Environmental DNA and environmental RNA: Current and prospective applications for biological monitoring

ABSTRACTOceanic islands are among the most unique and vulnerable ecosystems in the world. Biodiversity monitoring is crucial for the sustainable management of resources; however, the isolation of many islands makes routine assessment challenging. Environmental DNA (eDNA) provides a promising approach to enhance traditional marine biodiversity assessments, reducing the logistical and financial challenges of monitoring. This study employed eDNA to characterize marine vertebrate biodiversity at the world's most remote inhabited islands of Tristan da Cunha. Two 12 s rRNA gene metabarcoding assays targeting marine fish and vertebrates were applied to seawater samples from 18 sites across the archipelago. This multi‐assay approach detected 51 Operational Taxonomic Units (OTUs) encompassing 24 families, 28 genera, and 13 species. Comparison with existing results from traditional survey methods (SCUBA, pelagic BRUVS, and deep‐water camera drops) and fisheries by‐catch showed eDNA to successfully resolve the islands characteristic diversity profile. In addition, eDNA reported rare and vulnerable taxa underrepresented by the traditional surveys and detected species previously unrecorded at the islands. eDNA resolved greater species richness in kelp versus non‐kelp habitats. Dominant Tristanian taxa had the highest number of reads, adding to evidence linking reads and abundance. eDNA detection was robust to sampling technique, volume filtered, time between collection, filtration, and sequencing of samples, demonstrating the effectiveness of this technique for use in challenging remote locations. Community composition varied significantly between metabarcoding assays, with unique OTUs detected by each marker, highlighting the importance of assay selection for capturing the full depth and breadth of diversity. 23%–40% of OTUs were resolved to species level, emphasizing the need for the expansion of taxonomic and sequence databases for this region. The study demonstrates the potential of eDNA as a high‐resolution tool that can provide new insights into biodiversity around Tristan and can be operationalized to monitor future changes at these isolated islands.

Monitoring the diversity and distribution of species in an ecosystem is essential to assess the success of restoration strategies. Implementing biomonitoring methods, which provide a comprehensive assessment of species diversity and mitigate biases in data collection, holds significant importance in biodiversity research. Additionally, ensuring that these methods are cost-efficient and require minimal effort is crucial for effective environmental monitoring. In this study we compare the efficiency of species detection, the cost and the effort of two non-destructive sampling techniques: Baited Remote Underwater Video (BRUV) and environmental DNA (eDNA) metabarcoding to survey marine vertebrate species. Comparisons were conducted along the Sussex coast upon the introduction of the Nearshore Trawling Byelaw. This Byelaw aims to boost the recovery of the dense kelp beds and the associated biodiversity that existed in the 1980s. We show that overall BRUV surveys are more affordable than eDNA, however, eDNA detects almost three times as many species as BRUV. eDNA and BRUV surveys are comparable in terms of effort required for each method, unless eDNA analysis is carried out externally, in which case eDNA requires less effort for the lead researchers. Furthermore, we show that increased eDNA replication yields more informative results on community structure. We found that using both methods in conjunction provides a more complete view of biodiversity, with BRUV data supplementing eDNA monitoring by recording species missed by eDNA and by providing additional environmental and life history metrics. The results from this study will serve as a baseline of the marine vertebrate community in Sussex Bay allowing future biodiversity monitoring research projects to understand community structure as the ecosystem recovers following the removal of trawling fishing pressure. Although this study was regional, the findings presented herein have relevance to marine biodiversity and conservation monitoring programs around the globe.

Molecular technologies have facilitated the expansion of biodiversity assessments across a broad range of organisms and aquatic systems. Environmental DNA (eDNA) and environmental RNA (eRNA), collectively referred to as environmental nucleic acids (eNAs), have revolutionized biodiversity monitoring due to their noninvasive nature and high‐resolution capabilities when compared to traditional survey methods. While eNA applications have grown exponentially over the past decade, methodological inconsistencies hinder reproducibility and comparability. To assess the current state of eNA methodologies in aquatic ecology, we conducted a systematic review of 300 peer‐reviewed eNA studies that assess ecological communities across diverse aquatic systems. Of these papers, 291 examine eDNA, four examine eRNA, and five consider both eDNA and eRNA. The small number of eRNA studies compared to eDNA highlights the eRNA field is in its infancy. Of the eNA studies looked at, we found a clear geographic bias, with approximately 50% of all eNA studies occurring in six high‐income countries, while less than 10% of eNA studies occur across South America and Africa. Further, we report a lack of methodological standardization across eNA studies, showing high variability in water filtration volume, filter material, filter pore size, eNA extraction method, marker choice, and bioinformatic pipelines. We further highlight that incomplete reference sequence databases for both eRNA and eDNA limit taxonomic assignment and biodiversity inferences. Finally, we identify a systematic issue across eNA studies in community ecology: missing methodological details, which compromise reproducibility, especially in newly emerging eRNA applications. To facilitate the standardization of eNA monitoring across aquatic systems and permit the further integration of eNA applications in biodiversity monitoring, we recommend the improvement of reference databases alongside guidelines that encourage methodological transparency.

