DNA Metabarcoding Approach Research Articles

Assessing changes in stream macroinvertebrate communities across ecological gradients using morphological versus DNA metabarcoding approaches

Freshwater macroinvertebrates provide valuable indicators for biomonitoring ecosystem change in relation to natural and anthropogenic drivers. DNA metabarcoding is an efficient approach for estimating such indicators, but its results may differ from morphotaxonomic approaches traditionally used in biomonitoring. Here we test the hypothesis that despite differences in the number and identity of taxa recorded, both approaches may retrieve comparable patterns of community change, and detect similar ecological gradients influencing such changes. We compared results obtained with morphological identification at family level of macroinvertebrates collected at 80 streams under a Water Framework Directive biomonitoring program in Portugal, with results obtained with metabarcoding from the ethanol preserving the bulk samples, using either single (COI-M19BR2, 16S-Inse01, 18S-Euka02) or multiple markers. Metabarcoding recorded less families and different communities compared to morphotaxonomy, but community sensitivities to disturbance estimated with the IASPT index were more similar across approaches. Spatial variation in local community metrics and the factors influencing such variation were significantly correlated between morphotaxonomy and metabarcoding. After reducing random noise in the dissimilarity matrices, the spatial variation in community composition was also significantly correlated across methods. A dominant gradient of community change was consistently retrieved, and all methods identified a largely similar set of anthropogenic stressors strongly influencing such gradient. Overall, results confirm our initial hypothesis, suggesting that morphotaxonomy and metabarcoding can estimate consistent spatial patterns of community variation and their main drivers. These results are encouraging for macroinvertebrate biomonitoring using metabarcoding approaches, suggesting that they can be intercalibrated with morphotaxonomic approaches to recover equivalent spatial and temporal gradients of ecological change.

Are Genetic Reference Libraries Sufficient for Environmental DNA Metabarcoding of Mekong River Basin Fish?

Environmental DNA (eDNA) metabarcoding approaches to surveillance have great potential for advancing biodiversity monitoring and fisheries management. For eDNA metabarcoding, having a genetic reference sequence identified to fish species is vital to reduce detection errors. Detection errors will increase when there is no reference sequence for a species or when the reference sequence is the same between different species at the same sequenced region of DNA. These errors will be acute in high biodiversity systems like the Mekong River Basin, where many fish species have no reference sequences and many congeners have the same or very similar sequences. Recently developed tools allow for inspection of reference database coverage and the sequence similarity between species. These evaluation tools provide a useful pre-deployment approach to evaluate the breadth of fish species richness potentially detectable using eDNA metabarcoding. Here we combined established species lists for the Mekong River Basin, resulting in a list of 1345 fish species, evaluated the genetic library coverage across 23 peer-reviewed primer pairs, and measured the species specificity for one primer pair across four genera to demonstrate that coverage of genetic reference libraries is but one consideration before deploying an eDNA metabarcoding surveillance program. This analysis identifies many of the eDNA metabarcoding knowledge gaps with the aim of improving the reliability of eDNA metabarcoding applications in the Mekong River Basin. Genetic reference libraries perform best for common and commercially valuable Mekong fishes, while sequence coverage does not exist for many regional endemics, IUCN data deficient, and threatened fishes.

A metabarcoding tool to detect predation of the honeybee Apis mellifera and other wild insects by the invasive Vespa velutina

The invasive Vespa velutina has been widely referred as an effective predator of honeybees. Despite the potential risk to pollination services provision and honey production, there is no accurate quantification and assessment of its real consequences for honeybees. To date, the identification of the honeybee and other insects in the diet of V. velutina has been investigated by direct observation of adult foraging or examination of food pellets. To overcome these limitations, in this study we used a DNA metabarcoding approach to evaluate the usefulness of different types of sample (jaws and stomachs collected from workers and larval faecal pellets taken from the hornet comb) to investigate the predation of V. velutina upon honeybees, and potentially on other insects. Honeybee DNA was identified in all types of samples, but larval faecal pellets retrieved the higher number of reads of honeybee DNA and the largest diversity at all taxonomic levels. Over all samples we could identify 4 orders, 9 families, 6 genera and 1 species of prey. We estimate that collecting 6 workers is sufficient to identify honeybee predation by a colony using worker’s jaws. Stomachs were the least useful sample type to detect honeybee DNA. The presence of honeybee DNA in all analysed colonies irrespective of collection site, and the variety of insect orders detected in the diet support current concerns over the acknowledged negative impact of V. velutina on managed honeybees and its potential threat to pollination services provision.

