a riverine taxon-independent community index (TICI) that objectively assigns indicator values to amplicon sequence variants (ASVs), and significantly improves the statistical power and utility of eDNA-based bioassessments. The TICI model training step uses the Chessman iterative learning algorithm to assign health indicator scores to a large number of ASVs that are commonly encountered across a wide geographic range. New sites can then be evaluated for ecological health by averaging the indicator value of the ASVs present at the site. We trained a TICI model on an eDNA dataset from 53 well-studied riverine monitoring sites across New Zealand, each sampled with a high level of biological replication (n = 16). Eight short-amplicon metabarcoding assays were used to generate data from a broad taxonomic range, including bacteria, microeukaryotes, fungi, plants, and animals. Site-specific TICI scores were strongly correlated with historical stream condition scores from macroinvertebrate assessments (macroinvertebrate community index or MCI; R2 = 0.82), and TICI variation between sample replicates was minimal (CV = 0.013). Taken together, this demonstrates the potential for taxon-independent eDNA analysis to provide a reliable, robust and low-cost assessment of ecological health that is accessible to environmental managers, decision makers, and the wider community.

Biodiversity and distribution patterns of blooming jellyfish in the Bohai Sea revealed by eDNA metabarcoding.

BACKGROUND: The mass occurrence of scyphozoan jellyfish severely affects marine ecosystems and coastal economies, and the study of blooming jellyfish population dynamics has emerged in response. However, traditional ecological survey methods required for such research have difficulties in detecting cryptic life stages and surveying population dynamics owing to high spatiotemporal variations in their occurrence. The environmental DNA (eDNA) technique is an effective tool for overcoming these limitations. RESULTS: In this study, we investigated the biodiversity and spatial distribution characteristics of blooming jellyfish in the Bohai Sea of China using an eDNA metabarcoding approach, which covered the surface, middle, and bottom seawater layers, and sediments. Six jellyfish taxa were identified, of which Aurelia coerulea, Nemopilema nomurai, and Cyanea nozakii were the most dominant. These three blooming jellyfish presented a marked vertical distribution pattern in the offshore regions. A. coerulea was mainly distributed in the surface layer, whereas C. nozakii and N. nomurai showed a upper-middle and middle-bottom aggregation, respectively. Horizontally, A. coerulea and C. nozakii were more abundant in the inshore regions, whereas N. nomurai was mainly distributed offshore. Spearman's correlation analysis revealed a strong correlation between the eDNA of the three dominant blooming jellyfish species and temperature, salinity, and nutrients. CONCLUSIONS: Our study confirms the applicability of the eDNA approach to both biodiverstiy evaluation of blooming jellyfish and investigating their spatial distribution, and it can be used as a supplementary tool to traditional survey methods.

Predicting downstream transport distance of fish eDNA in lotic environments.

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.

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.

Development and Testing of Species-Specific Primers for Detecting the Presence of the Northern Pacific Sea Star (Asterias amurensis) from Environmental DNA.

The starfish Asterias amurensis, a well-known predator of molluscan species in intertidal ecosystems, has caused substantial ecological and economic losses in North China such as offshore Qingdao. Effective monitoring and prevention measures are urged to minimize its negative impacts. Compared with traditional biomonitoring methods, environmental DNA technology has emerged as a powerful and cost-efficient tool for inferring species' presence and abundance. In this study, we developed a pair of species-specific primers (i.e., Ast-F and Ast-R) for the A. amurensis mitochondrial COI gene and tested its utility in amplifying and quantifying the DNA fragments from environmental samples under both laboratory and field conditions. The results of controlled water tank experiments demonstrated that the amount of eDNA released by A. amurensis was positively related to its biomass; after the removal of the starfish, the eDNA degraded significantly in 24 h and remained detectable for 8 days. The number of eDNA copies enriched tended to increase with smaller pore size of filter membrane and larger volume of filtered water. For field tests, we confirmed the validation of our approach in six locations in Qingdao by filtering 1000 ml water per sample with a 0.45-µm pore size filtration. All the amplification products generated a single and bright band via gel electrophoresis, and the quantitative PCR results unveiled significant differences in eDNA copies. This study provided an eDNA-based approach for investigating the distribution and biomass of A. amurensis, which may help to formulate early warning and management strategies in coastal Qingdao and other regions.

Exploitation of environmental DNA (eDNA) for ecotoxicological research: A critical review on eDNA metabarcoding in assessing marine pollution.

