Search

International audience; Little is still known about the low dose effects of radiation on the microbial communities in the environment. Mineral springs are ecosystems than can be affected by natural radioactivity. These extreme environments are, therefore, observatories for studying the influence of chronic radioactivity on the natural biota. In these ecosystems we find diatoms, unicellular microalgae, playing an essential role in the food chain. The present study aimed to investigate, using DNA metabarcoding, the effect of natural radioactivity in two environmental compartments (i.e. spring sediments and water) on the genetic richness, diversity and structure of diatom communities in 16 mineral springs in the Massif Central, France. Diatom biofilms were collected during October 2019, and a 312 bp region of the chloroplast gene rbcL (coding for the Ribulose Bisphosphate Carboxylase) used as a barcode for taxonomic assignation. A total of 565 amplicon sequence variants (ASV) were found. The dominant ASV were associated with Navicula sanctamargaritae, Gedaniella sp., Planothidium frequentissimum, Navicula veneta, Diploneis vacillans, Amphora copulata, Pinnularia brebissonii, Halamphora coffeaeformis, Gomphonema saprophilum, and Nitzschia vitrea, but some of the ASVs could not be assigned at the species level. Pearson correlation failed to show a correlation between ASV’ richness and radioactivity parameters. Non-parametric MANOVA analysis based on ASVs occurrence or abundances revealed that geographical location was the main factor influencing ASVs distribution. Interestingly, 238U was the second factor that explained diatom ASV structure. Among the ASVs in the mineral springs monitored, ASV associated with one of the genetic variants of Planothidium frequentissimum was well represented in the springs and with higher levels of 238U, suggesting its high tolerance to this particular radionuclide. This diatom species may therefore represent a bio-indicator of high natural levels of uranium

Ciliates are unicellular heterotrophic organisms that play a key role in the planktonic and benthic food webs of lakes, and represent a great potential as bioindicator. In this study, we used the top-bottom paleolimnological approach to compare the recent and past (i.e. prior to major anthropogenic impacts) ciliate communities of 48 lakes located along an elevation gradient using metabarcoding techniques applied on sedimentary DNA (sed-DNA). Our results show an overall decline in the β-diversity in recent time, especially in lowland lakes which are more strongly expose to local human pressure. Analyses of the functional groups indicate important restructuration of the trophic food web and changes that are consistent with several well documented environmental changes such as the widespread increase in deep water anoxia, changes in thermal stability and nutrient cycling. Our study demonstrates the potential offered by sed-DNA to uncover information about past ciliate communities on a wide variety of lakes and the potential of using ciliates as valuable indicators, integrating information from the pelagic to the benthic profundal (and littoral) zones. Through trait-based functional community approach, the ciliates provide additional valuable information on ecosystem functioning, thus offering more a holistic view on the long-term changes of aquatic ecosystems.

Ciliates are unicellular heterotrophic organisms that play a key role in aquatic planktonic and benthic food webs. Advances in sedimentary DNA (sed-DNA) analysis offer the possibility to integrate these bioindicators in paleoenvironmental reconstructions. In this study, we used the top–bottom paleolimnological approach and metabarcoding techniques applied to sed-DNA to compare the recent and past (i.e. prior to major anthropogenic impacts) ciliate communities of 48 lakes located along an elevation gradient. Our results show an overall decline in the β-diversity in recent time, especially in lowland lakes, which are more strongly exposed to local human pressures. Analyses of the functional groups indicate important restructuration of the food web, including the recent increase in mixotrophs. Moreover, changes in the benthic ciliates were consistent with the widespread increase in deep water anoxia. Our results provided evidence that sed-DNA can uncover information about past ciliate communities on a wide variety of lakes. Overall, our study demonstrates the potential of using ciliates as new paleoindicators, integrating information from the pelagic to the benthic zones, and providing valuable insights into ecosystem functioning through a trait-based functional community approach. As paleoindicator, they thus offer a more holistic view on the long-term changes of aquatic ecosystems.

L’évaluation de l’état écologique des cours d’eau repose sur le calcul d’indices de qualité basés sur la sensibilité de certains groupes biologiques, dont les diatomées, à la pollution. La détermination et la quantification des espèces de diatomées reposent généralement sur des méthodes d’identification morphologique en microscopie qui peuvent paraître complexes, chronophages et relativement onéreuses. Au cours de la dernière décennie, une nouvelle méthode de biologie moléculaire basée sur l’ADN a été développée, permettant d’identifier les espèces sur la base de critères génétiques plutôt que sur des critères morphologiques : le métabarcoding. En combinaison avec les technologies de séquençage à haut débit, le métabarcoding permet d’identifier l’ensemble des espèces présentes au sein d’un échantillon environnemental et de traiter plusieurs centaines d’échantillons en parallèle. Cet article présente les résultats de deux études récentes menées sur les cours d’eau de Mayotte (2013-2018) et de France métropolitaine (2016-2018), visant à tester le potentiel d’application du métabarcoding pour la bio-indication au sens de la directive cadre sur l’eau (DCE). Nous abordons les différents développements méthodologiques et optimisations qui ont été réalisés pour fiabiliser les inventaires taxonomiques de diatomées produits en métabarcoding, notamment en matière de quantification des espèces basée sur l’abondance relative des séquences ADN. Nous présentons ensuite les résultats d’application de l’approche moléculaire pour l’évaluation de l’état écologique de plus de 500 sites de cours d’eau nationaux, en les confrontant avec les résultats obtenus via l’approche classique en morphologie. Finalement, nous discutons du potentiel d’application en routine du métabarcoding à l’échelle des réseaux de surveillance des cours d’eau, de ses limites d’application et proposons certaines recommandations pour une future implémentation complémentaire à l’approche morphologique actuellement prescrite dans les arrêtés réglementaires.

The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99-100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas.