Lampenflora in a Show Cave in the Great Basin Is Distinct from Communities on Naturally Lit Rock Surfaces in Nearby Wild Caves.

In show caves, artificial lighting is intended to illuminate striking cave formations for visitors. However, artificial lighting also promotes the growth of novel and diverse biofilm communities, termed lampenflora, that obtain their energy from these artificial light sources. Lampenflora, which generally consist of cyanobacteria, algae, diatoms, and bryophytes, discolor formations and introduce novel ecological interactions in cave ecosystems. The source of lampenflora community members and patterns of diversity have generally been understudied mainly due to technological limitations. In this study, we investigate whether members of lampenflora communities in an iconic show cave—Lehman Caves—in Great Basin National Park (GRBA) in the western United States also occur in nearby unlit and rarely visited caves. Using a high-throughput environmental DNA metabarcoding approach targeting three loci—the ITS2 (fungi), a fragment of the 16S (bacteria), and a fragment of 23S (photosynthetic bacteria and eukaryotes)—we characterized diversity of lampenflora communities occurring near artificial light sources in Lehman Caves and rock surfaces near the entrances of seven nearby “wild” caves. Most caves supported diverse and distinct microbial-dominated communities, with little overlap in community members among caves. The lampenflora communities in the show cave were distinct, and generally less diverse, from those occurring in nearby unlit caves. Our results suggest an unidentified source for a significant proportion of lampenflora community members in Lehman Caves, with the majority of community members not found in nearby wild caves. Whether the unique members of the lampenflora communities in Lehman Caves are related to distinct abiotic conditions, increased human visitation, or other factors remains unknown. These results provide a valuable framework for future research exploring lampenflora community assemblies in show caves, in addition to a broad perspective into the range of microbial and lampenflora community members in GRBA. By more fully characterizing these communities, we can better monitor the establishment of lampenflora and design effective strategies for their management and removal.

Diversity of Insects in Nature protected Areas (DINA): an interdisciplinary German research project

Insect declines and biodiversity loss have attracted much attention in recent years, but lack of comprehensive data, conflicting interests among stakeholders and insufficient policy guidance hinder progress in preserving biodiversity. The project DINA (Diversity of Insects in Nature protected Areas) investigates insect communities in 21 nature reserves in Germany. All selected conservation sites border arable land, with agricultural practices assumed to influence insect populations. We taught citizen scientists how to manage Malaise traps for insect collection, and subsequently used a DNA metabarcoding approach for species identification. Vegetation surveys, plant metabarcoding as well as geospatial and ecotoxicological analyses will help to unravel contributing factors for the deterioration of insect communities. As a pioneering research project in this field, DINA includes a transdisciplinary dialogue involving relevant stakeholders such as local authorities, policymakers, and farmers, which aims at a shared understanding of conservation goals and action pathways. Stakeholder engagement combined with scientific results will support the development of sound policy recommendations to improve legal frameworks, landscape planning, land use, and conservation strategies. With this transdisciplinary approach, we aim to provide the background knowledge to implement policy strategies that will halt further decline of insects in German protected areas.

