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Environmental filtering is often found to dominate assembly rules in diatoms. These microalgae are diverse, especially at subspecies level, and tend to exhibit a niche phylogenetic conservatism. Therefore, other rules, such as competition or mass effects, should be detectable when environmental gradients and dispersal barriers are limited. We used metabarcoding to analyse benthic littoral diatom communities in 153 sites in a large lake (Geneva) exhibiting weak geographical barriers and weak environmental gradients outside river estuaries. We assessed assembly rules using variance partitioning, phylogenetic and source tracking analyses. No phylogenetic over-dispersion of communities, indicative of exclusive competition, was detected. Instead, we found these communities to be phylogenetically over-clustered, indicating environmental filtering, which was even stronger near river estuaries where environmental gradients are stronger. Finally, using a Bayesian method (SourceTracker), we found that rivers flowing into the lake bring communities that settle, especially in sites close to estuaries. Rivers with the highest discharges are primarily responsible for immigration, explaining 27% of lake composition. Therefore, despite favourable conditions to observe other rules, our results support that diatom communities are prominently assembled by environmental filtering and immigration processes, in particular from rivers. However, this does not exclude that other assembly rules may be at play at a finer spatial, temporal and/or phylogenetic scale.

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

The study of environmental DNA released by aquatic organisms in their habitat offers a fast, non-invasive and sensitive approach to monitor their presence. Common eDNA sampling methods such as filtration and precipitation are time consuming, require human intervention and are not applicable to a wide range of habitats such as turbid waters and poorly-accessible environments. To circumvent these limitations, we propose to use the binding properties of minerals to create a passive eDNA sampler.We have designed 3D-printed samplers made of hydroxyapatite (HAp samplers), a mineral known for its high binding affinity with DNA. The shape and the geometry of the samplers have been designed to facilitate their handling in laboratory and field. Here we describe and test the ability of HAp samplers to recover artificial DNA and eDNA.We show that HAp samplers efficiently recover DNA and are effective even on small amounts of eDNA (By better understanding the physico-chemical interactions between DNA and the HAp sampler surface, one could improve the repeatability of the sampling process and provide an easy-to-use eDNA sampling tool for aquatic environments.

Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2021.151783, Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring., The work is the result of the DNAqua-Net workshop held in Rome, on June 10–12, 2019, organised by IRSA-CNR and supported by European Cooperation in Science and Technology (COST) grant CA15219 (DNAqua-Net). It largely benefited from the discussions with DNAqua-Net members during this event as well as other DNAqua-Net conferences.This study was carried out within the UNIVPM VIRIDE Project (n. 2017EKFA98) funded by the Ministry of University and Research of Italy (MUR) and the Italian National Project on the use of environmental DNA for the identification of marine non-indigenous species funded by the Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA). JP, TC, and LAPG were supported by the Swiss National Science Foundation (grant 31003A_179125) as well as FL with an Early Postdoc.Mobility grant (P2GEP3_171829). MS-M was supported by the Ministry of Education, Youth and Sports of the Czech Republic, European Regional Development Fund-Project CZ.02.1.01/0.0/0.0/16_019/0000845 and LTC 20063. KP was supported by the European Regional Development Fund within National Programme for Addressing Socio-Economic Challenges through R&D (L190003VLVI RITA1/02-60-05) and base financed project P190250PKKH of Estonian University of Life Sciences, IM was supported by the Slovenian Research Agency (ARRS) programme P4-0085. SD was supported by the Portuguese Foundation for Science and Technology (FCT) (CEECIND/00667/2017). LC was supported by the PON AIM 1854833 – PON Ricerca e Innovazione 2014–2020 – Azione I.2 – D.D. n. 407, 27/02/2018 – Attraction and International Mobility.VK was supported by grants PUT1389 (ETAg) and Institute of Technology, University of Tartu basic funding grant.

International audience; Herbicides are well known for unintended effects on freshwater periphyton communities. Large knowledge gaps, however, exist regarding indirect herbicide impacts on primary consumers through changes in the quality of periphyton as a food source (i.e., diet-related effects). To address this gap, the grazer Physella acuta (Gastropoda) was fed for 21 days with periphyton that grew for 15 days in the presence or absence of the herbicide diuron (8 mu g/L) to quantify changes in the feeding rate, growth rate, and energy storage (neutral lipid fatty acids; NLFAs) of P. acuta. Periphyton biomass, cell viability, community structure, and FAs served as proxies for food quality that support a mechanistic interpretation of the grazers' responses. Diuron changed the algae periphyton community and fatty acid profiles, indicating alterations in the food quality, which could explain differences in the snails' feeding rate compared to the control. While the snails' growth rate was, despite an effect size of 55%, not statistically significantly changed, NLFA profiles of P. acuta were altered. These results indicate that herbicides can change the food quality of periphyton by shifts in the algae composition, which may affect the physiology of grazers.