Recent metabarcoding work in European rivers, including our own studies of Mediterranean rivers in Catalonia (Pérez-Burillo et al. 2020), has shown promising agreement between morphology- (LM) and DNA-based (HTS) assessments of diatom periphyton for the Water Framework Directive. However, in 10 out of the 164 Catalan sites we analysed, the ecological status class was downgraded from “Good”/ “High” with LM to “Moderate”/ “Poor”/ “Bad”by HTS. We call these the “critical” sites and they are especially important because the WFD requires remedial action to be taken by water managers for any river stretch with Moderate or lower status. In order to discover the reasons for downgrading we investigated the contribution of each species to the Indice de Polluosensibilité Spécifique [Specific Pollution-sensitivity Index, IPS] using a “leave-one-out” sensitivity analysis, paying special attention to the critical sites. Discrepancies in IPS between LM and HTS were mainly due to the misidentification and overlooking in LM of a few species, which were better recovered by HTS. This bias was particularly important in the case of Fistulifera saprophila, whose clear underrepresentation in LM was important for explaining 8 out of the 10 critical sites and probably reflected destruction of its very weakly-silicified frustules during sample preparation for LM. Another important bias was brought about by differences between species in the rbcL copy number per cell, which for example affected the relative abundance obtained by HTS for Nitzschia inconspicua and Ulnaria ulna; these species were also identified by the sensitivity analysis as important for the WFD. However, blanket application of a correction factor based on cell volume did not lead to a clear improvement in the agreement between HTS and LM assessments. Only minor IPS discrepancies could be attributed to the incompleteness of the reference library, as most of the abundant and influential species (to the IPS) were well represented there. Finally, we propose that leave-one-out analysis is a valuable method for identifying priority species for isolation and barcoding that are currently not, or only poorly, represented in the DNA reference database.

Environmental DNA (eDNA) based methods (Fig. 1) are 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. Freshwater fish are actively involved in aquatic ecosystems functioning and diversity, contributing to the health, well-being and economy in every geographic realm. Unfortunately, many freshwater fish are experiencing critical population decline with risk of local or global extinction because of intense anthropogenic pressure. Within the EU project Eco-AlpsWater, advanced high throughput sequencing (HTS) techniques are used to improve the traditional WFD monitoring approaches by using environmental DNA (eDNA) collected in Alpine waterbodies. To evaluate the performance of the metabarcoding approach specifically designed to measure freshwater fish biodiversity in Alpine lakes and rivers, an intercalibration test was performed. This exercise forecasted the use of mock samples containing either tissue-extracted DNA of different target species or water collected from aquaculture tanks to mimic real environmental water sampling and processing. Moreover, three water samples collected in Lake Bourget (France) were used to compare the efficiency of taxonomic assignments in natural and mock community samples. Our results highlighted a good efficiency of the molecular laboratory protocols for HTS and a good amplification success of the selected primers, providing essential information concerning the taxonomic resolution of the 12S mitochondrial marker. As further confirmation, different concentration of species DNA in the mock samples were well represented by the relative read abundance. This preliminary test confirmed the applicability of eDNA metabarcoding analyses for the biomonitoring of freshwater fish inhabiting Alpine and perialpine lakes and rivers.

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems. Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring.

This book represents a synthesis of knowledge and best practice in the field of DNA-based biomonitoring at the time of writing. It has been written with end-users of molecular tools in mind, as well as those who are new to the field in research settings and are looking to gain an overall grounding in the subject area. For each of the main types of sample (water, soil / sediment, bulk invertebrates and diatoms), and for each stage of the field and laboratory processes, we outline key considerations, decisions that need to be made, factors that might influence those decisions, and trade-offs inherent in the choices made. We hope that this will help users, practitioners, and those commissioning DNA-based monitoring programmes to navigate this large field and critically evaluate the strengths and weaknesses of different analysis workflows based on context, project aims and available resources., ISBN:978-619-248-052-3, ISBN:978-619-248-053-0

International audience; Fish eDNA metabarcoding is usually performed from filtered water samples. The volume of filtered water depends on the study scope and can rapidly become time consuming according to the number of samples that have to be processed. To avoid time allocated to filtration, passive DNA samplers have been used to recover fish eDNA from marine environments faster. In freshwater ecosystems, aquatic biofilms were used to catch eDNA from macroinvertebrates. Here, we test the capacity of aquatic biofilms to entrap fish eDNA in a large lake and, therefore, the possibility to perform fish eDNA metabarcoding from this matrix compared to the traditional fish eDNA approach from filtered water samples. Methodological aspects of the use of aquatic biofilms for fish eDNA metabarcoding (e.g. PCR replicates, biological replicates, bioinformatics pipeline, reference database and taxonomic assignment) were validated against a mock community. When using biofilms from habitats sheltered from wind and waves, biofilm and water approach provided similar inventories. Richness and diversity were comparable between both approaches. Approaches differed only for rare taxa. Our results illustrate the capacity of aquatic biofilms to act as passive eDNA samplers of fish eDNA and, therefore, the possibility to use biofilms to monitor fish communities efficiently from biofilms. Furthermore, our results open up avenues of research to study a diversity of biological groups (among which bioindicators as diatoms, macroinvertebrates and fish) from eDNA isolated from a single environmental matrix reducing sampling efforts, analysis time and costs.