Natural contaminants in bee pollen: DNA metabarcoding as a tool to identify floral sources of pyrrolizidine alkaloids and fungal diversity

The use of bee pollen as a food supplement has increased in recent years as it contains several nutrients and phytochemicals. However, depending on floral composition, bee pollen can be contaminated by pyrrolizidine alkaloids (PAs), PA N-oxides (PANOs) and toxigenic fungi found in plants, which may pose a potential health risk for consumers. Thus, a DNA metabarcoding approach based on internal transcribed spacer 2 (ITS2) region was used to identify the plant sources of 17 PAs/PANOs detected by a validated method in liquid chromatography coupled to mass spectrometry (LC-MS/MS), as well as floral and fungal diversity in 61 bee pollen samples. According to LC-MS/MS analysis, 67% of the samples contained PAs/PANOs with mean concentration of 339 µg/kg. The contamination pattern was characterised by lycopsamine- and senecionine-type PAs/PANOs. PA/PANO-producing plants were identified in 54% of the PA/PANO-contaminated samples analysed by DNA metabarcoding, which also allowed identifying the overall floral and fungal composition of 56 samples. To evaluate the performance of the molecular approach, a subset of 25 samples was analysed by classical palynology. Palynological analysis partially confirmed the results of DNA metabarcoding, which had a better performance in distinguishing pollens of different genera from Asteraceae (76%) and Brassicaceae (88%). However, the molecular analysis did not identify pollens from Castanea, Eucalyptus, Hedera and Salix, which were abundant in 11 samples according to palynology. On the other hand, the molecular analysis allowed identifying several fungal genera in 33 samples, including the toxigenic fungi Alternaria and Aspergillus, which were positively correlated to the plant genus Hypericum. Despite limitations in identifying some pollen types, these preliminary results suggest that the DNA metabarcoding could be applied in a multidisciplinary approach to give a picture of floral and fungal diversity, which can be sources of natural contaminants in bee pollen and would help to control its safety.

The factors and scales shaping fungal assemblages in fallen spruce trunks: A DNA metabarcoding study

Understanding the distribution of biodiversity across forest landscapes is a key issue for the spatial planning of conservation management. Obtaining such spatial perspective is challenging because a large part of biodiversity remains hidden to the conventional survey approaches. High-productivity forests are probably the hotspots of hidden biodiversity and, at the same time, under severe timber harvesting pressure. We used DNA-metabarcoding approach to assess habitat quality of successional high productivity forests for wood-inhabiting fungi, focusing on fungal diversity that remains hidden in fruit-body surveys. We sampled a fixed amount of coarse fallen Norway spruce trunks in 40 naturally developing stands ranging from 44 to 140 years of age in hemi-boreal Estonia. We found three main habitat quality patterns. The total number of fungal OTUs in older (>80 years) stands exceeded that in younger stands due to the accumulation of rare fungi. The proportion of trunks hosting highest number of OTUs and trunk-scale richness of rare fungi were greatest in stands surrounded by extensive forest area. The average number of red-listed species per trunk increased mainly along with the volume of fallen dead wood; large volumes provided quality habitats already before the stands had reached 80 years of age. These results support the view that both substrate amount in the forest stand and habitat connectivity on a landscape scale support fungal diversity in dead wood. Earlier studies on wood-inhabiting fungi have reached similar conclusions based on observations of fungal fruit-bodies. Thus, the overall principle of focusing conservation efforts to remaining high quality habitats and landscapes extends to the fungi hidden to the conventional survey methods.

Falcon fuel: metabarcoding reveals songbird prey species in the diet of juvenile Merlins (Falco columbarius) migrating along the Pacific Coast of western North America

During autumn migration, bird‐eating raptors are thought to rely on flocks of migrant songbirds (Passeriformes) as a critical resource to fuel the energetic demands of long‐distance migration. However, this hypothesis has been challenging to investigate, and the foraging ecology during migration of most migrant raptors remains unexplored. To address these knowledge gaps, our objective was to document the diet of a bird‐eating falcon on active migration. We swabbed visible and trace prey residues from the exterior surface of beaks and talons of migrant juvenile Merlins Falco columbarius in the autumn of 2015 and 2016 at a raptor migration monitoring station positioned on the Pacific Coast of western North America. We used a DNA metabarcoding approach and detected the presence of 40 distinct prey species derived from 210 individual prey species detections on 63 of the 72 (87.5%) migrant juvenile Merlins sampled. We detected an average of 3.3 ± 1.6 prey species on individual Merlins. We found that juvenile males selected smaller prey on average compared with juvenile females. Of the prey species detected, over 80% were migratory songbird species within the Pacific Flyway. In 2015, we detected a greater proportion of irruptive migrants in juvenile Merlin diet compared with 2016. In 2016, we found that the proportion of annual migrants consumed by Merlins corresponded to the timing of peak annual songbird migration in the Pacific Flyway. This study represents one of the first detailed descriptions of songbird prey species consumed by a migrating raptor and supports the hypothesis that migrating juvenile Merlins rely on migrant songbirds to support the energetic demands of migration.