This paper explores the diversity and taxonomy of species within Fragilaria sensu stricto, an abundant and ecologically important diatom genus, taking advantage of cultured and DNA ‐barcoded material. The goal is to facilitate the identification of European taxa within this complex, providing a unified view on morphological and molecular diversity. There is a general agreement that the separation of species within the group of Fragilaria is difficult because morphological descriptions of species are not consistent between authorities, ongoing taxonomic revisions have resulted in species described with standards of the late 20th and 21st centuries alongside descriptions based on 19th century (light microscopical) criteria, and because not all diagnostic characters can be seen in all specimens encountered in routine analyses. Consequent confusion could blur potentially important ecological distinctions between species. Our study demonstrated that some species defined on morphological criteria could be confirmed using the rbc L chloroplast gene as a genetic marker, for example, Fragilaria gracilis , Fragilaria tenera , Fragilaria perminuta , and Fragilaria subconstricta . However, even for those species, preliminary identifications based on morphology often differed from identifications based on phylogenetic clustering combined with detailed morphological study. Clades were well‐defined by rbc L, but based on morphology, the terminal taxa of these clades did not match the currently described Fragilaria species. To clarify recognition of these taxa, we describe three new species: Fragilaria agnesiae , Fragilaria heatherae , and Fragilaria joachimii . info:eu-repo/semantics/acceptedVersion

International audience; Littoral benthic diatom communities are pivotal indicators which react to coastline sources of pollution. Littoral communities, however, may be also strongly influenced by other environmental factors: we assumed that even in lakes without anthropogenic pressure, communities could be heterogeneous. To investigate the natural causes of community heterogeneity along shoreline, we sampled 58 pristine high-altitude lakes in the French Alps. Inside each lake, three different littoral stations were sampled. Water chemical composition was measured with major environmental settings. Lakes with homogeneous communities along their shoreline were dominated by pioneer species adapted to strong physical disturbances. The water renewal time in these lakes was short (a few days) and this conveyed strong turbulences. Lakes with heterogeneous communities along their shore had longer water renewal time and were dominated by high-profile diatoms that were able to compete for light and nutrients. The within-lake heterogeneity could be explained by the variability in coastline terrestrial habitats. This study illustrates how water turbulences can limit the expression of some ecological processes like interspecific competition and reduce heterogeneity when it is an overriding stressor. Finally, implications for lake managers are given in terms of monitoring efforts.

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.

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.

Recent developments in the use of environmental DNA are opening up new horizons for the assessment of the quality of aquatic environments. These rapid and cost-effective methods, in very swift progress, will potentially offer the opportunity to identify all the taxa present in an environmental sample (water or biota) by the use of complementary markers. The produced inventories can then be used for the assessment of biodiversity and ecological quality. However, the inclusion of these new DNA-based methods in monitoring practices is not straightforward and requires harmonised actions in the coming years at national and international levels. In order to foresee and stimulate such a harmonised implementation, the European network DNAqua-Net (COST Action CA15219) brought together some of its members, experts of ECOSTAT and other environmental biomonitoring stakeholders from different European countries. Through workshops, bringing together 51 participants in 7 sub-groups in April 2020, an implementation roadmap was designed. The coordinated actions to be taken in the different countries, and the possible collaborations and steps to be taken at the EU level were identified. This presentation will give an overview of all discussions (Lefrançois et al. 2020) reflecting the diversity of situations in Europe, as well as common views. We will highlight important actions required for a successful implementation of DNA-based biomonitoring of aquatic ecosystems to the horizon of 2030.

The high-dispersal rates of microorganisms have driven to the expectation of their cosmopolitan geographic distribution. However, recent studies demonstrate that microorganisms instead show particular biogeography. Despite the existence of cosmopolitan species, geographically limited microbial groups have been found in aquatic and terrestrial environments worldwide. Diatoms are long time used model to study the biogeography of microorganisms. They are unicellular eukaryotic microalgae that contribute significantly to the aquatic primary production and have huge taxonomic diversity and marked species-specific ecological preferences. Several authors considered that diatoms have no limits in dispersion and are ubiquitously present. On the other hand, recent studies have shown that endemism exists for several genera, and species may have low dispersal capacity. However, all these studies are based on data obtained by microscopy and therefore suffer from the many well-identified biases associated with the optical identification of microorganisms at large scale. Metabarcoding technologies provide an access to taxonomic precision with a higher resolution compared to microscopy and open therefore the possibility of analyzing microbial diversity at genetic level. Recent bioinformatics tools allow reliable and standardized comparison of large datasets originating from distant geographic regions, overcoming issues related to biases in species identification. In this study we used metabarcoding data to revisit central questions in freshwater diatom biogeography. We assembled a large dataset of samples of benthic diatoms collected from rivers in seven different geographic regions. These regions cover the subpolar (Fennoscandia), temperate (France Mainland) and tropical (West Africa, French Guyana, New Caledonia, Tahiti island and Mayotte island) climate zones. The selected geographic regions can also be classified into four continental areas (Fennoscandia, France Mainland, West Africa, and French Guyana) and three islands (New Caledonia, Tahiti and Mayotte). We analyzed diatom alpha, beta and gamma diversity patterns in this dataset to address two main questions: 1) the presence of a latitudinal gradient in diatom diversity and 2) the cosmopolitanism of diatoms. Similarly to results previously reported by Soininen et al. 2016, our data showed a decrease in diatom richness with a decrease in latitude. However, testing the effect of land type (island vs. mainland) showed that this factor explains the actual variability of richness along the climatic gradient and the effect of latitude is not significant. Differences in community structure between regions and climate zones were significant. In multivariate analysis, tropical samples did not overlap with any of the other climate zones, suggesting the specificity of these communities.