Environmental DNA – An emerging tool in conservation for monitoring past and present biodiversity

Environmental DNA (eDNA) analysis has significantly transformed the way biodiversity assessment and monitoring are conducted in many environments. This review study synthesizes findings from multiple studies to provide a comprehensive overview of eDNA collection strategies in diverse settings. The review examines the techniques used for sampling eDNA in water, air, soil, sediment, and coral reef ecosystems. Water filtration, sediment sampling, and passive sampling devices are commonly used methods for collecting eDNA in aquatic environments. These techniques provide non-invasive ways to identify and track aquatic organisms, offering vital information about the interactions within the community and the global distribution of species. Similarly, the use of airborne eDNA sampling techniques is becoming increasingly promising for evaluating biodiversity on land, although there is room for improvement. Soil eDNA extraction techniques involve the use of soil coring to collect samples, followed by DNA extraction from these samples, and the application of metabarcoding methods. These techniques allow for thorough investigations of biodiversity in the soil. Specialized techniques for collecting eDNA are required for coral reef ecosystems due to their intricate habitat structure and fluctuating water conditions. The importance of choosing appropriate techniques for eDNA collection based on ecosystem parameters and research objectives is emphasized by comparative analysis. This mini-review consolidates knowledge from a selected body of recent studies and serves as a helpful resource for scholars and practitioners involved in biodiversity monitoring and conservation across diverse ecosystems.

Understanding biodiversity is crucial for protecting unique environments, but acquiring this knowledge is challenging in isolated areas due to limited availability of easy-to-implement biomonitoring tools. To determine optimal sampling strategies in remote regions, environmental DNA and RNA (eDNA and eRNA) sampling workflows were evaluated at 12 sites in three fiords within Fiordland National Park, Aotearoa-New Zealand. For filtration comparison, a modified cruising speed net was used to concentrate eDNA/eRNA onto 20μm nylon filters, while water from the net's cod-end was filtered through a 5μm Smith-Root self-preserving filter using the eDNA Citizen Scientist Sampler. To compare preservation methods, Smith-Root filters were cut in half, with one half preserved in the self-preserving unit and the other in DNA/RNA Shield™ buffer. Biodiversity screening was performed by sequencing the 18S rRNA gene for eukaryotes and two mitochondrial 16S rRNA genes for fish and marine vertebrates. Comparable amplicon sequence variant (ASVs) richness was observed between methods, yet samples preserved with buffer showed higher richness of fish and marine vertebrate taxa and higher PCR amplification success. There was little variation in community composition, except for 16S rRNA targeting fish, where distinct patterns emerged based on preservation methods. Overall, sampling workflows showed similar community composition and alpha diversity across both nucleic acids. These results confirm that enhancing eDNA/eRNA yields for sparse taxa requires consideration of collection and preservation methods. However, abundant taxa biodiversity is captured consistently, allowing for adjustments without compromising robustness. These insights support streamlined eDNA/eRNA sampling, emphasizing adaptive strategies based on targeted taxa.

The present study suggests that standardized methodology, careful site selection, and stratigraphy are essential for investigating ancient ecosystems in order to evaluate biodiversity and DNA-based time series. Based on specific keywords, this investigation reviewed 146 publications using the SCOPUS, Web of Science (WoS), PUBMED, and Google Scholar databases. Results indicate that environmental deoxyribose nucleic acid (eDNA) can be pivotal for assessing and conserving ecosystems. Our review revealed that in the last 12 years (January 2008–July 2021), 63% of the studies based on eDNA have been reported from aquatic ecosystems, 25% from marine habitats, and 12% from terrestrial environments. Out of studies conducted in aquatic systems using the environmental DNA (eDNA) technique, 63% of the investigations have been reported from freshwater ecosystems, with an utmost focus on fish diversity (40%). Further analysis of the literature reveals that during the same period, 24% of the investigations using the environmental DNA technique were carried out on invertebrates, 8% on mammals, 7% on plants, 6% on reptiles, and 5% on birds. The results obtained clearly indicate that the environmental DNA technique has a clear-cut edge over other biodiversity monitoring methods. Furthermore, we also found that eDNA, in conjunction with different dating techniques, can provide better insight into deciphering eco-evolutionary feedback. Therefore, an attempt has been made to offer extensive information on the application of dating methods for different taxa present in diverse ecosystems. Last, we provide suggestions and elucidations on how to overcome the caveats and delineate some of the research avenues that will likely shape this field in the near future. This paper aims to identify the gaps in environmental DNA (eDNA) investigations to help researchers, ecologists, and decision-makers to develop a holistic understanding of environmental DNA (eDNA) and its utility as a palaeoenvironmental contrivance.