The role of taxonomic expertise in interpretation of metabarcoding studies

Abstract The performance of DNA metabarcoding approaches for characterizing biodiversity can be influenced by multiple factors. Here, we used morphological assessment of taxa in zooplankton samples to develop a large barcode database and to assess the congruence of taxonomic identification with metabarcoding under different conditions. We analysed taxonomic assignment of metabarcoded samples using two genetic markers (COI, 18S V1–2), two types of clustering into molecular operational taxonomic units (OTUs, ZOTUs), and three methods for taxonomic assignment (RDP Classifier, BLASTn to GenBank, BLASTn to a local barcode database). The local database includes 1042 COI and 1108 18S (SSU) barcode sequences, and we added new high-quality sequences to GenBank for both markers, including 109 contributions at the species level. The number of phyla detected and the number of taxa identified to phylum varied between a genetic marker and among the three methods used for taxonomic assignments. Blasting the metabarcodes to the local database generated multiple unique contributions to identify OTUs and ZOTUs. We argue that a multi-marker approach combined with taxonomic expertise to develop a curated, vouchered, local barcode database increases taxon detection with metabarcoding, and its potential as a tool for zooplankton biodiversity surveys.

Unraveling the dietary diversity of Neotropical top predators using scat DNA metabarcoding: A case study on the elusive Giant Otter

AbstractLarge carnivores play a pivotal regulating role in maintaining healthy and balanced ecosystems; however, most of them are rare and elusive, and knowledge about their resource consumption is scarce. Traditional methods based on morphological identification of undigested remains are labor intensive and often not sufficiently accurate, leading to errors and biased ecological inferences. Here, we developed a multi‐marker DNA metabarcoding approach to analyze the dietary diversity of giant otters (Pteronura brasiliensis) from fecal DNA while controlling predator species identity. We combined two mitochondrial markers, 12S rRNA and cytochrome c oxidase 1 (COI) gene, that target the full range of potential vertebrate and invertebrate prey. We compiled a local reference database of DNA barcodes for most potentially ingested fish, which were used to evaluate the specificity of the metabarcoding primers in silico. Most prey are identified at the species level (>90%) and the dietary profiles provided independently by the two markers are highly similar, whether in terms of list of prey or frequency of occurrences, hence validating the approach. We detected a higher number of rare fish prey with the 12S primers that amplified solely Teleost species while the degenerate COI primers revealed non‐fish prey (e.g., amphibians, snakes, birds, and earthworms) and confirmed predator species identity. This study demonstrated that scat DNA metabarcoding is particularly useful to provide in‐depth information on elusive carnivorous dietary profile. Our methodology opens up new opportunities to understand how top carnivores diet cope with the effects of anthropogenic alteration of ecosystems and identify conflicts with humans and livestock.

Marine Fish Detection by Environmental DNA (eDNA) Metabarcoding Approach in the Pelabuhan Ratu Bay, Indonesia