The rise in worldwide population has led to a noticeable spike in the production, consumption, and transportation of energy and food, contributing to elevated environmental pollution. Marine pollution is a significant global environmental issue with ongoing challenges, including plastic waste, oil spills, chemical pollutants, and nutrient runoff, threatening marine ecosystems, biodiversity, and human health. Pollution detection and assessment are crucial to understanding the state of marine ecosystems. Conventional approaches to pollution evaluation usually represent laborious and prolonged physical and chemical assessments, constraining their efficacy and expansion. The latest advances in environmental DNA (eDNA) are valuable methods for the detection and surveillance of pollution in the environment, offering enhanced sensibility, efficacy, and involvement. Molecular approaches allow genetic information extraction from natural resources like water, soil, or air. The application of eDNA enables an expanded evaluation of the environmental condition by detecting both identified and unidentified organisms and contaminants. eDNA methods are valuable for assessing community compositions, providing indirect insights into the intensity and quality of marine pollution through their effects on ecological communities. While eDNA itself is not direct evidence of pollution, its analysis offers a sensitive tool for monitoring changes in biodiversity, serving as an indicator of environmental health and allowing for the indirect estimation of the impact and extent of marine pollution on ecosystems. This review explores the potential of eDNA metabarcoding techniques for detecting and identifying marine pollutants. This review also provides evidence for the efficacy of eDNA assessment in identifying a diverse array of marine pollution caused by oil spills, harmful algal blooms, heavy metals, ballast water, and microplastics. In this report, scientists can expand their knowledge and incorporate eDNA methodologies into ecotoxicological research.

Evaluating environmental DNA detection of a rare fish in turbid water using field and experimental approaches.

Detection sensitivity of aquatic species using environmental DNA (eDNA) generally decreases in turbid water but is poorly characterized. In this study, eDNA detection targeted delta smelt (Hypomesus transpacificus), a critically endangered estuarine fish associated with turbid water. eDNA sampling in the field was first paired with a trawl survey. Species-specific detection using a Taqman qPCR assay showed concordance between the methods, but a weak eDNA signal. Informed by the results of field sampling, an experiment was designed to assess how turbidity and filtration methods influence detection of a rare target. Water from non-turbid (5 NTU) and turbid (50 NTU) estuarine sites was spiked with small volumes (0.5 and 1 mL) of water from a delta smelt tank to generate low eDNA concentrations. Samples were filtered using four filter types: cartridge filters (pore size 0.45 µm) and 47 mm filters (glass fiber, pore size 1.6 µm and polycarbonate, pore sizes 5 and 10 µm). Prefiltration was also tested as an addition to the filtration protocol for turbid water samples. eDNA copy numbers were analyzed using a censored data method for qPCR data. The assay limits and lack of PCR inhibition indicated an optimized assay. Glass fiber filters yielded the highest detection rates and eDNA copies in non-turbid and turbid water. Prefiltration improved detection in turbid water only when used with cartridge and polycarbonate filters. Statistical analysis identified turbidity as a significant effect on detection probability and eDNA copies detected; filter type and an interaction between filter type and prefilter were significant effects on eDNA copies detected, suggesting that particulate-filter interactions can affect detection sensitivity. Pilot experiments and transparent criteria for positive detection could improve eDNA surveys of rare species in turbid environments.

eDNA-based detection reveals invasion risks of a biofouling bivalve in the world's largest water diversion project.

Environmental DNA (eDNA) has increasingly been used to detect rare species (e.g., newly introduced nonindigenous species) in both terrestrial and aquatic ecosystems, often with distinct advantages over traditional methods. However, whether water eDNA signals can be used to inform invasion risks remains debatable owing to inherent uncertainties associated with the methods used and the varying conditions among study systems. Here, we sampled eDNA from canals of the central route of the South-to-North Water Diversion Project (hereafter SNWDP) in China to investigate eDNA distribution and efficacy to inform invasion risks in a unique lotic system. We first conducted a total of 16 monthly surveys in this system (two sites in the source reservoir and four sites in the main canal) to test if eDNA could be applied to detect an invasive, biofouling bivalve, the golden mussel Limnoperna fortunei. Second, we initiated a one-time survey in a sub-canal of the SNWDP using refined sampling (12 sites in ~22 km canal) and considered a few environmental predictors. We found that detection of target eDNA in the main canal was achieved up to 1100 km from the putative source population but was restricted to the warmer months (May-November). Detection probability exhibited a significant positive relationship with average daily minimum air temperature and with water temperature, consistent with the expected spawning season. eDNA concentration in the main canal generally fluctuated across months and sites and was generally higher in warmer months. Golden mussel eDNA concentration in the sub-canal decreased significantly with distance from the source and with increasing water temperature and became almost undetectable at ~22 km distance. Given the enormity of the SNWDP, golden mussels may eventually expand their distribution in the main canal, with established "bridgehead" populations facilitating further spread. Our findings suggest an elevated invasion risk of golden mussels in the SNWDP in warm months, highlighting the critical period for spread and, possibly, management.

