eDNA Metabarcoding Methods Research Articles

Benthic diatom eDNA metabarcoding for ecological assessment of an urban river: A comparison with morphological method

Benthic diatoms have been widely utilized for assessing the ecological status of rivers, due to their ability to reflect the environmental changes from natural and anthropogenic disturbance. However, the recognized limitations of traditional morphological method for diatom identification hinder its widespread application in local department monitoring and assessment efforts. Recently, eDNA metabarcoding methods have emerged as a promising alternative for efficient diatom identification. To reveal the feasibility of eDNA metabarcoding methods in diatom-based evaluation, this study comprehensively compared them with the traditional morphological method. The results revealed that eDNA metabarcoding effectively reflected diatom composition at genus but not species level. It recognized 80.4 % of genera identified morphologically through rbcL identification and 66.7 % through 18S rDNA identification. Despite species-level discrepancies in taxonomic composition, eDNA metabarcoding accurately indicated alpha diversity of diatoms, while also provided better illustration of beta diversity. Additionally, comparisons were performed in taxonomy-based and trait-based indices. It was suggested that no significance was observed between identification methods in any index. Linear regression analyses further supported rbcL identification as a preferred option over 18S rDNA identification due to its stronger congruency with morphological method. In all, this study presented two key suggestions: 1) rbcL identification is preferable to 18S rDNA identification in diatom taxonomy, and 2) eDNA metabarcoding is proposed to be a promising technique to assess river ecological status.

Application of eDNA metabarcoding in the assessment of fish biodiversity in Philippine mangroves: Challenges and opportunities

Biodiversity assessments are important in designing mangrove conservation and restoration programs. In the Philippines, conventional biodiversity assessment methods (e.g., trap nets, fish visual census) can be time-consuming, labor-intensive, and expensive. In recent years, environmental DNA (eDNA) metabarcoding has been an emerging tool for rapid biodiversity monitoring as it is fast, non-intrusive, and can provide broader detection of fauna. But, it is still subject to various field sampling and laboratory analysis constraints. Here, we applied the eDNA metabarcoding method to document and assess fish biodiversity in mangroves from two biogeographic regions in the Philippines: Oriental Mindoro in the West Philippine Sea and Sorsogon in the Northern Philippine Sea. Using 12S genetic markers from eDNA water samples, we detected 89 fish species from 44 families. Only twelve species were commonly detected in both sites. Several species were found in the Philippines’ list of economically important aquatic organisms while one species (Epinephelus fuscoguttatus) was classified as Vulnerable in IUCN’s Red List of Threatened Species. Seventy-six percent more species were detected in Sorsogon probably because the sampling sites were within a seascape of mangroves, seagrasses, and coral reefs. In contrast, the lesser species detected in Oriental Mindoro could probably be because of the more limited sampling points to coastal fringes. Our results serve as baseline data and the first obtained using this method in the country. However, we observed some limitations that should be addressed to improve the method: (1) lack or absence of a comprehensive reference database specific to Philippine aquatic organisms; (2) low eDNA reads which could be attributed to insufficient on-site filtration due to turbid seawaters common in Philippine mangroves; and (3) possible cross-contaminations that can affect comparative analyses. Despite these limitations, we were able to demonstrate the usefulness of this technique in doing rapid assessments which could address knowledge gaps in Philippine mangrove biodiversity studies and contribute to its conservation programs.

An eDNA metabarcoding of brittle stars: Examples from the Seto Inland Sea, a semi-closed marine area

Marine ecosystem are facing a crisis due to climate change and anthropogenic activities, and many marine organisms are threatened by declining biodiversity. Regular and extensive surveys of these organisms are necessary to conserve the biodiversity. Conventional methods, however, rely on direct observation and collection of specimens by experts with identification and sampling skills. Using environmental DNA (eDNA) metabarcoding methods, we detected a total of 15 brittle star species at two sites in the Seto Inland Sea, a semi-enclosed marine area in Japan. This study showed that brittle stars are useful environmental indicator organisms, and that the eDNA metabarcoding method specific to brittle star is expected to be a useful tool for a wide-ranging and simple investigation method for marine ecosystems. Further investigations will need to verify whether species detected by metabarcoding without being confirmed by direct observations are due to false positives and whether this is due to the semi-enclosed character of the area.