Diversity studies in marine ecosystems have experienced many developments with molecular applications through the environmental DNA (eDNA) metabarcoding approach. This method has widely used in identifying rare species, invasive species, and biodiversity information in both freshwater and marine water environments. In this study, the eDNA metabarcoding approach was the first applied to estimate fish diversity surrounding the Pelabuhan Ratu Bay waters. Five liters of seawater samples have been collected from five spots of the surrounding port area. The metabarcoding technique was applied out by using combination DNA barcoding and the next-generation sequencing (NGS) method by MiSeq platform. The MiFish Pipeline for data analyzing to get species estimation and reads numbers for quantifying the fish abundance in this location. DNA successfully identified 20 species, which commercially in the traditional fish market in Pelabuhan Ratu. Then, the eDNA metabarcoding revealed that 44 marine fish species representing 36 genera, 24 families, and ten orders exist in Pelabuhan Ratu Bay's surrounding water. Several marine fish not included in economically but have ecological value in marine fish diversity. Furthermore, fish fall into the category of vulnerable fish (Thunnus obesus), poisonous fish (Acanthurus lineatus, Acanthurus xanthopterus, Siganus vermiculatus, and Siganus fuscescens), and migratory fish. Regular surveys to determine the fluctuations and effects of human activities are necessary to monitor marine diversity, both the effect of over-exploitation and potential pollution from the coastal areas around the Pelabuhan Ratu Bay.

Comprehensive coverage of human last meal components revealed by a forensic DNA metabarcoding approach

Stomach content analyses are a valuable tool in human forensic science to interpret perimortem events. While the identification of food components of plant and animal origin has traditionally been conducted by macro- and microscopical approaches in case of incomplete digestion, molecular methods provide the potential to increase sensitivity and taxonomic resolution. In particular, DNA metabarcoding (PCR-amplification and next generation sequencing of complex DNA mixtures) has seen a rapid growth in the field of wildlife ecology to assess species’ diets from faecal and gastric samples. Despite clear advantages, molecular approaches have not yet been established in routine human forensics to investigate the last meal components of deceased persons. In this pilot study we applied for the first time a DNA metabarcoding approach to assess both plant and vertebrate components of 48 human stomach content samples taken during medicolegal autopsies. We obtained a final dataset with 34 vertebrate and 124 vegetal unique sequences, that were clustered to 9 and 33 operational taxonomic units (OTUs), respectively. Our results suggest that this approach can provide crucial information about circumstances preceding death, and open promising perspectives for biomedical dietary surveys based on digested food items found in the gastrointestinal tract.

The diet of major predators of forest soils: A first analysis on syntopic species of Chilopoda through DNA metabarcoding

Centipedes (Chilopoda) represent a dominant group of predators in temperate forest soils, where multiple species often coexist in syntopy. However, their trophic ecology is largely unknown because traditional methodologies are hindered by the secretive habits of these animals and their partial fluid-feeding. We applied a DNA-metabarcoding approach with COI to assess and compare the prey spectrum of three centipede species (Clinopodes flavidus, Eupolybothrus tridentinus, Lithobius validus) living in a forest plot in the Southern Alps. We obtained DNA sequences from the guts of 79 specimens through Next-Generation Sequencing technology and applied a suite of stringent criteria of data filtering and taxonomic assignment. We found evidence for a similar rate of prey detection between predator species and for a broad prey spectrum, including most taxa of invertebrates living in the local soil biocoenosis and having subduable body size. We did not find evidence of diet variation in relation to sex and annual season, and instead slight but significant differentiation of diet between the geophilomorph C. flavidus and the other two lithobiomorph species.

Sedimentary Ancient DNA From the Subarctic North Pacific: How Sea Ice, Salinity, and Insolation Dynamics Have Shaped Diatom Composition and Richness Over the Past 20,000 Years

AbstractWe traced diatom composition and diversity through time using diatom‐derived sedimentary ancient DNA (sedaDNA) from eastern continental slope sediments off Kamchatka (North Pacific) by applying a short, diatom‐specific marker on 63 samples in a DNA metabarcoding approach. The sequences were assigned to diatoms that are common in the area and characteristic of cold water. SedaDNA allowed us to observe shifts of potential lineages from species of the genus Chaetoceros that can be related to different climatic phases, suggesting that pre‐adapted ecotypes might have played a role in the long‐term success of species in areas of changing environmental conditions. These sedaDNA results complement our understanding of the long‐term history of diatom assemblages and their general relationship to environmental conditions of the past. Sea‐ice diatoms (Pauliella taeniata [Grunow] Round & Basson, Attheya septentrionalis [Østrup] R. M. Crawford and Nitzschia frigida [Grunow]) detected during the late glacial and Younger Dryas are in agreement with previous sea‐ice reconstructions. A positive correlation between pennate diatom richness and the sea‐ice proxy IP25 suggests that sea ice fosters pennate diatom richness, whereas a negative correlation with June insolation and temperature points to unfavorable conditions during the Holocene. A sharp increase in proportions of freshwater diatoms at ∼11.1 cal kyr BP implies the influence of terrestrial runoff and coincides with the loss of 42% of diatom sequence variants. We assume that reduced salinity at this time stabilized vertical stratification which limited the replenishment of nutrients in the euphotic zone.