Diatoms (Bacillariophyta) are ubiquitous microalgae, which present a huge taxonomic diversity, changing in correlation with differing environmental conditions. This makes them excellent ecological indicators for various ecosystems and ecological problematics (ecotoxicology, biomonitoring, paleo-environmental reconstruction …). Current standardized methodologies for diatoms are based on microscopic determinations, which is time consuming and prone to identification uncertainties. DNA metabarcoding has been proposed as a way to avoid these flaws, enabling the sequencing of a large quantity of barcodes from natural samples. A taxonomic identity is given to these barcodes by comparing their sequences to a barcoding reference library. However, to identify environmental sequences correctly, the reference database should contain a representative number of reference sequences to ensure a good coverage of diatom diversity. Moreover, the reference database needs to be carefully taxonomically curated by experts, as its content has an obvious impact on species detection. Diat.barcode is an open-access library for diatoms linking diatom taxonomic identities to rbcL barcode sequences (a chloroplast marker suitable for species-level identification of diatoms), which has been maintained since 2012. Data are accumulated from three sources: (1) the NCBI nucleotide database, (2) unpublished sequencing data of culture collections and more recently (3) environmental sequences. Since 2017, an international network of experts in diatom taxonomy curate this library. The last version of the database (version 9.2), includes 8066 entries that correspond to more than 280 different genera and 1490 different species. In addition to the taxonomic information, morphological features (e.g. biovolumes, chloroplasts, etc.), life-forms (mobility, colony-type) and ecological features (taxa preferences to pollution) are given. The database can be downloaded from the website (www6.inrae.fr/carrtel-collection/Barcoding-database/) or directly through the R package diatbarcode. Ready-to-use files for commonly used metabarcoding pipelines (Mothur and DADA2) are also available.

International audience; Aquatic biofilms are heterogeneous assemblages of microorganisms surrounded by a matrix of extracellular polymeric substances (EPS). Recent studies suggest that aquatic biofilms can physically act as sorptive sponges of DNA. We took the opportunity from already available samples of stone biofilms and macroinvertebrates specimens collected in parallel at the same sites to test the capacity of biofilms to act as DNA samplers of macroinvertebrate communities in streams. Macroinvertebrate communities are usually studied with metabarcoding using the DNA extracted from their bodies bulk samples, which remains a time-consuming approach and involves the destruction of all individual specimens from the samples. The ability of biofilms to capture DNA was explored on 19 rivers sites of a tropical island (Mayotte Island, France). First, macroinvertebrate specimens were identified based on their morphological characteristics. Second, DNA was extracted from biofilms, and macroinvertebrate communities were targeted using a standard COI barcode. The resulting morphological and molecular inventories were compared. They provided comparable structures and diversities for macroinvertebrate communities when one is working with the unassigned OTU data. After taxonomic assignment of the OTU data, diversity and richness were no longer correlated. The ecological assessment derived from morphological bulk samples was conserved by the biofilms samples. We also showed that the biofilm method allows to detect a higher diversity for some organisms (Cnidaria), that is hardly accessible with the morphological method. The results of this study exploring the DNA signal captured by natural biofilms are encouraging. However, a more detailed study integrating more replicates and comparing the biodiversity signal based on both morphological and molecular bulk macroinvertebrate samples to the one captured by biofilms will be necessary. Better understanding how the DNA signal captured by natural biofilms represents the biodiversity of a given sampling site is necessary before considering its use for bioassessment applications.

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data, (undefined)

In freshwater ecosystems, periphytic biofilms include diatom assemblages that depend on environmental conditions (e.g., nutrient concentrations, salinity, temperature etc.). These assemblages respond rapidly to environmental changes, which makes diatoms valuable bioindicators. For this reason, they are currently used in freshwater biomonitoring programs (e.g., EU Water Framework Directive - WFD) (Foster et al., 2000). To date, diatom taxonomic identification is based on morphological criteria, which requires high taxonomic expertise to identify them to the species level needed for biomonitoring. Having this in mind, new strategies have been examined for the development of high-throughput, non-biased identification approaches. Human activities are the leading cause of environmental impairments and appropriate biomonitoring of ecosystems is needed to effectively assess the impact of their activities. In the last ten years, DNA metabarcoding combined with next-generation sequencing and bioinformatics, have been proposed as a complementary approach to morphological identification. In the past ten years, DNA metabarcoding coupled with next-generation sequencing and bioinformatics represents a complementary approach for diatom biomonitoring (Vasselon et al., 2019). In this study, this approach was used for the first time in Cyprus considering the association of environmental and anthropogenic pressures to diatom assemblages using the rbcL 312 bp barcode, next-generation sequencing (MiSeq Illumina), and bioinformatic evaluation (Mothur Software). Statistical analysis was then applied to identify the environmental (i.e., river types, geo-morphological) and anthropogenic (i.e., physical, chemical, human land-use pressures) variables' role in the observed diatom diversity. The Indice de Polluosensibilité Spécifique (IPS) index was used as it was shown to better respond to pressures that affect water quality in Cyprus rivers (WDD, 2014). Results indicate differences in diatom assemblages between intermittent and perennial rivers. Achnanthidium minutissimum was more abundant in intermittent rivers; whereas Amphora pediculus and Planothidium victorii (P. caputium) in perennial ones. Furthermore, we could demonstrate the correlation between nutrients (e.g., nitrogen, phosphorus), characteristics of the individual sampling sites (e.g., elevation), and land use activities on the observed differences in diatom diversity (Pissaridou, 2021). Additionally, results were compared to the morphotaxonomy-based approach which was conducted microscopically. Results show a positive correlation between morphological and molecular IPS scores. Points deviating from the norm are influenced by the limitations of both techniques. Fistulifera saprophila had a key role in this observation, as it negatively influences IPS scores. All in all, we conclude that DNA metabarcoding complements the morphological methodology for the ecological quality assessment of freshwaters in Cyprus. Multi-stressors and anthropogenic pressures have a significant statistical relationship to the observed diatom diversity and play a pivotal role in determining Cyprus' rivers' ecological status (Fig. 1). Foster, D., Wood, A., Griffiths, M., 2000. The Water Framework Directive (2000/60/EC) – An introduction Dave Foster – Policy Advisor (Europe), Aram Wood EP Scientist (Water), Dr Martin Griffiths – Head of Water Quality, Environment Agency, Head Office, Rio House, Waterside Drive, Aztec West, Almon 7–9. Pissaridou, P., Vasselon V., Christou A., Chonova T., Lacroix S., Papatheodoulou A., Drakou K., Tziortzis I., Dörflinger G., Rimet F., Bouchez A. and Vasquez MI. 2021 Deciphering Cyprus’ diatom diversity and the effects of environmental and anthropogenic influences for ecological assessment of rivers using DNA metabarcoding.Chemosphere (In Press) Vasselon, V., Frédéric, R., Isabelle, D., Olivier, M., Yorick, R., Agnès, B., 2019. Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: Experience and developments from France Water Framework Directive networks. Metabarcoding and Metagenomics 3, 101–115. https://doi.org/10.3897/mbmg.3.39646 WDD, 2014. Review and update of article 5 of Directive 2000/60/EC (Water reservoirs) & Classification of water status (Rivers, natural lakes and water reservoirs), That will establish baseline information and data for the 2nd cyprus river basin management plan.