Yangtze finless porpoise (Neophocaena asiaeorientalis) prey fish diversity based on environmental DNA metabarcoding

Abstract Knowledge of the feeding habits of the Critically Endangered Neophocaena asiaorientalis (Yangtze finless porpoise, YFP) is vital for the management and protection of this species. However, stringent protections implemented in 2020 dictate that only non‐invasive methods may be used to gain information. Therefore, environmental DNA (eDNA) metabarcoding was used to obtain YFP prey diversity and community structures from different environmental samples collected from YFP habitat in April 2021. Sampling strategies for using eDNA to monitor the YFP were explored. Of the 63 fish species detected, more species were detected in samples from surface water than in samples from deeper water and sediment, but the diversity and community structures of fish in the samples from surface water, deeper water, and sediment did not significantly differ. The number of YFP sequences in the main lake area of Poyang Lake was significantly higher than that in other sampling areas. The YFP sequence number, operational taxonomic unit (OTU) number, and OTU occurrence frequency in surface water samples were higher than those in deeper water samples. Eighteen previously verified species of YFP prey fishes were detected, with more species detected in water samples than in sediment samples. The sequence numbers of the YFP and their prey fishes Ctenopharyngodon idella, Pseudolaubuca engraulis, and Tachysurus argentivittatus were significantly positively correlated, indicating that YFP preferentially selected these three species. Surface water samples yielded good results for the convenient and non‐invasive eDNA metabarcoding method of monitoring the feeding habits of this flagship species and for identifying early signals of YFP responses to changes in prey fish communities. The creation of critical conservation areas, eDNA monitoring standards, and emergency rescue plans are vital for YFP conservation.

Conservation assessment based on large-scale monitoring of eDNA: Application to freshwater mussels

Environmental DNA (eDNA) analysis allows efficient surveys of freshwater species and is being increasingly used. However, because different authors use different methods, eDNA data cannot easily be summarised for a global overview. One promising approach is to standardise methods for long-term monitoring. Standardised eDNA metabarcoding methods have been implemented in France for an elusive group: freshwater bivalves. All French bivalve species can be detected and identified at each sampling site. Here, we analyse this comprehensive dataset (280 sites across all France; 40 species) to investigate detection probabilities, infer species distributions and reassess conservation status, and finally examine community patterns (richness) and their determinants.Our method gave over 95 % probability of detection for most species. Species distributions inferred from eDNA sampling were compared with currently known distributions. Four invasive species and one species of conservation concern were found outside their known range. Others were found in a surprisingly small number of localities, although they are currently thought to be widespread and not of conservation concern. Indeed, the databases currently used to determine conservation status are biased by the inclusion of old data, mainly based on shell records only, and may include misidentified species. The main drivers of species richness were latitude and distance from the spring. Co-occurrence of species was investigated for the first time. The results highlight the valuable data that eDNA analysis can provide when used on a large scale, for monitoring and ecological studies, as well as providing improved species distributions, which are essential for conservation policymaking.

A Comparative Analyzing of Zooplankton Community Diversity in Surface Layer Water of Reservoir Via eDNA Metabarcoding and Microscopy

We compared two sampling methods, eDNA metabarcoding and microscope identification (MSI), for the analysis of zooplankton diversity in reservoirs with its inflow and outflow streams. The dynamic patterns of Cladocera and Rotifera at different time points were similar between the two sampling methods, but there was a slight difference in the Copepoda. Specifically, the members of the Copepoda subclass could not be easily classified using the MSI method, whereas eDNA metabarcoding could detect minor taxa of Cladocera and Rotifera. Upon comparing the list of zooplankton communities in Korea with the gene database of NCBI, only ~56% of the zooplankton genera reported in Korea could be detected based on the 18S rRNA gene. However, eDNA metabarcoding detected a more diverse range of zooplankton despite the lack of genetic information. As water temperature increased after May, the zooplankton diversity decreased according to the MSI method but increased according to the eDNA metabarcoding method. Although eDNA metabarcoding has some limitations, it was able to detect a wider diversity of zooplankton compared to the MSI. eDNA metabarcoding provides a more reliable means to identify zooplankton.