Review: DNA metabarcoding approach for fisheries management

Increased publications on meta-barcoding promote interests in studies associated with fisheries biodiversity, diet, and biomonitoring. These studies primarily concentrated on an ecosystem that has implications for fisheries management with complex interpretation. Various theories have arisen to describe the interactions among aquatic species and eventually contribute to the roles they play in ecology. Identifying organisms is also a prerequisite for answering questions in the field of ecology, which shows that reliable and accurate knowledge is important and desirable. Therefore, as a modern molecular technique, metabarcoding provides a realistic approach that lets ecologists analyze organisms in complex environmental samples and rapidly solve the problem associated with the food web in the coral reef ecosystem. This research explores the use of metabarcoding in fisheries management research and highlights the drawback and value of the future process.

Deciphering the diet of a wandering spider (Phoneutria boliviensis; Araneae: Ctenidae) by DNA metabarcoding of gut contents.

Arachnids are the most abundant land predators. Despite the importance of their functional roles as predators and the necessity to understand their diet for conservation, the trophic ecology of many arachnid species has not been sufficiently studied. In the case of the wandering spider, Phoneutria boliviensis F. O. Pickard‐Cambridge, 1897, only field and laboratory observational studies on their diet exist. By using a DNA metabarcoding approach, we compared the prey found in the gut content of males and females from three distant Colombian populations of P. boliviensis. By DNA metabarcoding of the cytochrome c oxidase subunit I (COI), we detected and identified 234 prey items (individual captured by the spider) belonging to 96 operational taxonomic units (OTUs), as prey for this wandering predator. Our results broaden the known diet of P. boliviensis with at least 75 prey taxa not previously registered in fieldwork or laboratory experimental trials. These results suggest that P. boliviensis feeds predominantly on invertebrates (Diptera, Lepidoptera, Coleoptera, and Orthoptera) and opportunistically on small squamates. Intersex and interpopulation differences were also observed. Assuming that prey preference does not vary between populations, these differences are likely associated with a higher local prey availability. Finally, we suggest that DNA metabarcoding can be used for evaluating subtle differences in the diet of distinct populations of P. boliviensis, particularly when predation records in the field cannot be established or quantified using direct observation.

Community metabarcoding uncovers vast diversity and a lack of barcode references for aquatic invertebrates in Carpathian spring fens

Recent studies on diversity of stream amphipods proposed that the Western Carpathians have served as an important glacial refugium of freshwater fauna. If this scenario is true, a considerably high molecular diversity can be expected in this biogeographic region also for other taxa. In our project, we aimed to uncover and characterize molecular diversity of benthic macroinvertebrate fauna of spring fens, a well studied and hence convenient model community. Using a DNA metabarcoding approach, we sequenced a fragment of the COI gene of pooled spring fen invertebrate communities from 21 localities in the Western Carpathians. The preliminary analysis of the sequences revealed a substantially higher amount of operational taxonomic units (OTUs), when compared to the number of taxa identified in the same spring fens based on morphological characters. The lack of reference sequences in public databases (Barcoding of Life Database, GenBank) for the majority of the detected OTUs indicates that the Western Carpathian region is not yet sufficiently covered by barcoding efforts, and suggests that there indeed may be a considerable unrecognized diversity of macrozoobenthos. However, most newly uncovered diversity is concentrated in a few taxa such as the dipteran families Tabanidae and Ceratopogonidae, annelid species (complexes) Eiseniella tetraedra and Helobdella stagnalis or the amphipod genus Gammarus. In the future, these findings will be the basis for several levels of research: detailed investigation of seemingly hyperdiverse taxa (phylogeography, phylogeny, reference coverage) and comparison of overall molecular diversity of these communities in the Western Carpathians and neighbouring Bohemian Massif, a region where we expect much lower diversity due to different biogeographic history.