A wild river is a living river, which is at baseline, well-preserved, and which runs freely and is home to a rich biodiversity in its high quality waters and on its banks. In Europe there are very few rivers which could be considered “wild”, which function at a high ecological level, since wild rivers, in the true sense of the term, no longer exist. Based on the fact that these rivers remain threatened, and that the existing tools (technical, regulatory, and financial) are insufficient and not adapted to ensure their preservation over the long term, the Wild Rivers project was founded in 2007, through a meeting of environmental defenders, scientists, fishermen, managers of land and river natural resources, and elected officials, all of whom were anxious to save the last of the French rivers which were still preserved, with a human impact that would be compatible with the conservation of the ecosystem. In 2014 the “Wild Rivers Site” label was created in France, as a conservation tool for rivers, both voluntary and non-regulatory, which allows the support necessary to enable the territorial players to preserve their rivers in harmony with the activity in the surrounding valleys. It also identifies and highlights these unique watercourses. The Valserine in the Ain region was the first river to obtain the Wild Rivers Site label. Today 28 rivers in France are labelled “Wild Rivers Sites” and the 22 management structures of these rivers are members of the Wild Rivers Site Network. To obtain the label, a river must fulfill two sets of criteria 1. The criteria grid: The watercourse must obtain a mark over 70/100. The grid is composed of 47 criteria evaluating the quality of the area, of which 12 are eliminatory, 8 are unrated, and 9 are under a bonus/penalty scheme 2. The program of actions taken by local players: The local managers must put in place a system of governance built around actions to be taken over a period of years, shared among them, and ambitious, going beyond the regulatory objectives of the European Directive Framework. It allows for the restoration of penalty points and the establishment of innovative conservation activities. The Wild Rivers Sites are also an open air laboratory for the development and use of innovative methods in order to provide new information on aquatic environments, and to improve their management and conservation. Numerous steps have already been taken within the network, such as the Ecosystem Services Study (Costa and Hernandez 2019); on the study of the genetic makeup of the brown trout population. Recently, the use of genetic study using environmental DNA to complete biodiversity inventories has also been deployed to study benthic diatoms (DNA of Diatoms Project 2020-2022). This project seeks to use DNA metabarcoding to respond to a number of objectives: i) inventory of the species of diatoms and their community structure in these watercourses which are generally seldom studied; ii) complete ecological status studies; iii) develop new genetic metrics and taxonomies adapted to the conservation of wild river watercourses. It is in this spirit that the Wild Rivers program was developed, and has received numerous positive responses on the behalf of watercourse management in France. Thanks to this impetus, work has been conducted to extend this conservation label to water sources in other countries (Switzerland, Ireland, Spain), with the future plan of building a European network dedicated to the conservation of Wild Rivers.

Diatoms, macroinvertebrates and fish communities are widely used for the assessment of the ecological status of rivers and lakes. Metabarcoding studies of these communities are usually performed from “bulk” samples in the case of diatoms and macroinvertebrates; and from water samples in the case of fish. Recent studies, suggest that aquatic biofilms can physically act as environmental catchers of environmental DNA (eDNA) (e.g. Mariani et al. 2019). Thus, we propose an alternative metabarcoding approach to study macroinvertebrates and fishes directly from this matrix. The capacity of aquatic biofilms to catch macroinvertebrate eDNA was tested from a previous study in Mayotte Island were both biofilm samples and macroinvertebrate morphological inventories were available at same river sites (Rivera et al. 2021). First, macroinvertebrate specimens were identified based on their morphological characteristics. Second, DNA was extracted from biofilms, and macroinvertebrate communities were targeted using a standard COI barcode. The resulting morphological and molecular inventories were compared. Our results showed that both methods provided comparable structures and diversities for macroinvertebrate communities when using unassigned OTUs suggesting that macroinvertebrate DNA is present in biofilms and representative of the communities. However, after taxonomic assignment of OTUs, diversity and richness were no longer correlated. Indeed, many constraints were observed as the need for: a) more specific primers to avoid co-amplification of untargeted taxa inhabiting biofilms, b) primers targeting shorter barcodes to sequence more easily degraded eDNA that may be captured in biofilms, and c) a reference database well adapted to our tropical study sites. Finally, even if the results of this first study were encouraging, we wanted to test the biofilm approach on organisms that do not inhabit this environmental matrix in order to be able to distinguish between intra or extra-cellular DNA. Based on these observations, a second study looking for a fish eDNA signal in aquatic biofilms was performed. Environmental biofilm and water samples were collected in parallel at littoral sites at Lake Geneva. DNA was extracted from these samples, and fish communities were targeted using a standard 12S barcode. The molecular inventories derived from the biofilm and the water samples were compared. Both methods provide comparable floristic lists, providing a novel approach for ecological studies related to fish phenology using eDNA in biofilms. Our results open the door to the study of diatoms, macroinvertebrates and fish communities through metabarcoding from a single matrix reducing sampling efforts and costs.

