Environmental DNA analysis for macro-organisms: species distribution and more

In an era of severe biodiversity loss, biological monitoring is becoming increasingly essential. The analysis of environmental DNA (eDNA) has emerged as a new approach that could revolutionize the biological monitoring of aquatic ecosystems. Over the past decade, macro-organismal eDNA analysis has undergone significant developments and is rapidly becoming established as the golden standard for non-destructive and non-invasive biological monitoring. In this review, I summarize the development of macro-organismal eDNA analysis to date and the techniques used in this field. I also discuss the future perspective of these analytical methods in combination with sophisticated analytical techniques for DNA research developed in the fields of molecular biology and molecular genetics, including genomics, epigenomics, and single-cell technologies. eDNA analysis, which to date has been used primarily for determining the distribution of organisms, is expected to develop into a tool for elucidating the physiological state and behaviour of organisms. The fusion of microbiology and macrobiology through an amalgamation of these technologies is anticipated to lead to the future development of an integrated biology.     

Environmental DNA methylation of Lymnaea stagnalis varies with age and is hypermethylated compared to tissue DNA

Environmental DNA (eDNA) approaches contributing to species identifications are quickly becoming the new norm in biomonitoring and ecosystem assessments. Yet, information such as age and health state of the population, which is vital to species biomonitoring, has not been accessible from eDNA. DNA methylation has the potential to provide such information on the state of a population. Here, we measured the methylation of eDNA along with tissue DNA (tDNA) of Lymnaea stagnalis at four life stages. We demonstrate that eDNA methylation varies with age and allows distinguishing among age classes. Moreover, eDNA was globally hypermethylated in comparison to tDNA. This difference was age-specific and connected to a limited number of eDNA sites. This differential methylation pattern suggests that eDNA release with age is partially regulated through DNA methylation. Our findings help to understand mechanisms involved in eDNA release and shows the potential of eDNA methylation analysis to assess age classes. Such age class assessments will encourage future eDNA studies to assess fundamental processes of population dynamics and functioning in ecology, biodiversity conservation and impact assessments.     

The bug in a teacup—monitoring arthropod–plant associations with environmental DNA from dried plant material

Environmental DNA analysis (eDNA) has revolutionized the field of biomonitoring in the past years. Various sources have been shown to contain eDNA of diverse organisms, for example, water, soil, gut content and plant surfaces. Here we show that dried plant material is a highly promising source for arthropod community eDNA. We designed a metabarcoding assay to enrich diverse arthropod communities while preventing amplification of plant DNA. Using this assay, we analysed various commercially produced teas and herbs. These samples recovered ecologically and taxonomically diverse arthropod communities, a total of over a thousand species in more than 20 orders, many of them specific to their host plant and its geographical origin. Atypically for eDNA, arthropod DNA in dried plants shows very high temporal stability, opening up plant archives as a source for historical arthropod eDNA. Considering these results, dried plant material appears excellently suited as a novel tool to monitor arthropods and arthropod–plant interactions, detect agricultural pests and identify the geographical origin of imported plant material. The simplicity of our approach and the ability to detect highly diverse arthropod communities from all over the world in tea bags also highlights its utility for outreach purposes and to raise awareness about biodiversity.     

Revealing hidden plant diversity in arid environments

Estimating total plant diversity in extreme or hyperarid environments can be challenging, as adaptations to pronounced climate variability include evading prolonged stress periods through seeds or specialized underground organs. Short‐term surveys of these ecosystems are thus likely poor estimators of actual diversity. Here we develop a multimethod strategy to obtain a more complete understanding of plant diversity from a community in the Atacama Desert. We explicitly test environmental DNA‐based techniques (eDNA) to see if they can reveal the observed and ‘hidden' (dormant or locally rare) species. To estimate total plant diversity, we performed long‐term traditional surveys during eight consecutive years, including El Niño and La Niña events, we then analyzed eDNA from soil samples using high‐throughput sequencing. We further used soil pollen analysis and soil seed bank germination assays to identify ‘hidden' species. Each approach offers different subsets of current biodiversity at different taxonomic, spatial and temporal resolution, with a total of 92 taxa identified along the transect. Traditional field surveys identified 77 plant species over eight consecutive years. Observed community composition greatly varies interannually, with only 22 species seen every year. eDNA analysis revealed 37 taxa, eight of which were ‘hidden' in our field surveys. Soil samples contain a viable seed bank of 21 taxa. Soil pollen (27 taxa) and eDNA analysis show affinities with vegetation at the landscape scale but a weak relationship to local plot diversity. Multimethod approaches (including eDNA) in deserts are valuable tools that add to a comprehensive assessment of biodiversity in such extreme environments, where using a single method or observations over a few years is insufficient. Our results can also explain the resilience of Atacama plant communities as ‘hidden' taxa may have been active in the recent past or could even emerge in the future as accelerated global environmental change continues unabated.     