Alpine freshwater fish biodiversity assessment: an inter-calibration test for metabarcoding method set up

The analysis of environmental DNA (eDNA) by high throughput sequencing (HTS) is proving to be a promising tool for freshwater fish biodiversity assessment in Europe within the Water Framework Directive (WFD, 2000/60/EC), especially for large rivers and lakes where current fish monitoring techniques have known shortcomings. These new biomonitoring methods based on eDNA show several advantages compared to classical morphological methods. The sampling procedures are easier and cheaper and eDNA metabarcoding is non-invasive and very sensitive, allowing for the detection of traces of DNA. However, eDNA metabarcoding methods need careful standardization to make the results of different surveys comparable. The aim of the EU project Eco-AlpsWater is to test and validate molecular biodiversity monitoring tools for aquatic ecosystems (i.e., eDNA metabarcoding) to improve the traditional WFD monitoring approaches in Alpine waterbodies. To this end, an inter-calibration test was performed using fish mock community samples containing either tissue-extracted DNA, eDNA collected from aquaculture tanks and eDNA samples collected from Lake Bourget (France). Samples were analysed using a DNA metabarcoding approach, relying on the amplification and HTS of a 12S rDNA marker, in two separate laboratories, to evaluate if different laboratory and bioinformatic protocols can provide a reliable and comparable description of the fish communities in both mock and natural samples. Our results highlight good replicability of the molecular laboratory protocols for HTS and good amplification success of selected primers, providing essential information concerning the taxonomic resolution of the 12S mitochondrial marker in describing the Alpine fish communities. Interestingly, different concentrations of species DNA in the mock samples were well represented by the relative DNA reads abundance. These tests confirm the reproducibility of eDNA metabarcoding analyses for the biomonitoring of freshwater fish inhabiting Alpine and peri-Alpine lakes and rivers.

Application of eDNA Metabarcoding for Detecting Anura on a Tropical Island

As anuran biodiversity quickly declines, it is important to understand local patterns of anuran occurrence. However, the limitations of traditional sampling methods make anuran biodiversity surveys inadequate. Tropical environments are rich in anuran species, which makes biodiversity measurements more difficult. Therefore, it is important to develop a rapid, inexpensive and nondestructive method to measure anuran biodiversity in tropical environments. We used eDNA metabarcoding to measure anuran diversity at 288 sites in 18 regions of Hainan Island. We also used traditional methods and compared the results with those obtained through the eDNA metabarcoding methods. We detected 9 anuran species by traditional sampling methods. We produced 626 million reads by eDNA metabarcoding and assigned them to 15 anuran species. Therefore, eDNA metabarcoding can be used for rapid and large-scale anuran biodiversity surveys.

Environmental DNA metabarcoding as a useful tool for evaluating terrestrial mammal diversity in tropical forests.

