Environmental DNA最新文献

Analysis of environmental DNA (eDNA) through DNA metabarcoding has become an important technique for environmental science as it allows precise reconstructions of species communities in a fast, cheap and non-invasive way. In this study, we scrutinize how environmental reconstructions derived from metabarcoding data may be affected by a process in which sample specific labels (tags), added to sequences for identification of individual samples, are changed unintentionally during adapter ligation causing translocation of sequences between samples (‘tag jumping’). We compare animal and plant communities reconstructed using sedimentary eDNA records processed according two different protocols: (i) a twin-tagging approach (control) where all amplicons received the same tag on both sides (N = 102); and (ii) a combinatorial tagging protocol (affected by tag jumps) where each amplicon received a unique combination, but where some tags on each side were reused to form new combinations (N = 102). We analyzed six different sediment matrices and observed higher average number of taxa in the combinatorial tagging dataset in comparison to our twin-tagged dataset serving as a reference for results unaffected by tag jumps. In the control dataset with twin tagged amplicons, reconstructed animal communities were statistically different in 14 out of 15 pairwise comparisons, while only 8 out of 15 of the comparisons were different when samples were analyzed using the combinatorial tagging protocol. All of the inferred plant communities were statistically different when analyzed with a twin-tagging approach, while 20% of these plant communities were not different in our combinatorial tagged dataset. Our results clearly show that tag jumps added species to samples where they were not originally present and affects interpretations of species diversity and time-trends for whole communities. We conclude that tag jumping, being rarely discussed in metabarcoding studies, constitutes a concern in parity with direct sample contamination.

Efficient and scalable methods for monitoring marine biodiversity are critical for understanding ecological change in coastal environments, given the limited resources available. Environmental DNA (eDNA) metabarcoding shows promise for monitoring coastal taxa, but its ability to differentiate communities from different locations remains insufficiently understood, particularly in dynamic marine environments. Here, we evaluate the effectiveness and resolution capacity of eDNA metabarcoding in detecting rocky intertidal taxa across three spatial scales—national, regional, and local—in the United Kingdom. Onshore surface-water samples were collected from 32 sites across five UK Regional Seas from rockpools in high and low shore zones, as well as directly from the sea. We detected 1026 target taxa within 442 families and 19 phyla using two established markers targeting invertebrates (COI) and macroalgae (18S). Distinct eDNA signals were found at all spatial scales, indicating local discreteness even between vertical shore heights within the same sites. Communities were more discrete at larger scales (i.e., between regions) than at smaller scales (i.e., between shore heights). eDNA signals were more strongly structured by geographical location than by vertical shore height as a probable consequence of greater DNA homogenization over the tidal cycle at smaller spatial scales. Established ecological zonation patterns were reflected in eDNA signals, with higher richness at lower shore heights, reflecting abiotic stress gradients. Detections of cold-affinity boreal species increased with latitude, while warm-affinity lusitanian species declined with latitude. Our work supports the utility of eDNA metabarcoding for multiscale biodiversity monitoring in dynamic marine environments and for detections beyond this study's target taxa. We recommend the adoption of scale-appropriate sampling protocols to optimize the benefits of eDNA, such as prioritizing open water sampling at high tide for broad-scale assessments and rockpool sampling at low tide for capturing local-scale patterns. Future work should validate detections through direct visual comparisons.

The European green toad (Bufotes viridis) is currently in decline and considered endangered across the northern extent of its native range, with large investments in ongoing conservation and translocation efforts. To assist conservation efforts, survey methods must be established that are cost-effective, non-invasive, and rapidly deployable. Here we evaluated the effectiveness of eDNA metabarcoding for amphibian conservation across three objectives: (1) Test B. viridis probability of detection before and after translocation efforts in 3 ponds in Öland, Sweden. (2) Assess pond biodiversity and biotic interactions across Öland and Kalmar using eDNA metabarcoding. (3) Determine which surveyed sites are suitable for future translocation efforts. We found that the detection probability of B. viridis increased 100% 24 h after the translocation was initiated, whereby they were undetected prior to release. Additionally, we detected 11 fish species, 14 bird species, 9 mammal species, and 4 amphibian species across the translocated sites. The results from the 37 pond eDNA surveys resulted in the detection of 15 fish species, 38 bird species, 8 amphibian species, and 17 mammal species. Species richness of the surveyed ponds ranged from 1 to 24, with an average richness of 8. Co-occurrence analysis found significant associations between several species, including a significant negative association between amphibian occurrence and cattle and gray heron and positive associations with duck and common crane. Multi-Criteria Decision Analysis (MCDA) suggests 6 sites had consistent lower site rankings, indicating them as more favorable locations for future amphibian translocation efforts. Overall, these findings showcase eDNA high-throughput sequencing as a viable means to non-invasively assess European green toads and simultaneously assess wider community dynamics that may help evaluate the sustainability of reintroduced and endemic populations.