International audience; Human activities are the leading cause of environmental impairments. Appropriate biomonitoring of ecosystems is needed to assess these activities effectively. In freshwater ecosystems, periphytic and epilithic biofilms have diatom assemblages. These assemblages respond rapidly to environmental changes, making diatoms valuable bioindicators. For this reason, freshwater biomonitoring programs are currently using diatoms (e.g., Water Framework Directive). In the past ten years, DNA metabarcoding coupled with next-generation sequencing and bioinformatics represents a complementary approach for diatom biomonitoring. In this study, this approach is used for the first time in Cyprus by considering the association of environmental and anthropogenic pressures to diatom assemblages. Statistical analysis was then applied to identify the environmental (i.e., river types, geo-morphological) and anthropogenic (i.e., physicochemical, human land-use pressures) variables' role in the observed diatom diversity. Results indicate differences in diatom assemblages between intermittent and perennial rivers. Achnanthidium minutissimum was more abundant in intermittent rivers; whereas Amphora pediculus and Planothidium caputium in perennial ones. Additionally, we could demonstrate the correlation between nutrients (e.g., nitrogen, phosphorus), stations' local characteristics (e.g., elevation), and land use activities on the observed differences in diatom diversity. Finally, we conclude that multi-stressors and anthropogenic pressures together as multiple stressors have a significant statistical relationship to the observed diatom diversity and play a pivotal role in determining Cyprus' rivers' ecological status.

This protocol is part of the DNA workflow applied in the Eco-ALpsWater Project, here in particular to characterize the diversity of diatom assemblage in biofilms or plankton samples. Different studies have already revealed the potential of diatom metabarcoding applications for freshwater quality assessment (Kermarrec et al. 2014; Vasselon et al. 2017ab; Visco et al. 2015). The choice of the marker gene and barcode region is key for obtaining relevant inventories of diversity and precise taxonomic assignment. For benthic diatoms, the rbcL gene has proved to be an appropriate taxonomic marker for biomonitoring (Kermarrec et al. 2013, 2014; Vasselon et al. 2017a,b) and a well‐curated barcode reference library is already available to assign species names to rbcL sequences (R‐Syst::diatom, Rimet et al. 2016). For the Eco-AlpsWater project, biolfilms sampled in rivers and lakeshores are collected as described in the dedicated protocols (“Lake plankton sample collection ...” and “Biofilms sample collection ...”) and DNA is extracted as described in the protocol “Biofilms DNA extraction” ; all these protocols are part of the Deliverable D.T1.1.2. We present here the following step in the DNA workflow (i.e. PCR amplification of selected barcodes, and wet lab methods to prepare DNA library for downstream MiSeq Sequencing). This protocol has been used in recent studies (e.g. Keck et al 2018 ; Vasselon et al 2018) where diatoms metabarcoding has been used for ecological assessment of rivers. Several primers were proposed in the literature to characterize Diatom communities through environmental DNA metabarcoding approaches, including the 18S, COI and rbcL barcodes. Following the recommendation provided by Kermarrec et al. 2014, who compared the efficiency of those 3 barcodes to accurately characterize diatom communities from freshwater samples (lakes and rivers), the rbcL barcode will be used within the Eco-AlpsWater project as he provides a good taxonomical resolution.

This protocol is part of the DNA workflow applied in the Eco-AlpsWater Project. The methodological step described here is the extraction of DNA, this is a critical step for obtaining relevant results since molecular inventories might be influenced by the DNA extraction method used. The choice of the methodology for biofilms DNA extraction is based on previous studies and in particular on the work done by Vasselon et al. (2017) where the authors have tested 5 DNA extraction methods combining various types of cell lysis and DNA purification to extract DNA (from pure diatom cultures and biofilms samples from streams and lakes). For the Eco-AlpsWater project, after being sampled in lakes or rivers, biofilms are stored in 50 mL tubes in ethanol at 4°C, and for a maximum of 3 months before DNA extraction (the extraction should preferably be done in the month following the sampling). The DNA extraction protocol presented below has been used in several recent studies (e.g. Vasselon et al 2017ab, 2018) focussed on the application of diatoms metabarcoding; this extraction is based on a protocol adapted from the NucleoSpin® Soil kit (MACHEREY-NAGEL) with specific modifications for biofilm DNA extraction.

International audience; Recently, several studies demonstrated the usefulness of diatom eDNA metabarcoding as an alternative to assess the ecological quality of rivers and streams. However, the choice of the taxonomic marker as well as the methodology for data analysis differ between these studies, hampering the comparison of their results and effectiveness. The aim of this study was to compare two taxonomic markers commonly used in diatom metabarcoding and three distinct analytical approaches to infer a molecular diatom index. We used the values of classical morphological diatom index as a benchmark for this comparison. We amplified and sequenced both a fragment of the rbcL gene and the V4 region of the 18S rRNA gene for 112 epilithic samples from Swiss and French rivers. We inferred index values using three analytical approaches: by computing it directly from taxonomically assigned sequences, by calibrating de novo the ecovalues of all metabarcodes, and by using a supervised machine learning algorithm to train predictive models. In general, the values of index obtained using the two "taxonomy-free" approaches, encompassing molecular assignment and machine learning, were closer correlated to the values of the morphological index than the values based on taxonomically assigned sequences. The correlations of the three analytical approaches were higher in the case of rbcL compared to the 18S marker, highlighting the importance of the reference database which is more complete for the rbcL marker. Our study confirms the effectiveness of diatom metabarcoding as an operational tool for rivers ecological quality assessment and shows that the analytical approaches by-passing the taxonomic assignments are particularly efficient when reference databases are incomplete.