Environmental DNA metabarcoding: Transforming how we survey animal and plant communities

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High‐throughput sequencing (“HTS”) platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed “environmental DNA” or “eDNA”). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called “eDNA metabarcoding” and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.     

Riverine distribution of mussel environmental DNA reflects a balance among density,transport, and removal processes

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Environmental DNA (eDNA) metabarcoding reveals strong discrimination among diverse marine habitats connected by water movement

While in recent years environmental DNA (eDNA) metabarcoding surveys have shown great promise as an alternative monitoring method, the integration into existing marine monitoring programs may be confounded by the dispersal of the eDNA signal. Currents and tidal influences could transport eDNA over great distances, inducing false‐positive species detection, leading to inaccurate biodiversity assessments and, ultimately, mismanagement of marine environments. In this study, we determined the ability of eDNA metabarcoding surveys to distinguish localized signals obtained from four marine habitats within a small spatial scale (<5 km) subject to significant tidal and along‐shore water flow. Our eDNA metabarcoding survey detected 86 genera, within 77 families and across 11 phyla using three established metabarcoding assays targeting fish (16S rRNA gene), crustacean (16S rRNA gene) and eukaryotic (cytochrome oxidase subunit 1) diversity. Ordination and cluster analyses for both taxonomic and OTU data sets show distinct eDNA signals between the sampled habitats, suggesting dispersal of eDNA among habitats was limited. Individual taxa with strong habitat preferences displayed localized eDNA signals in accordance with their respective habitat, whereas taxa known to be less habitat‐specific generated more ubiquitous signals. Our data add to evidence that eDNA metabarcoding surveys in marine environments detect a broad range of taxa that are spatially discrete. Our work also highlights that refinement of assay choice is essential to realize the full potential of eDNA metabarcoding surveys in marine biodiversity monitoring programs.     

环境DNA在湖泊生物多样性研究中的应用

环境DNA(EnvironmentalDNA,eDNA)可用于监测湖泊生物多样性,该技术对湖泊生态环境破坏性小,对于开展湖泊生态保护具有重要意义.湖泊流速较为缓慢,相对于河流更容易富集DNA,更适合于应用eDNA方法开展生物多样性研究.文章对eDNA在湖泊生物多样性上的应用进行了回顾,综述了其实验设计,分析了该技术存在...     

eDNA metabarcoding as a new surveillance approach for coastal Arctic biodiversity

Because significant global changes are currently underway in the Arctic, creating a large‐scale standardized database for Arctic marine biodiversity is particularly pressing. This study evaluates the potential of aquatic environmental DNA (eDNA) metabarcoding to detect Arctic coastal biodiversity changes and characterizes the local spatio‐temporal distribution of eDNA in two locations. We extracted and amplified eDNA using two COI primer pairs from ~80 water samples that were collected across two Canadian Arctic ports, Churchill and Iqaluit, based on optimized sampling and preservation methods for remote regions surveys. Results demonstrate that aquatic eDNA surveys have the potential to document large‐scale Arctic biodiversity change by providing a rapid overview of coastal metazoan biodiversity, detecting nonindigenous species, and allowing sampling in both open water and under the ice cover by local northern‐based communities. We show that DNA sequences of ~50% of known Canadian Arctic species and potential invaders are currently present in public databases. A similar proportion of operational taxonomic units was identified at the species level with eDNA metabarcoding, for a total of 181 species identified at both sites. Despite the cold and well‐mixed coastal environment, species composition was vertically heterogeneous, in part due to river inflow in the estuarine ecosystem, and differed between the water column and tide pools. Thus, COI‐based eDNA metabarcoding may quickly improve large‐scale Arctic biomonitoring using eDNA, but we caution that aquatic eDNA sampling needs to be standardized over space and time to accurately evaluate community structure changes.     