Environmental DNA and remote sensing datasets reveal the spatial distribution of aquatic insects in a disturbed subtropical river system.

Biodiversity datasets with high spatial resolution are critical prerequisites for river protection and management decision-making. However, traditional morphological biomonitoring is inefficient and only provides several site estimates, and there is an urgent need for new approaches to predict biodiversity on fine spatial scales throughout the entire river systems. Here, we combined the environmental DNA (eDNA) and remote sensing (RS) technologies to develop a novel approach for predicting the spatial distribution of aquatic insects with high spatial resolution in a disturbed subtropical Dongjiang River system of southeast China. First, we screened thirteen RS-based vegetation indices that significantly correlated with the eDNA-inferred richness of aquatic insects. In particular, the green normalized difference vegetation index (GNDVI) and normalized difference red-edge2 (NDRE2) were closely related to eDNA-inferred richness. Second, using the gradient boosting decision tree, our data showed that the spatial pattern of eDNA-inferred richness could achieve a high spatial resolution to 500 m reach and accurate prediction of more than 80%, and the prediction efficiency of the headwater streams (Strahler stream order = 1) was slightly higher than the downstream (Strahler stream order >1). Third, using the random forest algorithm, the spatial distribution of aquatic insects could reach a prediction rate of over 70% for the presence or absence of specific genera. Overall, this study provides a new approach to achieving high spatial resolution prediction of the distribution of aquatic insects, which supports decision-making on river diversity protection under climate changes and human impacts.

Monitoring of multiple fish species by quantitative environmental DNA metabarcoding surveys over two summer seasons.

Periodic monitoring can provide important information for the protection of endangered fish, sustainable use of fishery resources and management of alien species. Previous studies have attempted to monitor fish using non-invasive environmental DNA (eDNA) technology, generally employing quantitative PCR to quantify the eDNA concentration. However, the throughput was limited. High-throughput metabarcoding technology can detect the DNA of multiple species simultaneously in a single experiment but does not provide sufficient quantification. In this study, we applied a quantitative metabarcoding approach to simultaneously quantify the eDNA concentration of an entire fish assemblage in a small reservoir over two summer seasons. Traditional surveys were also conducted to investigate the individuals of fish. The eDNA concentrations were quantified using quantitative metabarcoding, and the fish species detected using this approach were highly consistent with the results of traditional fish monitoring. A significant positive relationship was observed between the eDNA concentration and fish species abundance. Seasonal changes in fish community structure were estimated using eDNA concentrations, which may reveal the activity seasons of different fish. The eDNA concentrations of different fish species peaked at different water temperatures, reflecting the differential responses of fish species to this environmental factor. Finally, by detecting outlier eDNA concentrations, the spawning activities of 13 fish species were estimated, 12 of which were roughly consistent with the current knowledge of fish spawning periods. These results indicate that quantitative eDNA metabarcoding with dozens of sampling times is useful for the simultaneous ecological monitoring of multiple fish species.

Environmental DNA reveals patterns of biological invasion in an inland sea.

Non-native species have the potential to cause ecological and economic harm to coastal and estuarine ecosystems. Understanding which habitat types are most vulnerable to biological invasions, where invasions originate, and the vectors by which they arrive can help direct limited resources to prevent or mitigate ecological and socio-economic harm. Information about the occurrence of non-native species can help guide interventions at all stages of invasion, from first introduction, to naturalization and invasion. However, monitoring at relevant scales requires considerable investment of time, resources, and taxonomic expertise. Environmental DNA (eDNA) metabarcoding methods sample coastal ecosystems at broad spatial and temporal scales to augment established monitoring methods. We use COI mtDNA eDNA sampling to survey a diverse assemblage of species across distinct habitats in the Salish Sea in Washington State, USA, and classify each as non-native, native, or indeterminate in origin. The non-native species detected include both well-documented invaders and species not previously reported within the Salish Sea. We find a non-native assemblage dominated by shellfish and algae with native ranges in the temperate western Pacific, and find more-retentive estuarine habitats to be invaded at far higher levels than better-flushed rocky shores. Furthermore, we find an increase in invasion level with higher water temperatures in spring and summer across habitat types. This analysis contributes to a growing understanding of the biotic and abiotic factors that influence invasion level, and underscores the utility of eDNA surveys to monitor biological invasions and to better understand the factors that drive these invasions.