Organisms continuously release DNA into their environments via shed cells, excreta, gametes and decaying material. Analysis of this 'environmental DNA' (eDNA) is revolutionizing biodiversity monitoring. eDNA outperforms many established survey methods for targeted detection of single species, but few studies have investigated how well eDNA reflects whole communities of organisms in natural environments. We investigated whether eDNA can recover accurate qualitative and quantitative information about fish communities in large lakes, by comparison to the most comprehensive long-term gill-net data set available in the UK. Seventy-eight 2L water samples were collected along depth profile transects, gill-net sites and from the shoreline in three large, deep lakes (Windermere, Bassenthwaite Lake and Derwent Water) in the English Lake District. Water samples were assayed by eDNA metabarcoding of the mitochondrial 12S and cytochrome b regions. Fourteen of the 16 species historically recorded in Windermere were detected using eDNA, compared to four species in the most recent gill-net survey, demonstrating eDNA is extremely sensitive for detecting species. A key question for biodiversity monitoring is whether eDNA can accurately estimate abundance. To test this, we used the number of sequence reads per species and the proportion of sampling sites in which a species was detected with eDNA (i.e. site occupancy) as proxies for abundance. eDNA abundance data consistently correlated with rank abundance estimates from established surveys. These results demonstrate that eDNA metabarcoding can describe fish communities in large lakes, both qualitatively and quantitatively, and has great potential as a complementary tool to established monitoring methods.

This study aimed to determine the practical efficacy of passive eDNA samplers (PEDS) for monitoring fish diversity in riverine ecosystems. It investigated the utility of environmental DNA (eDNA) in accurately depicting fish composition and diversity within the Lancang River. Environmental DNA technology, particularly PEDS, may be used as a substitute for traditional water filtration techniques. However, its effectiveness in natural water ecosystems remains to be proven. The filter materials included mixed cellulose acetate and nitrate (MCE), nylon (NL), glass fiber (GF), and polyvinyl chloride filter membrane (PVC). This study used four different types of filters, each with identical pore sizes and dimensions but constructed from various materials, to assess eDNA capture under laboratory and field conditions in the water samples. The filter materials included mixed cellulose acetate and nitrate (MCE), nylon (NL), glass fiber (GF), and polyvinyl chloride filter membrane (PVC). Environmental DNA macrobarcoding was used to analyze fish biodiversity and to understand the environmental effects on species distribution. Our study identified 50 fish species inhabiting the Lancang River, with equal representation of exotic and native species. A comparative analysis of four filter-based environmental DNA samplers and traditional environmental DNA sampling methods demonstrated comparable species richness. Redundancy analysis indicated that environmental variables, elevation, electrical conductivity, salinity, and chlorophyll-a significantly influenced the distribution patterns of both non-native and native fish species in the river. This study highlights the significance of eDNA technology in evaluating fish diversity across diverse habitats, thereby establishing a theoretical framework for the sustained monitoring and management of fish biodiversity in protected areas.