Innovative techniques, such as environmental DNA (eDNA) metabarcoding, are now promoting broader biodiversity monitoring at unprecedented scales, because of the reduction in time, presumably lower cost, and methodological efficiency. Our goal was to assess the efficiency of established inventory techniques (live-trapping grids, pitfall traps, camera trapping, mist netting) as well as eDNA for detecting Amazonian mammals. For terrestrial small mammals, we used 32 live-trapping grids based on Sherman and Tomahawk traps (total effort of 10,368 trap-nights); in addition to 16 pitfall traps (1,408 trap-nights). For bats, we used mist nets at 8 sites (4,800 net hours). For medium and large mammals, we used 72 camera trap stations (5,208 camera-days). We identified vertebrate and mammal taxa based on eDNA analysis (12S region, with V05 and Mamm01 markers) from water samples, including a total of 11 3-km transects for stagnant water sampling and seven small streams for running water sampling. A total of 106 mammal species were recorded. Building on sample-based rarefaction and extrapolation curves, both trapping grids and pitfall were successful, recording 91.16% and 82.1% of the expected species for these techniques (~22 and ~9 species), and 16.98% and 6.60% of the total recorded mammal species, respectively. Mist nets recorded 83.2% of the expected bat species (~48), and 34.91% of the total recorded species. Camera trapping recorded 99.2% of the predicted large- and medium-sized species (~31), and 33.02% of the total recorded species. eDNA recorded 75.4% of the expected mammal species for this technique (~68), and 47.0% of the total recorded species. eDNA resulted in a useful tool that saves on effort and reduces sampling costs. This study is among the first to show the large potential of eDNA metabarcoding for assessing Amazonian mammal communities, providing, in combination with conventional techniques, a rapid overview of mammal diversity with broad applications to monitoring, management and conservation. By including appropriate genetic markers and updated reference databases, eDNA metabarcoding method can be extended to the whole vertebrate community.

Evaluating the ecological status of mountain streams from a megacity (Mexico City) with diatoms: development and implementation of morphological and eDNA-based methods

In central Mexico, streams flow from mountain protected areas at high elevations, i.e. abive 2,400 m a.s.l., being exposed to different human activities along their course. The main human threats of these aquatic ecosystems include the presence of hydraulic infrastructure (e.g. canals and dams), the construction of bridges or roads, fish farming, agriculture, livestock, unregulated tourism or irregular human settlements, resulting in nutrient enrichment, organic pollution and low hydromorphological quality. Diatoms, widely recognized as valuable indicators of environmental conditions, offer a unique opportunity to assess the ecological quality of the streams in and around this megacity, as they respond directly and sensitively to specific physical, chemical and biological changes. To conduct ecological assessment, the composition of diatom communities has traditionally been characterized by morphological methods and most recently by environmental DNA (eDNA) metabarcoding. However, the diatom communities of only a few of these streams in the Basin of Mexico have been characterized morphologically at low resolution light microscopy (LM), and no eDNA-based studies have been conducted yet. In this context, our aims are to 1) implement morphological and eDNA metabarcoding methods for the identification and quantification of epilithic diatoms and to assess their seasonal and spatial response in three peri-urban mountain streams from the Basin of Mexico, and 2) develop a metric to assess and monitor the ecological quality in these streams. For this purpose, we characterized a) the physical, environmental and hydro-morphological conditions related to ecosystem quality, and b) the diatom diversity and community structure of nine sites per stream (27 sites). The sites include headwaters, as well as middle and lower reaches with different land-use and anthropologic pressures, and were investigated over three hydrological periods, i.e. post-rainy, dry-cold and dry-warm. Species identification was first conducted under LM and scanning electron microscopy (SEM), and is currently been done by eDNA metabarcoding of the V4 region of the 18S rRNA gene and a fragment of the chloroplast marker rbcL. Our study has resulted in 520 infrageneric taxa identified by LM and SEM, the largest diversity ever found for freshwater diatoms of Mexico. From this data, we found a clear environmental gradient from almost pristine conditions towards heavily polluted states as the streams enter the city. These changes are well reflected by variations in the diatom community structure, as revealed by the Diatom Ecological Quality Index (DEQI), newly developed to evaluate the ecological quality of the streams in and around this megacity.

New trends in bioassessment of aquatic ecosystems: from organisms to DNA-based metrics