Plant–animal interactions (PAIs) are critical in natural and agricultural ecosystems, mediating energy flow with both positive and negative interactions. Traditional methods of tracking PAIs, such as morphological identification and camera trapping, are limited in speed and scalability, posing challenges for comprehensive biodiversity monitoring. Recently, environmental DNA (eDNA) metabarcoding has emerged as a promising technique for detecting species interactions non-destructively. This pilot study explores the application of eDNA metabarcoding to investigate frugivorous interactions involving 18 partially consumed and three intact fruits of each Carica papaya and Ananas comosus. Metabarcoding of mitochondrial COI gene fragments generated 796,234 paired-end reads representing 117 ASVs spanning diverse taxonomic groups, including Metazoans, Protozoans, Algae, Fungi, and Bacteria. After filtering for animal taxa, 41 ASVs were retained, dominated by Arthropoda (~97%). Major frugivores included Drosophila, Zaprionus, and Bactrocera species. Additional detections included beetles, ants, parasitoid wasps, and vertebrates such as Acridotheres javanicus, Callosciurus erythraeus, and Bandicota indica. Moreover, consumed fruits showed high insect (~90%–95%) and mammal (~4%–5%) DNA, while intact fruits were dominated by rotifers (~75%–80%). Distinct communities were found between pineapple and papaya, with 15 and 11 unique ASVs, respectively, and only one ASV was unique to intact fruits. Alpha and beta diversity analyses confirmed the differences in community structure between fruit types. Despite the limited sample size, our findings demonstrate the potential of fruit-surface eDNA to monitor frugivory and species interactions. Future studies should scale this approach across seasons and crop types to assess its potential in long-term biodiversity and pest management monitoring.

Effective conservation of threatened species relies on precise detection methods to inform conservation management strategies. Here, we developed a new environmental DNA (eDNA) assay to detect the endangered Booroolong frog (Litoria booroolongensis) in river water samples throughout their range in Australia. The suitability of the assay to detect Booroolong frog eDNA was first validated ex situ from a population of captive-bred frogs at Taronga Zoo, New South Wales. We then established the detectability of Booroolong frog DNA from river water samples collected in situ. The new assay successfully detected Booroolong frog eDNA in water samples collected from the breeding enclosure, from water samples exposed to adult frogs and frog egg masses. Testing in the field confirmed the effectiveness of the assay at detecting Booroolong frog eDNA in environmental water samples collected in situ from sites with known presence of the species determined from prior nocturnal surveys. Our results demonstrate the efficacy of eDNA for detecting Booroolong frogs from river water samples across its geographic range. Ongoing monitoring utilizing eDNA has the potential to support conservation efforts by enabling more efficient and widespread surveys for this endangered species.

Environmental DNA metabarcoding (eDNA) is emerging as a pivotal tool for assessing and monitoring marine biodiversity, exhibiting significant promise for the detection of marine mammals. The primary objective of this study was to evaluate various protocols for eDNA sampling of seawater from a small boat in tropical environment, under conditions devoid of cold chain storage or laboratory facilities and constrained by limited financial resources. Our focus was on optimizing the capture of eDNA and the subsequent detection of marine mammals in a replicable way. This investigation involved a comparative analysis between marine mammal detections via eDNA metabarcoding and traditional visual monitoring. Sampling was primarily conducted in close proximity to marine mammal sightings, off Réunion Island to evaluate the performance of eDNA detections. Réunion Island is located in the tropical western Indian Ocean and serves as a relevant model for this study, where long-term monitoring of cetaceans has been conducted since 2008, thereby enabling a robust comparison between visual sightings and molecular detections. Two sets of primers designed to target the hypervariable regions of mitochondrial 12S rRNA genes for vertebrates and mammals were used. Positive eDNA detections were identified in seven of the nine samples associated with visual sightings of one or more cetacean species. Marine mammal DNA was successfully amplified for three families (Balaenopteridae, Delphinidae, and Kogiidae) and found to be almost ubiquitously present for Delphinidae. Additionally, we investigated the potential influence of particle drift on the dispersal of eDNA. To better understand the spatial dynamics and persistence of eDNA in the marine environment, the Lagrangian model ICHTHYOP was used to simulate particle drift and assess how oceanographic processes might influence eDNA dispersal patterns around Réunion Island. Our study explores the potential of utilizing eDNA for monitoring cetaceans in tropical regions offering a valuable comparison to traditional visual surveys, and provides recommendations for further enhancements in future eDNA studies.