Our study of 164 diatom samples from Catalonia (NE Spain) is the first to evaluate the applicability of DNA metabarcoding, based on high throughput sequencing (HTS) using a 312-bp rbcL marker, for biomonitoring Mediterranean rivers. For this, we compared the values of a biotic index (IPS) and the ecological status classes derived from them, between light microscope-based (LM) and HTS methods. Very good correspondence between methods gives encouraging results concerning the applicability of DNA metabarcoding for Catalan rivers for the EU Water Framework Directive (WFD). However, in 10 sites, the ecological status class was downgraded from “Good”/“High” obtained by LM to “Moderate”/“Poor”/“Bad” by HTS; these “critical” sites are especially important, because the WFD requires remedial action by water managers for any river with Moderate or lower status. We investigated the contribution of each species to the IPS using a “leave-one-out” sensitivity analysis, paying special attention to critical sites. Discrepancies in IPS between LM and HTS were mainly due to the misidentification and overlooking in LM of a few species, which were better recovered by HTS. This bias was particularly important in the case of Fistulifera saprophila, whose clear underrepresentation in LM was important for explaining 8 out of the 10 critical sites and probably reflected destruction of weakly-silicified frustules during sample preparation. Differences between species in the rbcL copy number per cell affected the relative abundance obtained by HTS for Achnanthidium minutissimum, Nitzschia inconspicua and Ulnaria ulna, which were also identified by the sensitivity analysis as important for the WFD. Only minor IPS discrepancies were attributed to the incompleteness of the reference library, as most of the abundant and influential species (to the IPS) were well represented there. Finally, we propose that leave-one-out analysis is a good method for identifying priority species for isolation and barcoding. info:eu-repo/semantics/acceptedVersion

DNA metabarcoding has been introduced as a revolutionary way to identify organisms and monitor ecosystems. However, the potential of this approach for biomonitoring remains partially unfulfilled because a significant part of the sampled DNA cannot be affiliated to species due to incomplete reference libraries. Thus, biotic indices, which are based on the estimated abundances of species in a community and their ecological profiles, can be inaccurate. We propose to compute biotic indices using phylogenetic imputation of operational taxonomic units (OTUs') ecological profiles (OTU-PITI approach). First, OTUs sequences are inserted within a reference phylogeny. Second, OTUs' ecological profiles are estimated on the basis of their phylogenetic relationships with reference species whose ecology is known. Based on these ecological profiles, biotic indices can be computed using all available OTUs. Using freshwater diatoms as a case study, we show that short DNA barcodes can be placed accurately within a phylogeny and their ecological preferences estimated with a satisfactory level of precision. In the light of these results, we tested the approach with a data set of 139 environmental samples of benthic river diatoms for which the same biotic index (specific sensitivity index) was calculated using (a) traditional microscopy, (b) OTUs with taxonomic assignment approach, (c) OTUs with phylogenetic estimation of ecological profiles (OTU-PITI) and (d) OTU with taxonomic assignment completed by the phylogenetic approach (OTU-PITI) for unclassified OTUs. Using traditional microscopy as a reference, we found that the combination of the OTUs' taxonomic assignment completed by the phylogenetic method performed satisfactorily and substantially better than the other methods tested.

International audience; Diatoms are a compulsory biological quality element in the ecological assessment of rivers according to the Water Framework Directive. The application of current official indices requires the identification of individuals to species or lower rank under a microscope based on the valve morphology. This is a highly time-consuming task, often susceptible of disagreements among analysts. In alternative, the use of DNA metabarcoding combined with High-Throughput Sequencing (HTS) has been proposed. The sequences obtained from environmental DNA are clustered into Operational Taxonomic Units (OTUs), which can be assigned to a taxon using reference databases, and from there calculate biotic indices. However, there is still a high percentage of unassigned OTUs to species due to the incompleteness of reference libraries. Alternatively, we tested a new taxonomy-free approach based on diatom community samples to assess rivers. A combination of three machine learning techniques is used to build models that predict diatom OTUs expected in test sites, under reference conditions, from environmental data. The Observed/Expected OTUs ratio indicates the deviation from reference condition and is converted into a quality class. This approach was never used with diatoms neither with OTUs data. To evaluate its efficiency, we built a model based on OTUs lists (HYDGEN) and another based on taxa lists from morphological identification (HYDMORPH), and also calculated a biotic index (IPS). The models were trained and tested with data from 81 sites (44 reference sites) from central Portugal. Both models were considered accurate (linear regression for Observed and Expected richness: R2 ≈ 0.7, interception ≈ 0.8) and sensitive to global anthropogenic disturbance (Rs2 > 0.30 p < 0.006 for global disturbance). Yet, the HYDGEN model based on molecular data was sensitive to more types of pressures (such as, changes in land use and habitat quality), which gives promising insights to its use for bioassessment of rivers.

This report presents the outcome of the joint work of PhD students and senior researchers working with DNA-based biodiversity assessment approaches with the goal to facilitate others the access to definitions and explanations about novel DNA-based methods. The work was performed during a PhD course (SLU PNS0169) at the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden. The course was co-organized by the EU COST research network DNAqua-Net and the SLU Research Schools Focus on Soils and Water (FoSW) and Ecology - basics and applications. DNAqua-Net (COST Action CA15219, 2016-2020) is a network connecting researchers, water managers, politicians and other stakeholders with the aim to develop new genetic tools for bioassessment of aquatic ecosystems in Europe and beyond. The PhD course offered a comprehensive overview of the paradigm shift from traditional morphology-based species identification to novel identification approaches based on molecular markers. We covered the use of molecular tools in both basic research and applied use with a focus on aquatic ecosystem assessment, from species collection to the use of diversity in environmental legislation. The focus of the course was on DNA (meta)barcoding and aquatic organisms. The knowledge gained was shared with the general public by creating Wikipedia pages and through this collaborative Open Access publication, co-authored by all course participants.