Traditionally, the biological quality of aquatic ecosystems is assessed using selected groups of organisms that can be identified morphologically. Recent advances in high-throughput genomic approaches offered new opportunities to monitor biodiversity and assess ecological status using DNA barcoding and metabarcoding. The DNA-based tools have been used in three different ways: (1) to replace morphological identification of biological quality elements in existing biotic indices, (2) to develop new molecular indices based on morphologically inconspicuous groups of potential environmental indicators, and (3) to predict biotic indices from environmental DNA datasets using machine learning methods (Pawlowski et al. 2018). The next steps need to take advantages and challenges of these different approaches into account in view of their future application in routine bioassessment.The Working Group 2 of DNAqua-Net, Biotic Indices & Metrics, has worked with several task forces tackling different organism groups (fish, macroinvertebrates, diatoms, bacteria, protists, meiofauna), because challenges have been shown to be quite different dependent on the target organisms Kahlert et al. 2019. For the fish the eDNA-metabarcoding methods are well developed and give very good results in terms of species detection. The important question is to see if the semi-quantitative data retrieved from the metabarcoding (proportion in eDNA sequences) could be translated to proportions in biomass/numbers that are now used in many indices. The fish researchers are trying to fit these data in, but some correction factors might be needed to correct for differences between molecular and conventional methodsRegarding the macroinvertebrates, much discussion regarding index development was focusing on the importance of abundance measurements, and it was tested how existing indices would perform if barcoding data would be used instead of morphological data. Still discussion is ongoing on several technical issues, including the use of preservative for DNA extraction from bulk samples, the choice of primers for PCR amplification and the incompleteness of reference databases which impedes the correct assignment of eDNA sequences. Also minimum standards for routine operation are still missing.The diatom group has worked much on practical issues, starting a large initiative to compare diatom metabarcoding protocols used in routine freshwater biomonitoring for standardization (Bailet et al. 2019, Keck et al. 2018, Vasselon et al. 2017). With diatoms, all three approaches to develop molecular indices have been tested and seem promising, i.e. using existing indices with taxa names derived by matching sequences with reference databases, developing new indices based on molecular data only with traditional fixed scores, and using machine-learning techniques (Bailet et al. 2020, Vasselon et al. 2018, Tapolczai et al. 2019, Keck et al. 2018) The micro- and meiobiota group has worked towards an inclusion of microorganisms into aquatic assessment, because the microbial community dynamics are a missing link important for our understanding of rapid changes in the structure and function of aquatic ecosystems, and should be addressed in the future environmental monitoring of freshwater ecosystems (Sagova-Mareckova et al. 2021). Another focus was on how sediment DNA analysis can be integrated into stated goals of routine monitoring applications. It has been an interesting journey, and we WG2 coordinators would like to thank all the people for their engagement! Keep up the good work!

EDNA metabarcoding fish in relation to the Water Framework Directive indices

The Water Framework Directive (WFD) recognizes fish as a biological quality element to determine the ecological health and quality of waterbodies. Therefore fish monitoring is carried out using conventional methods like electrofishing and seine fishing following standardized protocols. This data is then used in metrics that calculate ecological quality ratios (EQR) for waterbodies. eDNA-metabarcoding could prove an alternative to this conventional WFD-monitoring by offering high sensitivity, possibly lower costs and less invasive sampling. A large project was set up in the Netherlands together with STOWA and the waterboards to explore the possibilities to use eDNA-metabarcoding as an alternative way to carry out the WFD-monitoring for fish. In 2015 in a total of 55 WFD transects (250 meter) were sampled using eDNA-metabarcoding in parallel to the conventional WFD-monitoring. On average 60% mores species were detected with eDNA-metabarcoding. In 2016 a total of 9 whole waterbodies (small rivers/ lakes / canals) were samples in parallel. A sample strategy that mixed water collected from 3 long transects was compared to a strategy that sampled the 3 transects individually. The results showed that the strategy with mixed water samples resulted in nearly the same results at far lower costs. Furthermore a first comparison was made between the relative abundance in eDNA sequences and the relative abundance in biomass and numbers caught. Building on this project between 2018 and 2020 a total of 73 waterbodies were sampled with eDNA-metabarcoding in parallel to conventional WFD-monitoring. Those included streams, rivers, canals, lakes and brackish waterbodies. The final goal is to assess if eDNA-metabarcoding fish can provide similar EQR-scores compared to the conventional WFD-monitoring. This can be done by either renovating the current metrics used or to build new ones based on eDNA data. This presentation will discuss the advantages and disadvantages of both options and gives some insight in the steps still needed for implementation of eDNA-metabarcoding methods in standardized WFD-monitoring.