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.     

环境DNA宏条形码在生物多样性研究与监测中的应用

环境DNA宏条形码(eDNA metabarcoding)技术通过提取水体、土壤、空气中的环境DNA,使用引物PCR扩增与高通量测序,进行物种鉴定与生物多样性评估.作为一种新的监测技术,相比于传统监测技术更加快捷、准确以及对自然环境的破坏小,因此在一定程度上改变了我们调查地球生物多样性的方式.本文综述了环境DNA宏条形...     

Assessing the potential of environmental DNA metabarcoding for monitoring Neotropical mammals: a case study in the Amazon and Atlantic Forest,Brazil

The application of environmental DNA (eDNA) metabarcoding as a biomonitoring tool has greatly increased, but studies have focused on temperate aquatic macro-organisms. We apply eDNA metabarcoding to detecting the mammalian community in two high-biodiversity regions of Brazil: the Amazon and Atlantic Forests. We identified Critically Endangered and Endangered mammalian species and found overlap with species identified via camera trapping. We highlight the potential for using eDNA monitoring for mammals in biodiverse regions and identify challenges: we need a better understanding of the ecology of eDNA within variable environments and more appropriate reference sequences for species identification in these anthropogenically impacted biomes.     

Assessment of fish communities using environmental DNA: Effect of spatial sampling design in lentic systems of different sizes

Freshwater fish biodiversity is quickly decreasing and requires effective monitoring and conservation. Environmental DNA (eDNA)‐based methods have been shown to be highly sensitive and cost‐efficient for aquatic biodiversity surveys, but few studies have systematically investigated how spatial sampling design affects eDNA‐detected fish communities across lentic systems of different sizes. We compared the spatial patterns of fish diversity determined using eDNA in three lakes of small (SL; 3 ha), medium (ML; 122 ha) and large (LL; 4,343 ha) size using a spatially explicit grid sampling method. A total of 100 water samples (including nine, 17 and 18 shoreline samples and six, 14 and 36 interior samples from SL, ML and LL, respectively) were collected, and fish communities were analysed using eDNA metabarcoding of the mitochondrial 12S region. Together, 30, 35 and 41 fish taxa were detected in samples from SL, ML, and LL, respectively. We observed that eDNA from shoreline samples effectively captured the majority of the fish diversity of entire waterbodies, and pooled samples recovered fewer species than individually processed samples. Significant spatial autocorrelations between fish communities within 250 m and 2 km of each other were detected in ML and LL, respectively. Additionally, the relative sequence abundances of many fish species exhibited spatial distribution patterns that correlated with their typical habitat occupation. Overall, our results support the validity of a shoreline sampling strategy for eDNA‐based fish community surveys in lentic systems but also suggest that a spatially comprehensive sampling design can reveal finer distribution patterns of individual species.     

A review of applications of environmental DNA for reptile conservation and management

Reptile populations are in decline globally, with total reptile abundance halving in the past half century, and approximately a fifth of species currently threatened with extinction. Research on reptile distributions, population trends, and trophic interactions can greatly improve the accuracy of conservation listings and planning for species recovery, but data deficiency is an impediment for many species. Environmental DNA (eDNA) can detect species and measure community diversity at diverse spatio‐temporal scales, and is especially useful for detection of elusive, cryptic, or rare species, making it potentially very valuable in herpetology. We aim to summarize the utility of eDNA as a tool for informing reptile conservation and management and discuss the benefits and limitations of this approach. A literature review was conducted to collect all studies that used eDNA and focus on reptile ecology, conservation, or management. Results of the literature search are summarized into key discussion points, and the review also draws on eDNA studies from other taxa to highlight methodological challenges and to identify future research directions. eDNA has had limited application to reptiles, relative to other vertebrate groups, and little use in regions with high species richness. eDNA techniques have been more successfully applied to aquatic reptiles than to terrestrial reptiles, and most (64%) of studies focused on aquatic habitats. Two of the four reptilian orders dominate the existing eDNA studies (56% Testudines, 49% Squamata, 5% Crocodilia, 0% Rhynchocephalia). Our review provides direction for the application of eDNA as an emerging tool in reptile ecology and conservation, especially when it can be paired with traditional monitoring approaches. Technologies associated with eDNA are rapidly advancing, and as techniques become more sensitive and accessible, we expect eDNA will be increasingly valuable for addressing key knowledge gaps for reptiles.     