International audience; Freshwater ecosystems are continuously affected by anthropogenic pressure. One of the main sources of contamination comes from wastewater treatment plant (WWTP) effluents that contain wide range of micro- and macropollutants. Chemical composition, toxicity levels and impact of treated effluents (TEs) on the recipient aquatic ecosystems may strongly differ depending on the wastewater origin. Compared to urban TEs, hospital ones may contain more active pharmaceutical substances. Benthic diatoms are relevant ecological indicators because of their high species and ecological diversity and rapid response to human pressure. They are routinely used for water quality monitoring. However, there is a knowledge gap on diatom communities' development and behavior in treated wastewater in relation to prevailing micro- and macropollutants. In this study, we aim to (1) investigate the response of diatom communities to urban and hospital TEs, and (2) evaluate TEs effect on communities in the recipient river. Environmental biofilms were colonized in TEs and the recipient river up- and downstream from the WWTP output to study benthic diatoms using DNA metabarcoding combined with high-throughput sequencing (HTS). In parallel, concentrations of nutrients, pharmaceuticals and seasonal conditions were recorded. Diatom metabarcoding showed that benthic communities differed strongly in their diversity and structure depending on the habitat. TE sites were generally dominated by few genera with polysaprobic preferences belonging to the motile guild, while river sites favored diverse communities from oligotrophic and oligosaprobic groups. Seasonal changes were visible to lower extent. To categorize parameters important for diatom changes we performed redundancy analysis which suggested that communities within TE sites were associated to higher concentrations of beta-blockers and non-steroidal anti-inflammatory drugs in urban effluents vs. antibiotics and orthophosphate in hospital effluents. Furthermore, indicator species analysis showed that 27% of OTUs detected in river downstream communities were indicator for urban or hospital TE sites and were absent in the river upstream. Finally, biological diatom index (BDI) calculated to evaluate the ecological status of the recipient river suggested water quality decrease linked to the release of TEs. Thus, in-depth assessment of diatom community composition using DNA metabarcoding is proposed as a promising technique to highlight the disturbing effect of pollutants in Alpine rivers.

Diatoms (Bacillariophyta) are ubiquitous microalgae which produce a siliceous exoskeleton and which make a major contribution to the productivity of oceans and freshwaters. They display a huge diversity, which makes them excellent ecological indicators of aquatic ecosystems. Usually, diatoms are identified using characteristics of their exoskeleton morphology. DNA-barcoding is an alternative to this and the use of High-Throughput-Sequencing enables the rapid analysis of many environmental samples at a lower cost than analyses under microscope. However, to identify environmental sequences correctly, an expertly curated reference library is needed. Several curated libraries for protists exists; none, however are dedicated to diatoms. Diat.barcode is an open-access library dedicated to diatoms which has been maintained since 2012. Data come from two sources (1) the NCBI nucleotide database and (2) unpublished sequencing data of culture collections. Since 2017, several experts have collaborated to curate this library for rbcL, a chloroplast marker suitable for species-level identification of diatoms. For the latest version of the database (version 7), 605 of the 3482 taxonomical names originally assigned by the authors of the rbcL sequences were modified after curation. The database is accessible at https://www6.inra.fr/carrtel-collection_eng/Barcoding-database.

International audience; Extensive studies on the taxonomic resolution required for bioassessment purposes have determined that resolution above species level (genus, family) is sufficient for their use as indicators of relevant environmental pressures. The high‐throughput se - quencing (HTS) and meta‐barcoding methods now used for bioassessment tradition - ally employ an arbitrary sequence similarity threshold (SST) around 95% or 97% to cluster sequences into operational taxonomic units, which is considered descriptive of species‐level resolution. In this study, we analyzed the effect of the SST on the resulting diatom‐based ecological quality index, which is based on OTU abundance distribution along a defined environmental gradient, ideally avoiding taxonomic as - signments that could result in high rates of unclassified OTUs and biased final values. A total of 90 biofilm samples were collected in 2014 and 2015 from 51 stream sites on Mayotte Island in parallel with measures of relevant physical and chemical param - eters. HTS sequencing was performed on the biofilms using the rbcL region as the genetic marker and diatom‐specific primers. Hierarchical clustering was used to group sequences into OTUs using 20 experimental SST levels (80%–99%). An OTU‐ based quality index (Idx OTU ) was developed based on a weighted average equation using the abundance profiles of the OTUs. The developed Idx OTU revealed significant correlations between the Idx OTU values and the reference pressure gradient, which reached maximal performance using an SST of 90% (well above species level delimi - tation). We observed an interesting and important trade‐off with the power to dis - criminate between sampling sites and index stability that will greatly inform future applications of the index. Taken together, the results from this study detail a thor - oughly optimized and validated approach to generating robust, reproducible, and complete indexes that will greatly facilitate effective and efficient environmental monitoring

Benthic diatoms are routinely used as ecological indicators in rivers. A standardized methodology is based on biofilm sampling, species identification, and counting under microscope. DNA-metabarcoding is an alternative methodology that can identify species and assess their proportion based on high-throughput DNA sequencing. Sequence data is analyzed with bioinformatics tools, and several strategies can be chosen. The strategy choice can affect communities composition and structure, and therefore the resulting ecological assessment. We wanted to optimize the bioinformatics strategy to obtain the closest results to microscopy. This was done in the framework of the Mothur pipeline. Here, 447 samples from French rivers were analyzed in the monitoring context of the European Water Framework Directive. Samples were analyzed both with DNA metabarcoding and microscopy. A usual bioinformatics strategy in Mothur includes clustering DNA-sequences into Operational Taxonomic Units (OTUs). Different algorithms exist for this. From a subsample of 142 samples, we showed that some strategies (Furthest neighbor) gave closer results to microscopy than others (Opticlust) in terms of community structure and diatom index values. However, we showed that OTU clustering was not necessary for ecological monitoring: Direct taxonomic assignment of individual sequence units (ISU) gave similar results to those obtained in microscopy. Interestingly, direct assignment enabled the detection of more species 2 to 3 times faster in terms of computation time compared to the OTU strategy. However, it remained important to remove low quality and chimeric sequences; if not, biomonitoring results differed greatly from microscopy. We showed that it was preferable to have a loose taxonomical identification threshold instead of a stringent one. This allowed detecting more species, which could participate in the index calculation and increased its performance. Indeed, in diatoms, phylogenetically neighbor species often have similar ecologies, and this explains why it is preferable, in a biomonitoring framework, to identify more species with less stringency instead of identifying few species with stringency. Finally, the best strategy (direct assignment of filtered ISU with a loose taxonomical threshold of 60%) was applied to the 447 samples covering a large diversity of ecological qualities. These data were then used to produce quality index values, using a quantification correction factor taking into account species biovolumes. Compared to microscopy, the DNA-based method assigned the same quality class for 66% of the samples, and 72% of the samples had an index value (ranging from 0 to 20) with less than one point difference from microscopy.