Applicability of DNA-based identifications for the WFD-guided monitoring using macroinvertebrates: a large-scale DNA metabarcoding study for implementing routine ecological status assessments in Iberian rivers

Over the last decade, remarkable improvements have been made in the field of metabarcoding-based tools for routine ecological status assessments. However, important issues are yet to be solved to fulfil the European Water Framework Directive (WFD) requirements and standards. These limitations, which include problems related to e.g. the lack of a complete COI macroinvertebrate barcode database available for the Iberian Peninsula Murria 2020, or the scarce recovery of specific taxa due to DNA extraction and/or PCR amplification bias, are especially difficult to overcome for routine freshwater macroinvertebrate monitoring. For that purpose, a large-scale study is on going to test how metabarcoding data can infer existing macroinvertebrate morphotaxonomy-based biotic indexes and ecological status of Iberian rivers. Freshwater macroinvertebrates were selected as a Biological Quality Element and identified by using both morphological and metabarcoding approaches. The mitochondrial gene for cytochrome c oxidase subunit I (COI) was used as a DNA Barcode. Taxonomic coverage, taxonomic composition metrics and ecological status obtained from both approaches were analysed. Physical and chemical variables obtained during the routine biomonitoring, as well as other ecological parameters including biodiversity indexes, were also assessed. Multivariate data analysis of these environmental and biotic data obtained from both approaches were compared. Results seem to support the hypothesis Kuntke 2019 that the DNA-metabarcoding approach might deliver similar quality assessments results to the morphological approach, though some refinement must be done at the different steps of the process prior to establish a reliable procedure allowing the alternative use of both methods giving similar results for the ecological status classes marked by the WFD.

A pilot for implementing environmental DNA (eDNA) based methods into environmental and biomonitoring

Environmental DNA (eDNA) and other molecular based approaches are revolutionizing the field of biomonitoring. These approaches undergo rapid modifications, and it is crucial to develop the best practices by sharing the newest information and knowledge. In our ongoing project we: assess the state-of-the-art of eDNA methods at Finnish Environment Institute SYKE; identify concrete next steps towards the long-term aim of implementing eDNA methods into environmental and biomonitoring; promote information exchange on eDNA methods and advance future research efforts both within SYKE and with our national and international partners. assess the state-of-the-art of eDNA methods at Finnish Environment Institute SYKE; identify concrete next steps towards the long-term aim of implementing eDNA methods into environmental and biomonitoring; promote information exchange on eDNA methods and advance future research efforts both within SYKE and with our national and international partners. Scientific background Well-functioning and intact natural ecosystems are essential for human well-being, provide a variety of ecosystem services and contain a high diversity of organisms. However, human activities such as eutrophication, pollution, land-use or invasive species, are threatening the state and functioning of ecosystems from local to global scale (e.g. Benateau et al. 2019; Reid et al. 2018; Vörösmarty et al. 2010). New molecular techniques in the field and in the laboratory have enabled sampling and identification of much of terrestrial, marine and freshwater biodiversity. These include environmental DNA (eDNA, e.g. Valentini et al. 2016) and bulk-sample DNA metabarcoding approaches (e.g. Elbrecht et al. 2017) and targeted RNA-based methods (e.g. Mäki and Tiirola 2018). The eDNA technique uses DNA that is released from organisms into their environment, from which a signal of organisms’ presence in the system can be obtained. For example, in aquatic ecosystems, eDNA is typically extracted from sediment or filtered water samples (e.g. Deiner et al. 2016), and this approach is distinguished from bulk DNA metabarcoding, where organisms are directly identified from e.g. complete biological monitoring samples (e.g. Elbrecht et al. 2017). Despite the demonstrated potential of environmental and bulk-sample DNA metabarcoding approaches in recent years, there are still significant bottlenecks to their routine use that need to be addressed (e.g. Pawlowski et al. 2020). Methods and implementati on The project is divided into three work packages: WP1 Gathering existing knowledge, identifying knowledge gaps and proposing best practices, WP2 Roadmap to implementation and WP3 eDNA monitoring pilot. Please see more details in the Fig. 1