Exploring video and eDNA metabarcoding methods to assess oyster aquaculture cages as fish habitat

Multi-tiered oyster aquaculture cages may provide habitat for fish assemblages similar to natural structured seafloor. Methods were developed to assess fish assemblages associated with aquaculture gear and boulder habitat using underwater video census combined with environmental DNA (eDNA) metabarcoding. Action cameras were mounted on 3 aquaculture cages at a commercial eastern oysterCrassostrea virginicafarm (‘cage’) and among 3 boulders on a natural rock reef (‘boulder’) from June to August 2017 in Long Island Sound, USA. Interval and continuous video recording strategies were tested. During interval recording, cameras collected 8 min video segments hourly from 07:00 to 19:00 h on cages only. Continuous video was also collected for 2-3 h on oyster cages and boulders. Data loggers recorded light intensity and current speed. Seawater was collected for eDNA metabarcoding on the reef and farm. MaxN measurements of fish abundance were calculated in video, and 7 fish species were observed. Black sea bassCentropristis striata, cunnerTautogolabrus adspersus, scupStenotomus chrysops, and tautogTautoga onitiswere the most abundant species observed in both oyster cage and boulder videos. In continuous video, black sea bass, scup, and tautog were observed more frequently and at higher abundance on the cage farm, while cunner were observed more frequently and at higher abundance on boulders within the rock reef. eDNA metabarcoding detected 42 fish species at the farm and reef. Six species were detected using both methods. Applied in tandem, video recording and eDNA provided a comprehensive approach for describing fish assemblages in difficult to sample structured oyster aquaculture and boulder habitats.

Determining an effective sampling method for eDNA metabarcoding: a case study for fish biodiversity monitoring in a small, natural river

In recent years, biodiversity loss has become one of the most serious environmental issues worldwide, especially in aquatic ecosystems. To avoid diversity loss, it is necessary to monitor biological communities, and environmental DNA (eDNA) metabarcoding has been developed as a rapid, noninvasive, and cost-effective method for aquatic biodiversity monitoring. Although this method has been applied to various environments and taxa, a detailed assessment of the efficient sampling methods for monitoring is still required. In this study, we explored eDNA metabarcoding sampling methods for fish at a single site to maximize the number of detected species using realistic effort in a natural, small river. We considered the following three parameters: sample type (water or sediment), sample position at a site (right and left shore and center of the river), and water volume (10–4000 mL). The results suggested that the number of detected species from sedimentary eDNA was equivalent to that from aqueous eDNA, although the species composition was different. The number of detected species could be saturated by collecting a 1000 mL water sample, regardless of sampling position within a survey site. However, sedimentary eDNA showed a spatially heterogeneous species composition between sampling positions within a survey site despite the short distance (5 m) between positions, without apparent differences in physical properties such as velocity and sediment particle distribution. By completing eDNA biodiversity monitoring of fish with 1000 mL water samples across the whole river, we detected more fish species than in previous traditional surveys conducted at the same sites. Thus, the aqueous eDNA metabarcoding method is as efficient as traditional surveys, while sedimentary eDNA metabarcoding could complement the results of aqueous eDNA metabarcoding.

Detecting fish assemblages with environmental DNA: Does protocol matter? Testing eDNA metabarcoding method robustness

AbstractEnvironmental DNA (eDNA) metabarcoding has recently gain much attention to assess aquatic environment biodiversity. A great variety of protocols have been developed to collect, extract, and analyze eDNA, some of which are continuously evolving and optimized with technological improvements. Such technological shifts might deprecate the biological data produced with earlier protocols, leading to a loss of biological knowledge. Here, we investigated the robustness of an aquatic eDNA metabarcoding method through the comparison of two biodiversity datasets generated by two optimized protocols with different collection and extraction steps. To this end, we compared fish community richness and composition of 12 streams and 3 rivers from French Guiana, sampled with two distinct protocols commonly used in aquatic eDNA studies. Although sample collection with each protocol was not achieved the same year, our results show that species richness and species composition were only slightly affected by the protocol choice, both protocols producing similar fish assemblages at each sample site. Both protocols had a higher replicability in streams than in rivers, strengthening the importance to adapt sampling effort to waterbody type as rivers host a larger number of species than small streams. Despite the need for a standardized approach in eDNA metabarcoding studies, testing the robustness of datasets to protocol variations remains crucial to valorize old data, time series, or data collected in difficult to access locations.