Aquatic ecosystems across the globe are under significant threat, suffering from various forms of anthropogenic disturbances, which is greatly impacting global biodiversity, economy and human health. Reliable monitoring of species is crucial for data-driven management actions in this context, but remains a challenge owing to non-standardized and selective methods relaying on physical identification of species by visual surveys and counting of individuals. However, traditional monitoring techniques remain problematic due to difficulties associated with correct identification of cryptic species or juvenile life stages, These traditional methods depend on practical and taxonomic expertise, which is steadily declining. In an attempt to come up with new objective solutions for monitoring biodiversity, recent and ongoing studies in Qatari waters, uses and further develops novel environmental DNA (eDNA) methods for Monitoring aquatic biodiversity in the Gulf and elsewhere.Previous studies have shown that diversity of rare and threatened European freshwater animals - representing amphibians, fish, mammals, insects and crustaceans - can be detected and quantified based on environmental DNA (eDNA) obtained directly from small water samples of lakes, ponds and streams (Thomsen et al. 2012a).Subsequently, for the first time, we investigated the potential of using metabarcoding of eDNA obtained directly from seawater samples to account for marine fish and mammal biodiversity. We show that such marine eDNA can account for fish biodiversity using high-throughput sequencing. Promisingly, eDNA covered the fish diversity better than any of 9 methods, conventionally used in marine fish surveys. Additionally, we show that even short fish eDNA sequences in seawater degrades beyond detectable levels within days, in accordance with results obtained from freshwater eDNA (Thomsen et al. 2012b). Controlled mesocosm experiments have also shown that eDNA becomes undetectable within 2 weeks after removal of animals, indicating that eDNA traces are near contemporary with species presence. Our findings underpin the ubiquitous nature of eDNA traces in the environment and support the use of eDNA as a tool for monitoring rare, threatened and economically important species across a wide range of taxonomic groups.A particularly interesting study focuses on the large whale shark aggregation at the Al Shaheen Oil Field, located in the offshore area called “Block 5”, in the NE of the Exclusive Economic Zone (EEZ) of Qatar. Maersk Oil Qatar is currently operating several oil and gas production platforms within this field, and also take active part in a large whale shark research project studying many aspect about the aggregation. Quantitative PCR (qPCR) and high-throughput sequencing of sea water from the area have successfully been employed to gain new information about the occurrence, abundance and biology of the aggregation. More specifically, data from 2013 and 2014 showed that whale shark eDNA concentrations follows visual observations of abundance, and that degradation of eDNA occur rapidly, supporting that the obtained genetic material is of local origin. Our findings confirm that seawater samples can contain key information on populations of oceanic species, and demonstrate a general potential of eDNA for studying populations of marine organisms.Although further studies are needed to validate the eDNA approach under varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of aquatic biodiversity and resources in the Gulf.ReferencesThomsen PF, Kielgast J, Iversen LL, Wiuf C, Rasmussen M, Gilbert MTP, Orlando L, Willerslev E (2012a). Monitoring Endangered Freshwater Biodiversity using Environmental DNA. Molecular Ecology 21, 2565–2573.Thomsen PF, Kielgast J, Iversen LL, Møller PR, Rasmussen M, Willerslev E (2012b). Detection of a Diverse Marine Fish Fauna using Environmental DNA from Seawater Samples. PLOS ONE 7(8), e41732.

We present results demonstrating species-specificity and sub-species resolution by novel, automatically-designed metabarcoding primers for environmental DNA analysis. Conventional metabarcoding remains a cornerstone of rapid, high-throughput environmental DNA (eDNA) community analysis and biodiversity assessment. Standard barcodes such as 16S (prokaryotes) and ITS1 (fungi/oomycetes) have been instrumental in identifying the complex composition of communities using total eDNA. However, standard barcodes have limitations in terms of resolution and quantitation and, though genus-level identification can be reliable, species-level identification is often not possible. To overcome the limitations of resolution, we implemented extensions to the diagnostic primer design tool pdp (https://github.com/widdowquinn/find_differential_primers) that enable automated design of metabarcoding markers and corresponding primers that are (i) specific to a prescribed taxon at species level and (ii) capable of discriminating between members of the same species. This allows for rapid, high-throughput measurement of diversity below species level for a target organism. We aimed to survey geographical distribution and pathogen transfer of the widepread plant pathogenic bacterium Pectobacterium atrosepticum (Pba). This organism has considerable sub-species taxonomic structure identifiable using MLST and with whole-genome methods, but which is not accessible using standard barcodes. We designed metabarcoding primers (202bp) specific to Pba using `pdp`, and established that these have resolution comparable to eight-gene MLST, revealing sub species-level diversity within single fields, and on the same individual plant host.