Detection of rare and invasive freshwater fish species using eDNA pyrosequencing: Lake Iznik ichthyofauna revised

Assessment of fish biodiversity in freshwater environments is challenging, especially when rare species or species with low population densities exist. Environmental DNA is becoming a common tool in molecular ecology to detect target species found in the environment. Moreover, eDNA metabarcoding is now used to determine a complete list of target organisms without any prior knowledge on the species inhabiting the environment. This study is the first environmental DNA study designed to assess complete ichthyofauna of the largest lake in Marmara Region of Turkey. For this purpose, an eDNA metabarcoding approach enhanced with tagged primers according to sampling stations for a station specific species listing was used to revise the ichthyofauna of Lake Iznik. Results of pyrosequencing data indicate the presence of 23 species in the lake, five of which are reported for the first time. Short fragment of cytochrome b gene sequences amplified in this study were able to make identifications at species level and the eDNA metabarcoding approach was more cost effective and precise compared to conventional surveys. More molecular data from further studies will enhance the reference databases and eDNA metabarcoding could be used more efficiently as an important molecular tool in biodiversity assessment studies.     

Individual haplotyping of whale sharks from seawater environmental DNA

Population genetic data can provide valuable information on the demography of a species. For rare and elusive marine megafauna, samples for generating the data are traditionally obtained from tissue biopsies, which can be logistically difficult and expensive to collect and require invasive sampling techniques. Analysis of environmental DNA (eDNA) offers an alternative, minimally invasive approach to provide important genetic information. Although eDNA approaches have been studied extensively for species detection and biodiversity monitoring in metabarcoding studies, the potential for the technique to address population-level questions remains largely unexplored. Here, we applied “eDNA haplotyping” to obtain estimates of the intraspecific genetic diversity of a whale shark (Rhincodon typus) aggregation at Ningaloo reef, Australia. Over 2 weeks, we collected seawater samples directly behind individual sharks prior to taking a tissue biopsy sample from the same animal. Our data showed a 100% match between mtDNA sequences recovered in the eDNA and tissue sample for all 28 individuals sampled. In the seawater samples, >97% of all reads were assigned to six dominant haplotypes, and a clear dominant signal (~99% of sample reads) was recovered in each sample. Our study demonstrates accurate individual-level haplotyping from seawater eDNA. When DNA from one individual clearly dominates each eDNA sample, it provides many of the same opportunities for population genetic analyses as a tissue sample, potentially removing the need for tissue sampling. Our results show that eDNA approaches for population-level analyses have the potential to supply critical demographic data for the conservation and management of marine megafauna.     

Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring

Aquatic environmental DNA (eDNA) surveys are transforming how marine ecosystems are monitored. The time-consuming preprocessing step of active filtration, however, remains a bottleneck. Hence, new approaches that eliminate the need for active filtration are required. Filter-feeding invertebrates have been proven to collect eDNA, but side-by-side comparative studies to investigate the similarity between aquatic and filter-feeder eDNA signals are essential. Here, we investigated the differences among four eDNA sources (water; bivalve gill-tissue; sponges; and ethanol in which filter-feeding organisms were stored) along a vertically stratified transect in Doubtful Sound, New Zealand using three metabarcoding primer sets targeting fish and vertebrates. Combined, eDNA sources detected 59 vertebrates, while concurrent diver surveys observed eight fish species. There were no significant differences in alpha and beta diversity between water and sponge eDNA and both sources were highly correlated. Vertebrate eDNA was successfully extracted from the ethanol in which sponges were stored, although a reduced number of species were detected. Bivalve gill-tissue dissections, on the other hand, failed to reliably detect eDNA. Overall, our results show that vertebrate eDNA signals obtained from water samples and marine sponges are highly concordant. The strong similarity in eDNA signals demonstrates the potential of marine sponges as an additional tool for eDNA-based marine biodiversity surveys, by enabling the incorporation of larger sample numbers in eDNA surveys, reducing plastic waste, simplifying sample collection, and as a cost-efficient alternative. However, we note the importance to not detrimentally impact marine communities by, for example, nonlethal subsampling, specimen cloning, or using bycatch specimens.     