Integrated eDNA metabarcoding and morphological analyses assess spatio-temporal patterns of airborne fungal spores

Fungi represent relevant allergens and plant pathogens that can disperse on long ranges, potentially producing severe consequences on public health and agriculture. Up to 11% of the bioaerosol particles are fungal spores and mycelium fragments. Estimation of fungal species diversity in time and space is decisive but may be biased by abiotic conditions and sampling methods. Traditional morphological analyses of fungal spores have been widely applied in aerobiology in the past, while recently eDNA metabarcoding can complement these studies. Here, we used both morphological analysis (spore count and taxon identification) and high-throughput sequencing to disentangle spatio-temporal variation of fungi across Northern and Central Italy and to evaluate the detection efficiency of the two approaches. Our results showed that eDNA metabarcoding detects about three times more genera and has a higher detection efficiency than the morphological analyses. However, the efficiency is high in both spore count and eDNA metabarcoding methods when the most abundant or the rarest genera are considered but it can substantially vary between the two approaches when moderately abundant genera are analyzed. Furthermore, morphological spore determination resulted in higher variance explained by PERMANOVA analysis with respect to eDNA metabarcoding (26% and 13%, respectively), which leads to a better spatio-temporal characterization of the fungal genera. As both morphological analyses and eDNA metabarcoding methods capture significant interactions between seasons and sites, they could be preferably used as complementing approaches to reliably study airborne fungal diversity and variation.

Using environmental DNA for biomonitoring of freshwater fish communities: Comparison with established gillnet surveys in a boreal hydroelectric impoundment

AbstractAccurate data characterizing species distribution and abundance are critical for conservation and management of aquatic resources. Inventory methods, such as gillnet surveys, are widely used to estimate distribution and abundance of fish. However, gillnet surveys can be costly in terms of material and human resources, may cause unwanted mortality in the fish communities being studied, and is subject to size and species selection bias. Detecting allochthonous DNA released by species in their environment (i.e., environmental DNA, hereafter eDNA) could be used as a noninvasive and less costly alternative. In this study, we directly compare eDNA metabarcoding and gillnets for monitoring freshwater fish communities in terms of species richness and relative species abundance. Metabarcoding was performed with the 12S Mifish primers. We also used species‐specific quantitative PCR (qPCR) for the most abundant species, the walleye (Sander vitreus), to compare estimated relative abundance with metabarcoding and gillnet captures. Water sample collection, prior to gillnet assessment, was performed on 17 sites in the hydroelectric impoundment of the Rupert River (James Bay, Canada), comparing two water filtration methods. After controlling for amplification biases and repeatability, we show that fish communities’ complexity is better represented using eDNA metabarcoding than previously recorded gillnet data and that metabarcoding read count correlates with qPCR (r = 0.78, p < .001) in reflecting walleye abundance. Finally, based on partial redundancy analysis, we identified alpha chlorophyll, pH, and dissolved oxygen as environmental variable candidates that may influence differences in fish relative abundance between metabarcoding and gillnets. Altogether, our study demonstrates that the proposed eDNA metabarcoding method can be used as an efficient alternative or complementary technique adapted to the biomonitoring of the fish communities in boreal aquatic ecosystems.

Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis.