The work was done in collaboration with V. Lecomte (GRAIE). We thank B. Montuelle, F. Rimet, R. Sommaruga, and anonymous reviewers for their constructive comments on the manuscript.; International audience; Wastewater treatment plants (WWTP) are the main sources of a broad spectrum of pharmaceuticals found in freshwater ecosystems. These pollutants raise environmental health concerns because of their highly bioactive nature and their chronic releases. Despite this, pharmaceuticals' effects on aquatic environments are poorly defined. Biofilms represent a major part of the microbial life in rivers and streams. They can drive key metabolic cycles and their organizations reflect exposures to changing chemical, physical, and biological constraints. This study estimated the concentrations, over a 3-year period, of ten pharmaceuticals and five nutrients in a river contaminated by a conventional WWTP fed by urban and hospital wastewaters. Variations in these concentrations were related to biofilm bacterial community dynamics. Rock biofilms had developed over defined periods and were harvested at four locations in the river from the up- and downstream WWTP discharge point. Pharmaceuticals were found in all locations in concentrations ranging from not being detected to 192 ng L-1. Despite the high dilution factor of the WWTP effluents by the receiving river, pharmaceuticals were found more concentrated downstream than upstream the WWTP. Shifts in bacterial community structures linked to the environmental emission of pharmaceuticals were superior to seasonal community changes. A community structure from a site located downstream but close to the WWTP was more strongly associated with high pharmaceutical loads and different from those of biofilm samples from the WWTP upstream or far downstream sites. These latter sites were more strongly associated with high nutrient contents. Low environmental concentrations of pharmaceuticals can thus be transferred from WWTP effluents to a connected stream and induce bacterial aquatic community changes over time.

International audience; Diatoms are a species-diverse phylum of microalgae often presenting high biomass in aquatic habitats. This makes them excellent ecological indicators in rivers and lakes. They are routinely used to assess ecological quality of rivers and lakes using microscopy, which is time consuming. An alternative is to determine species in samples based on short DNA barcodes and high-throughput sequencing (HTS). Former studies showed that community structure and water quality assessments based on diatoms deliver similar results with both methods. But, none evaluated if diversities were assessed in the same way despite the importance of this ecological metric. Based on littoral benthic samplings carried out in 56 pristine alpine lakes, we compared different diversity indices measured with microscopy and metabarcoding. Each lake was sampled in three different places of its littoral. We showed that α (diversity measured in a single sampling site of a given lake) and ϒ (total diversity in a lake where three independent samples were considered) diversities obtained with HTS were higher than those obtained with microscopy. This may be explained by the capacity of HTS to detect morphologically cryptic species and to better detect rare taxa. On the other hand, β diversity obtained with HTS was smaller, which may be explained by the capacity of HTS to detect very rare species and free-floating extracellular DNA. Nevertheless, diversity indices obtained with both methodologies were well correlated each other. This study validates the possibility to assess diatom diversity with HTS in a comparable way to the classical microscopic analysis.

Diatoms are used routinely to assess pollution level in rivers and lakes. Current methods are based on identification by light microscopy, which is laborious. An alternative is to identify species based on short DNA fragments and High-Throughput Sequencing (HTS). However a potential limitation is the incomplete coverage of species in reference barcode libraries. Usually these libraries are compiled by isolating cells, before culturing and sequencing them, which is tedious and often unsuccessful. Here we propose the use of rbcL sequences from environmental samples analysed by HTS. We set several criteria to ensure good sequence quality and correspondence with the target species observed in microscopy: the sequence needed to be abundant in the sample, and with no insertions nor deletions or stop codon, phylogenetic neighbour taxa had to correspond to neighbour taxonomic taxa expected from morphological observations. Four species from tropical rivers are given as examples, including one that is new to science. info:eu-repo/semantics/publishedVersion

International audience; Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior.

International audience; 1. In recent years, remarkable progress has been made in developing environmental DNA metabarcoding. However, its ability to quantify species relative abundance remains uncertain, limiting its application for biomonitoring. In diatoms, although the rbcL gene appears to be a suitable barcode for diatoms, providing relevant qualitative data to describe taxonomic composition, improvement of species quantifica-tion is still required. 2. Here, we hypothesized that rbcL copy number is correlated with diatom cell biovol-ume (as previously described for the 18S gene) and that a correction factor (CF) based on cell biovolume should be applied to improve taxa quantification. We carried out a laboratory experiment using pure cultures of eight diatom species with contrasted cell biovolumes in order to (1) verify the relationship between rbcL copy numbers (estimated by qPCR) and diatom cell biovolumes and (2) define a potential CF. In order to evaluate CF efficiency, five mock communities were created by mixing different amounts of DNA from the eight species, and were sequenced using HTS and targeting the same rbcL barcode. 3. As expected, the correction of DNA reads proportions by the CF improved the congruence between morphological and molecular inventories. Final validation of the CF was obtained on environmental samples (metabarcoding data from 80 ben-thic biofilms) for which the application of CF allowed differences between molecular and morphological water quality indices to be reduced by 47%. 4. Overall, our results highlight the usefulness of applying a CF factor, which is effective in reducing over-estimation of high biovolume species, correcting quantitative biases in diatom metabarcoding studies and improving final water quality assessment. K E Y W O R D S benthic diatom, biovolume correction factor, freshwater ecosystems, gene copy number variation, quantitative metabarcoding