Validation of river typologies using molecular information from diatoms

The European Water Framework Directive (2000/60/EC) includes biological assessment of water bodies that has been implemented for many years. Indicator organisms such as diatoms respond to geological and hydrological features of rivers by modifying their structure. Therefore, when implementing the WFD, it was necessary to establish type-specific reference conditions to be able to measure the deviations of sampled communities due to anthropogenic impact.HTS-related eDNA metabarcoding has been developed to complement or even replace traditional approaches for its rapid, low-cost and highly accurate identification of communities for assessment of rivers’ ecological status (e.g. Mortágua et al., 2019; Pérez-Burillo et al. 2020) and proved to provide even more in-depth information about biological elements. The use of this information without assignment to species is being addressed once it eliminates the limiting factor of the reference database incompleteness and may provide new ecological information (e.g. Feio et al., 2020; Rivera et al., 2020). Since WFD requires the establishment of reference conditions for each water body type, for eDNA methods’ implementation it will be essential to review, confirm or reformulate, and perhaps create new typologies. Hereupon, the aim of this study is to analyze diatom communities from different typologies of Portuguese rivers resulting from DNA metabarcoding data and compare it with current typology system. To do so, we will verify the consistency of biological groups included in each type, validate the molecular data, analyze the correspondence of OTU/ISU/ESV to environmental characteristics of rivers. A total of 154 sampling sites were selected from central Portugal and northern Portugal in 2017 and 2019. The biofilm was collected for morphological identification and DNA sequencing of diatoms. Reference sites were selected for 4 river types (mountain, littoral, small and medium-large northern rivers) based on a set of pressure information (water quality, hydromorphology, land use and riparian zones). Diatom inventories were obtained from molecular and morphological analysis. DNA sequences were treated using Mothur software which processed two bioinformatic strategies in order to obtain the final ISU and OTU tables, while ESVs were treated with DADA2 package from R. Identification and counting of diatom valves took place under the light microscope concerning the morphological approach. We expect results to validate the molecular data for each typology either when assigning to species or not, and to understand whether it is necessary to establish new typologies for future use of the molecular approach in ecological assessment of rivers. Directive, W. F. (2000). Water Framework Directive. Journal reference OJL, 327, 1-73. Feio, M. J., Serra, S. R., Mortágua, A., Bouchez, A., Rimet, F., Vasselon, V., & Almeida, S. F. P. (2020). A taxonomy-free approach based on machine learning to assess the quality of rivers with diatoms. Science of the Total Environment, 722, 137900. https://doi.org/10.1016/j.scitotenv.2020.137900 Mortágua, A., Vasselon, V., Oliveira, R., Elias, C., Chardon, C., Bouchez, A., ... & Almeida, S. F. P. (2019). Applicability of DNA metabarcoding approach in the bioassessment of Portuguese rivers using diatoms. Ecological indicators, 106, 105470. https://doi.org/10.1016/j.ecolind.2019.105470 Pérez-Burillo, J., Trobajo, R., Vasselon, V., Rimet, F., Bouchez, A., & Mann, D. G. (2020). Evaluation and sensitivity analysis of diatom DNA metabarcoding for WFD bioassessment of Mediterranean rivers. Science of the Total Environment, 727, 138445. https://doi.org/10.1016/j.scitotenv.2020.138445 Rivera, S. F., Vasselon, V., Bouchez, A., & Rimet, F. (2020). Diatom metabarcoding applied to large scale monitoring networks: Optimization of bioinformatics strategies using Mothur software. Ecological indicators, 109, 105775. https://doi.org/10.1016/j.ecolind.2019.105775