Particle size influences decay rates of environmental DNA in aquatic systems

Environmental DNA (eDNA) analysis is a powerful tool for remote detection of target organisms. However, obtaining quantitative and longitudinal information from eDNA data is challenging, requiring a deep understanding of eDNA ecology. Notably, if the various size components of eDNA decay at different rates, and we can separate them within a sample, their changing proportions could be used to obtain longitudinal dynamics information on targets. To test this possibility, we conducted an aquatic mesocosm experiment in which we separated fish-derived eDNA components using sequential filtration to evaluate the decay rate and changing proportion of various eDNA particle sizes over time. We then fit four alternative mathematical decay models to the data, building towards a predictive framework to interpret eDNA data from various particle sizes. We found that medium-sized particles (1–10 μm) decayed more slowly than other size classes (i.e., <1 and > 10 μm), and thus made up an increasing proportion of eDNA particles over time. We also observed distinct eDNA particle size distribution (PSD) between our Common carp and Rainbow trout samples, suggesting that target-specific assays are required to determine starting eDNA PSDs. Additionally, we found evidence that different sizes of eDNA particles do not decay independently, with particle size conversion replenishing smaller particles over time. Nonetheless, a parsimonious mathematical model where particle sizes decay independently best explained the data. Given these results, we suggest a framework to discern target distance and abundance with eDNA data by applying sequential filtration, which theoretically has both metabarcoding and single-target applications.     

Estimating the spawning activity of fish species using nuclear and mitochondrial environmental DNA concentrations and their ratios

1. During spawning activity, fish release large amounts of sperm and eggs into the water, which has been assumed to cause an increase in environmental DNA (eDNA) levels and nuclear DNA/mitochondrial DNA ratios. To test whether these assumptions are valid and whether nuclear and mitochondrial eDNA analysis can be used to monitor the spawning activity of freshwater fish, we conducted field eDNA surveys and traditional surveys using common carp (Cyprinus carpio), largemouth bass (Micropterus salmoides) and bluegill sunfish (Lepomis macrochirus) as model species.
2. Fish spawning periods were estimated based on age, as estimated using the body lengths of juveniles collected in the Miharu reservoir in Fukushima, Japan. The results showed that the main spawning periods of largemouth bass and bluegill sunfish were from April to July and from July to August, respectively.
3. Field eDNA surveys were conducted in the Hebisawagawa front reservoir, which is connected to the Miharu reservoir. From March to August 2019 and 2020, weekly eDNA sampling was conducted at three sites, and daily sampling was conducted at six sites from 23 June to 3 July 2020. The eDNA concentrations of the nuclear internal transcribed spacer 1 (ITS1) and mitochondrial cytochrome B (CytB), as well as the ITS1/CytB ratio, were measured for each of the three fish in each water sample. Water temperature had a statistically significant effect on eDNA concentration, probably reflecting the relationship between water temperature and spawning.
4. We created generalised additive mixed models to estimate spawning activity periods based on weekly eDNA data. The estimated periods of spawning activity for common carp, largemouth bass and bluegill sunfish were March to May, May to July, and May to August, respectively. The estimated spawning periods coincided with known fish ecology or the results of traditional methods. This method also has been applied to daily eDNA samples, showing the feasibility of high-resolution estimation of spawning activity.
5. For common carp and bluegill sunfish, we were able to estimate the spawning period using this method. Although the method is affected by biomass and the diffusion and degradation of eDNA, it has the potential to accurately estimating spawning activities. These then can be estimated without conducting laborious traditional surveys, facilitating the monitoring of reproduction by rare, invasive or important fishery species. Further research on the diffusion distance and degradation time of the eDNA concentration peak caused by fish spawning activity may improve the accuracy of monitoring.

     

数字化植物园的理论与技术思考—以华南植物园为例

植物园是通过人工模拟区域自然环境和群落结构,实现物种多样性高度富集并进行相关科学研究的机构,也是生物多样性保育、科普教育、资源储存和开发利用的基地。随着信息技术的发展及其在植物园中的应用,将产生数字化植物园。在研究数字化植物园发展历史的基础上,提出广义和狭义的数字化植物园定义,并以华南植物园的数字化建设内容为例,探讨了数字化植物园的信息技术体系、虚拟植物和专类园智能化管理技术等理论与技术体系结构。