A qPCR assay for the rapid and specific detection of Shining ram's-horn snail (Segmentina nitida) eDNA from Stodmarsh National Nature Reserve, UK.

Segmentina nitida Müller 1774 is a freshwater snail which was formerly widespread throughout England and south Wales. Since the 1840s it has seen a rapid decline in its range which has been attributed to deteriorating water quality due to nutrient enrichment, lowering of water tables and over-management of the ditches in which it resides. S. nitida has therefore been identified as a UK Biodiversity Action Plan (UKBAP) priority species which recommends further research for its conservation. Here we have developed a Taqman based qPCR eDNA assay for the detection of S. nitida at the Stodmarsh National Nature Reserve and compared the results with a manual survey of the ditches at this location. 32 ditches were surveyed in November 2020 (22 at Stodmarsh) and February 2021 (10 outside the known range of S.nitida). Our eDNA analysis exhibited an observed percentage agreement of 84% with a kappa coefficient of agreement between manual and eDNA surveys of 0.56 (95% CI 0.22 to 0.92). Three ditches determined to be negative for S. nitida by eDNA analysis were manual survey positive, and a further two ditches that were negative by manual survey were positive by eDNA analysis revealing the potential for improved overall detection rates using a combination of manual and eDNA methodologies. eDNA analysis could therefore augment manual survey techniques for S. nitida as a relatively quick and inexpensive tool for collecting presence and distribution data that could be used to inform manual surveys and management of ditches.

Mock community experiments can inform on the reliability of eDNA metabarcoding data: a case study on marine phytoplankton.

Environmental DNA metabarcoding is increasingly implemented in biodiversity monitoring, including phytoplankton studies. Using 21 mock communities composed of seven unicellular diatom and dinoflagellate algae, assembled with different composition and abundance by controlling the number of cells, we tested the accuracy of an eDNA metabarcoding protocol in reconstructing patterns of alpha and beta diversity. This approach allowed us to directly evaluate both qualitative and quantitative metabarcoding estimates. Our results showed non-negligible rates (17-25%) of false negatives (i.e., failure to detect a taxon in a community where it was included), for three taxa. This led to a statistically significant underestimation of metabarcoding-derived alpha diversity (Wilcoxon p = 0.02), with the detected species richness being lower than expected (based on cell numbers) in 8/21 mock communities. Considering beta diversity, the correlation between metabarcoding-derived and expected community dissimilarities was significant but not strong (R2 = 0.41), indicating suboptimal accuracy of metabarcoding results. Average biovolume and rDNA gene copy number were estimated for the seven taxa, highlighting a potential, though not exhaustive, role of the latter in explaining the recorded biases. Our findings highlight the importance of mock communities for assessing the reliability of phytoplankton eDNA metabarcoding studies and identifying their limitations.

Environmental DNA-based biomonitoring of Cuban Crocodylus and their accompanying vertebrate fauna from Zapata Swamp, Cuba.

Crocodylians globally face considerable challenges, including population decline and extensive habitat modification. Close monitoring of crocodylian populations and their habitats is imperative for the timely detection of population trends, especially in response to management interventions. Here we use eDNA metabarcoding to identify the Critically Endangered Crocodylus rhombifer and the Vulnerable C. acutus, as well as vertebrate community diversity, in Cuba's Zapata Swamp. We tested four different primer sets, including those used previously in Crocodylus population genetic and phylogenetic research, for their efficiency at detecting crocodylian eDNA. We detected C. rhombifer eDNA in 11 out of 15 sampled locations within its historical geographic distribution. We found that data analyses using the VertCOI primers and the mBRAVE bioinformatics pipeline were the most effective molecular marker and pipeline combination for identifying this species from environmental samples. We also identified 55 vertebrate species in environmental samples across the four bioinformatics pipelines- ~ 85% known to be present in the Zapata ecosystem. Among them were eight species previously undetected in the area and eight alien species, including known predators of hatchling crocodiles (e.g., Clarias sp.) and egg predators (e.g., Mus musculus). This study highlights eDNA metabarcoding as a powerful tool for crocodylian biomonitoring within fragile and diverse ecosystems, particularly where fast, non-invasive methods permit detection in economically important areas and will lead to a better understanding of complex human-crocodile interactions and evaluate habitat suitability for potential reintroductions or recovery programs for threatened crocodylian species.