Recent advances in environmental DNA (eDNA) analysis using high-throughput sequencing (HTS) enable evaluation of intraspecific genetic diversity in a population. As the intraspecific genetic diversity provides invaluable information for wildlife conservation and management, there is an increasing demand to apply eDNA analysis to population genetics and the phylogeography by quantitative evaluation of intraspecific diversity. However, quantitative evaluations of intraspecific genetic diversity using eDNA is not straightforward because the number of eDNA sequence reads obtained by HTS may not be an index of the quantity of eDNA. In this study, to quantitatively evaluate genetic diversity using eDNA analysis, we applied a quantitative eDNA metabarcoding method using the internal standard DNAs. We targeted Ayu (Plecoglossus altivelis altivelis) and added internal standard DNAs with known copy numbers to each eDNA sample obtained from three rivers during the library preparation process. The sequence reads of each Ayu haplotype were successfully converted to DNA copy numbers based on the relationship between the copy numbers and sequence reads of the internal standard DNAs. In all rivers, the calculated copy number of each haplotype showed a significant positive correlation with the haplotype frequency estimated by a capture-based survey. Furthermore, estimates of genetic indicators such as nucleotide diversity based on the eDNA copy numbers were comparable with those estimated based on a capture-based study. Our results demonstrate that eDNA analysis with internal standard DNAs enables reasonable quantification of intraspecific genetic diversity, and this method could thus be a promising tool in the field of population genetics and phylogeography.

Effects of sampling seasons and locations on fish environmental DNA metabarcoding in dam reservoirs.

Environmental DNA (eDNA) analysis has seen rapid development in the last decade, as a novel biodiversity monitoring method. Previous studies have evaluated optimal strategies, at several experimental steps of eDNA metabarcoding, for the simultaneous detection of fish species. However, optimal sampling strategies, especially the season and the location of water sampling, have not been evaluated thoroughly. To identify optimal sampling seasons and locations, we performed sampling monthly or at two‐monthly intervals throughout the year in three dam reservoirs. Water samples were collected from 15 and nine locations in the Miharu and Okawa dam reservoirs in Fukushima Prefecture, respectively, and five locations in the Sugo dam reservoir in Hyogo Prefecture, Japan. One liter of water was filtered with glass‐fiber filters, and eDNA was extracted. By performing MiFish metabarcoding, we successfully detected a total of 21, 24, and 22 fish species in Miharu, Okawa, and Sugo reservoirs, respectively. From these results, the eDNA metabarcoding method had a similar level of performance compared to conventional long‐term data. Furthermore, it was found to be effective in evaluating entire fish communities. The number of species detected by eDNA survey peaked in May in Miharu and Okawa reservoirs, and in March and June in Sugo reservoir, which corresponds with the breeding seasons of many of fish species inhabiting the reservoirs. In addition, the number of detected species was significantly higher in shore, compared to offshore samples in the Miharu reservoir, and a similar tendency was found in the other two reservoirs. Based on these results, we can conclude that the efficiency of species detection by eDNA metabarcoding could be maximized by collecting water from shore locations during the breeding seasons of the inhabiting fish. These results will contribute in the determination of sampling seasons and locations for fish fauna survey via eDNA metabarcoding, in the future.

Environmental DNA metabarcoding to detect pathogenic Leptospira and associated organisms in leptospirosis-endemic areas of Japan

Leptospires, which cause the zoonotic disease leptospirosis, persist in soil and aqueous environments. Several factors, including rainfall, the presence of reservoir animals, and various abiotic and biotic components interact to influence leptospiral survival, persistence, and pathogenicity in the environment. However, how these factors modulate the risk of infection is poorly understood. Here we developed an approach using environmental DNA (eDNA) metabarcoding for detecting the microbiome, vertebrates, and pathogenic Leptospira in aquatic samples. Specifically, we combined 4 sets of primers to generate PCR products for high-throughput sequencing of multiple amplicons through next-generation sequencing. Using our method to analyze the eDNA of leptospirosis-endemic areas in northern Okinawa, Japan, we found that the microbiota in each river shifted over time. Operating taxonomic units corresponding to pathogenic L. alstonii, L. kmetyi, and L. interrogans were detected in association with 12 nonpathogenic bacterial species. In addition, the frequencies of 11 of these species correlated with the amount of rainfall. Furthermore, 10 vertebrate species, including Sus scrofa, Pteropus dasymallus, and Cynops ensicauda, showed high correlation with leptospiral eDNA detection. Our eDNA metabarcoding method is a powerful tool for understanding the environmental phase of Leptospira and predicting human infection risk.