环境DNA技术是一种非侵入、高灵敏、高效率且对环境无破坏性，对生物体无损伤的调查工具.为了筛选适合于蚌类环境DNA生物多样性研究的宏条形码引物，本研究通过对鄱阳湖流域24种常见蚌的基因组DNA进行普通扩增和高通量测序筛选了11对引物（设计了9对通用引物及从相关文献中引用了2对引物），结果显示引物cyt b和16S rRNA具有良好的扩增效果和高辨别度.进一步用环境样本（n=6）并结合传统采样技术对这2对引物进行验证，结果表明：使用引物16S rRNA共注释到蚌科物种6属8种，蚌科物种序列占总序列数的26.69%；而使用引物cyt b共注释到蚌科物种4属6种，蚌科物种序列占总序列数的6.60%.引物16S rRNA更适合用于蚌类环境DNA生物多样性研究的宏条形码引物.在生物多样性监测中环境DNA技术可以作为传统方法的有效补充，且同时使用多对引物，可增加可信度和检测率.;Environmental DNA (eDNA) technology is a non-invasive, highly sensitive, efficient and non-destructive to the environment, no damage to the organism investigation tool. To select suitable metabarcoding primers for freshwater mussel's environmental DNA biodiversity research, 11 pairs of primers (with 9 pairs of universal primers designed and 2 pairs cited from relevant literature) were screened by general amplification and high-throughput sequencing of genomic DNA of 24 common mussels in the Lake Poyang Basin. The results showed that the primer cyt b and 16S rRNA had good amplification effect and high discrimination. The two pairs of primers were further verified with environmental samples (n=6) and traditional sampling. It was found that 8 mussels species belonging to 6 genera were annotated by 16S rRNA which accounted for 26.69% of the total sequences, and 5 mussels species belonging to 4 genera were annotated by cyt b which accounted for 6.60% of the total sequences. The primer 16S rRNA was more suitable used for metabarcoding primer for the research of environmental DNA biodiversity in freshwater mussels. Environmental DNA technology can be used as an effective supplement to traditional methods in biodiversity monitoring. The reliability and detection rate can be increased by using multiple pairs of primers at the same time.

Abstract. Bautista JA, Manubag JJ, Sumaya NH, Martinez JG, Tabugo SR. 2023. Environmental DNA (eDNA) metabarcoding and fish visual census reveals the first record of Doboatherina magnidentatain the Philippines. Biodiversitas 24: 3063-3072. Biodiversity monitoring is the cornerstone for conserving marine fish species. However, classical methods, like the Fish Visual Census (FVC), are often limited due to sampling difficulties, the occurrence of rare and cryptic organisms, and reliance on a taxonomic expert for species identification. Recently, environmental DNA (eDNA) metabarcoding has been suggested as a non-invasive, powerful tool for biomonitoring. This study evaluates the eDNA approach as complementary tool for the FVC data in species detection and identification of important marine fishes from the marine sanctuary of Dalipuga, Iligan City, Philippines. The findings obtained through the eDNA approach provide insights into identifying significant fish species. Notably, the presence of the Hippocampus kuda Bleeker, 1852 (yellow seahorse), categorized as a vulnerable and threatened species, was detected. Additionally, the study identified Herklotsichthys quadrimaculatus Rüppell, 1837 (bluestripe herring), a native species to the Philippines that may pose potential risks to humans and the ecological balance. Furthermore, two demersal fish species, namely Large-scale whiting (Sillaginops macrolepis Bleeker, 1858) and Large-scale mullet (Planiliza macrolepis Smith, 1846), were also detected. The eDNA approach also delineated the morphologically cryptic fishes from Scaridae (parrotfishes) and Mugilidae (mullet fish) taxa to the species level. The highlight of this study was the detectionof the new Indo-pacific atherinomorine fish species Doboatherina magnidentata, which to the best of our knowledge, was the first record in the Philippine marine waters. Despite the efficiency of the eDNA metabarcoding in fish species detection and identification, the viability of eDNA in the marine environment and biases of the primer limit this method. Thus, the classical method must complement the molecular approach for better taxonomic resolution and community analysis. Future studies were also recommended to use a multigene eDNA approach to improve taxonomic sensitivity and reduce primer biases.

The future of biodiversity monitoring and conservation utilizing environmental DNA

Traditional methods of biodiversity monitoring are often logistically challenging, time-consuming, require experienced experts on species identification, and sometimes include destruction of the targeted specimens. Here, we investigated a non-invasive approach of combining the use of drones and environmental DNA (eDNA) to monitor insect biodiversity on vegetation. We aimed to assess the efficiency of this novel method in capturing insect diversity and comparing insect composition across different vegetation types (grassland, shrub and forest) in Switzerland. A commercial, off-the-shelf drone was equipped with a specialised probe that autonomously swabbed vegetation and collected eDNA. Then, samples were processed using rapid third-generation Oxford Nanopore sequencing. The obtained data were analysed for insect diversity, comparing taxonomic richness, evenness and community composition across the three habitat types using statistical techniques. Sequencing of the samples yielded 76 hexapod taxa, revealing an insect community with notable differences in taxonomic richness but not in evenness across grassland, shrub and forest habitats. Our study demonstrates the potential of drone-based sampling integrated with eDNA and nanopore sequencing for biodiversity monitoring, offering a non-destructive method for detecting insect occurrence on plant surfaces. Integrating robotics and eDNA technology provides a promising solution for fast, large-scale, non-invasive biodiversity monitoring, potentially improving conservation efforts and ecosystem management.