Integrating citizen science and environmental DNA metabarcoding to study biodiversity of groundwater amphipods in Switzerland.

Groundwater is the physically largest freshwater ecosystem, yet one of the least explored habitats on earth, both because of accessing difficulties and the scarcity of the organisms inhabiting it. Here, we demonstrate how a two-fold approach provides complementary information on the occurrence and diversity of groundwater amphipods. Firstly, we used a citizen science approach in collaboration with municipal water providers who sampled groundwater organisms in their spring catchment boxes over multiple weeks, followed by DNA barcoding. Secondly, we collected four 10 L water samples at each site, in one sampling event, for environmental DNA (eDNA) metabarcoding. We found that citizen science was very effective in describing the distribution and abundance of groundwater amphipods. Although the single time-point of eDNA sampling did not detect as many amphipods, it allowed the assessment of the entire groundwater community, including microorganisms. By combining both methods, we found different amphipod species co-occurring with distinct sequences from the eDNA-metabarcoding dataset, representing mainly micro-eukaryotic species. We also found a distinct correlation between the diversity of amphipods and the overall biodiversity of groundwater organisms detected by eDNA at each site. We thus suggest that these approaches can be used to get a better understanding of subterranean biodiversity.

Environmental DNA metabarcoding reveals distinct fish assemblages supported by seagrass (Zostera marina and Zostera pacifica) beds in different geographic settings in Southern California.

Seagrass beds are disappearing at a record pace despite their known value to our oceans and coastal communities. Simultaneously, our coastlines are under the constant pressure of climate change which is impacting their chemical, physical and biological characteristics. It is thus pertinent to evaluate and record habitat use so we can understand how these different environments contribute to local biodiversity. This study evaluates the assemblages of fish found at five Zostera beds in Southern California using environmental DNA (eDNA) metabarcoding. eDNA is a powerful biodiversity monitoring tool that offers key advantages to conventional monitoring. Results from our eDNA study found 78 species of fish that inhabit these five beds around Southern California representing embayment, open coastal mainland and open coastal island settings. While each bed had the same average number of species found throughout the year, the composition of these fish assemblages was strongly site dependent. There were 35 fish that were found at both open coast and embayment seagrass beds, while embayment seagrass sites had 20 unique fish and open coast sites had 23 unique fish. These results demonstrate that seagrass fish assemblages are heterogenous based on their geographic positioning and that marine managers must take this into account for holistic conservation and restoration efforts.

Harnessing environmental DNA to reveal biogeographical patterns of non-indigenous species for improved co-governance of the marine environment in Aotearoa New Zealand.

Aotearoa New Zealand's Northern region is a major gateway for the incursion and establishment of non-indigenous species (NIS) populations due to high numbers of recreational and commercial vessels. This region also holds a unique marine ecosystem, home to many taonga (treasured) species of cultural and economic importance. Regular surveillance, eradication plans and public information sharing are undertaken by local communities and governmental organizations to protect these ecosystems from the impact of NIS. Recently, considerable investments went into environmental DNA (eDNA) research, a promising approach for the early detection of NIS for complementing existing biosecurity systems. We applied eDNA metabarcoding for elucidating bioregional patterns of NIS distributions across a gradient from harbors (NIS hotspots) to open seas (spreading areas). Samples were collected during a research cruise sailing across three Aotearoa New Zealand harbors, Waitemata, Whangarei and Pewhairangi (Bay of Islands), and their adjacent coastal waters. The small-ribosomal subunit (18S rRNA) and mitochondrial cytochrome c oxidase I (COI) genes were screened using the online Pest Alert Tool for automated detection of putative NIS sequences. Using a probabilistic modelling approach, location-dependent occupancies of NIS were investigated and related to the current information on species distribution from biosecurity surveillance programs. This study was collaboratively designed with Maori partners to initiate a model of co-governance within the existing science system.