Environmental DNA (eDNA) is a powerful tool for biological monitoring that may overcome limitations of conventional macroinvertebrate surveys in running waters. However, the ability of eDNA to detect macroinvertebrate community changes immediately downstream of a perturbation, particularly in upstream–downstream comparisons, has not been adequately explored. To address this, we compared eDNA-based assessments with macroinvertebrate surveys in a river receiving inflow from a metal-contaminated tributary. Results from both eDNA-based assessment and benthic macroinvertebrate collection revealed distinctly lower richness of taxa and zero-radius operational taxonomic units (ZOTUs) at the metal-contaminated tributary site compared to other study sites. Results from the collection of macroinvertebrates indicated that most richness and abundance metrics were significantly reduced at three metal-contaminated sites located 150–1350 m downstream from the inflow of the tributary, compared with an upstream reference site. In contrast, the eDNA-based assessment revealed similar ZOTU richness at the reference site and the three contaminated sites. Although statistically not significant because sample sizes were small, eDNA-based nonmetric multidimensional scaling (NMDS) revealed some separation between the reference site and two downstream sites. However, no separation was apparent between the reference site and the site immediately downstream. This result suggested that eDNA at a site 150 m downstream from the inflow was likely affected by downstream drift of eDNA from the upstream reference area. That drift complicated the assessment of the community a short distance from the perturbation. The site separation detected by eDNA-based assessment was promising, but the ZOTUs that contributed to the separation were mainly from dipteran taxa rather than from metal-sensitive mayflies, which were significantly lower in abundance at the downstream, contaminated sites. Developing reliable local DNA barcoding information, particularly for these mayflies, may help overcome the limitations of making evaluations over relatively small spatial scales, such as upstream–downstream comparisons.

Environmental DNA (eDNA) is increasingly used to infer species abundance, but uncertainty remains about how well individual water samples reflect system-wide eDNA concentrations. Currently, high levels of variation in estimated eDNA concentration among samples, even under similar conditions, limit the utility of eDNA estimates of abundance, especially in conservation and management scenarios. To investigate the sources and magnitude of variation in eDNA estimates, we analyzed eDNA from replicate water samples taken from mesocosms housing Lithobates sylvaticus tadpoles. We partitioned the variance of observed eDNA concentrations between biological replicates (distinct water samples) and technical replicates. We further tested whether stochastic variation in extraction efficiency (e.g., silica-column DNA binding and elution) introduced variance using internal control DNA. We then trialed modifications of sampling methods that could be applied to improve precision, including increased water volume, spatially pooled subsamples, and modified filtration. The observed variance in eDNA concentrations was substantial, ~75% of which was attributable to variation among replicate samples from the same mesocosm and < 5% related to technical replicates; extraction-related variance was negligible. Sampling modifications, like increasing sample volume and taking combined samples of multiple scoops, improved precision, while others highlighted potential trade-offs between precision and accuracy. Our results suggest that sample-to-sample variation, even from seemingly homogeneous, controlled environments, can be substantial even under highly controlled conditions. This inherent variability imposes limits on the precision of abundance estimates derived from eDNA and underscores the importance of replication and protocol optimization in study design.

Environmental DNA (eDNA)-based monitoring has become an established and efficient method for surveying biodiversity in aquatic systems. However, there is a need to compare and standardize sampling methods across different ecosystem types, particularly complex ecosystems such as estuaries, where unique challenges exist for monitoring fish populations due to fluctuating environmental factors. Here, we compare species richness obtained from eDNA metabarcoding data using four different eDNA filtration methods: three manual filtration methods with different pore sizes (0.45, 1.2, and 5 μm) 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 μm filter recovered the highest richness (39 species), then the metaprobe method (35), followed by the 1.2 μm (34) and 5 μm (33) filters. Filter performance between salinity gradients revealed that the 0.45 μm and the 1.2 μm methods recovered the highest species richness across all sampled zones. The 0.45 μm also had the most consistent detection probabilities using representative species from each zone. While the 0.45 μm method appeared to be the optimal method, each of the methods can be considered 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 optimizing fish diversity assessments using eDNA metabarcoding in estuarine ecosystems, providing a valuable framework for future monitoring efforts in similar systems worldwide.

Biodiversity monitoring is crucial for understanding ecosystem dynamics and species distributions, particularly in the context of anthropogenic impacts and climate change. Cetaceans, as key indicator species of marine ecosystems, face increasing threats from human activities, highlighting the need for effective, non-invasive monitoring methods. In the present study, we developed novel Cetacea-specific primer sets to enhance the detection efficiency of cetacean species through environmental DNA (eDNA) metabarcoding, while minimizing the amplification of non-target vertebrates, such as fish and humans. We retrieved mitochondrial genomes of 71 cetacean species from a public database and designed 20 candidate primer sets, which were assessed in silico for their specificity and capacity to differentiate cetacean sequences. Four primer sets with the best in silico performance were selected for empirical validation using DNA from tissue samples and eDNA from seawater collected from aquarium pools and Hong Kong coastal waters. All four primer sets effectively amplified cetacean DNA from tissue samples. However, in the aquarium pool tests, three primer sets failed to accurately identify one or more cetacean species due to a lack of interspecific variation within the amplified region. From these, we selected one primer set targeting a 267 bp region of the mitochondrial 12S rRNA gene, named μCeta, and applied it to water samples collected from Hong Kong coastal waters, where the iconic Indo-Pacific humpback dolphin (Sousa chinensis) was observed. μCeta successfully detected S. chinensis eDNA while avoiding amplification of non-target species such as fish or humans. Our results demonstrate that μCeta is a reliable tool for cetacean eDNA detection in Hong Kong waters, contributing to cetacean conservation and enhancing our understanding of marine biodiversity.