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.

International audience; Bioindication has become an indispensable part of water quality monitoring in most countries of the world, with the presence and abundance of bioindicator taxa, mostly multicellular eukaryotes, used for biotic indices. In contrast, microbes (bacteria, archaea and protists) are seldom used as bioindicators in routine assessments, although they have been recognized for their importance in environmental processes. Recently, the use of molecular methods has revealed unexpected diversity within known functional groups and novel metabolic pathways that are particularly important in energy and nutrient cycling. In various habitats, microbial communities respond to eutrophication, metals, and natural or anthropogenic organic pollutants through changes in diversity and function. In this review, we evaluated the common trends in these changes, documenting that they have value as bioindicators and can be used not only for monitoring but also for improving our understanding of the major processes in lotic and lentic environments. Current knowledge provides a solid foundation for exploiting microbial taxa, community structures and diversity, as well as functional genes, in novel monitoring programs. These microbial community measures can also be combined into biotic indices, improving the resolution of individual bioindicators. Here, we assess particular molecular approaches complemented by advanced bioinformatic analysis, as these are the most promising with respect to detailed bioindication value. We conclude that microbial community dynamics are a missing link important for our understanding of rapid changes in the structure and function of aquatic ecosystems, and should be addressed in the future environmental monitoring of freshwater ecosystems.

The objective of this protocol is to provide a reliable and replicable method for the sampling of lake micro-phytobenthos and associated microbes in biofilms, to be used in both downstream DNA analysis and algal microscopic counts. The field protocol is optimized for routine sampling and is in agreement with CEN guidance (NF EN 13946) and CEN technical report (CEN/TR 17245) for the analysis of benthic diatoms from rivers and lakes. The application proposed here in the context of EcoAlpsWater aims at comparing DNA inventories to traditional inventories (microscopy). The sampling strategy has been designed to assess localized hotspots pollutions/perturbations impacts on the shoreline, taking into account shoreline’s heterogeneity. This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium have contributed to the optimization of this protocol. The use of microalgae to assess ecological quality of lakes is traditionally based on phytoplankton that allow to evaluate the trophic status. A single depth-integrated water sample from one sampling site above the deepest point of the lake is chosen, and samplings are carried out in the euphotic zone at different seasons. Though not always included as biological quality indicator in lake monitoring, phytobenthos has relevance for lake ecological quality assessment and might be used as a complementary indicator beside phytoplankton in the future (e.g. DeNicola & Kelly 2013). Phytobenthic communities of the lake’s shorelines are generally heterogeneous. The reason for this heterogeneity can be due to many factors, including substrate characteristics as well as anthropogenic factors such as eutrophication, the presence of pollution hotspots, like arrivals of polluted rivers in the lakes or arrivals of wastewaters through drains (e.g. Crossetti et al. 2013, Rimet et al. 2015/2016/2018, Cicek & Yamuc, 2017). But it can also be caused by the nature of the neighboring terrestrial habitats and land use (for instance closed forest vs open fields, or pasture vs steep rock falls). The objective of this protocol is to provide a reliable and replicable method for the sampling of lake micro-phytobenthos and associated microbes biofilms to be used in both downstream DNA analysis and algal microscopic counts. Examples of applications to assess localized hotspots pollutions impact on the shoreline, taking into account shoreline’s heterogeneity, will be provided. The field protocol is based on routine methods used for biofilms sampling and is in agreement with : - CEN 2014. Water quality - NF EN 13946 - Guidance for the routine sampling and preparation of benthic diatoms from rivers and lakes. Afnor, 1-23. - CEN 2018. Water quality - CEN/TR 17245 - Technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for metabarcoding analyses. CEN, 1-8.

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.

Dear participants of DNAQUA2021 International Conference, Undoubtedly, DNAQUA2021 is a major highlight of the EU COST Action DNAqua-Net (CA15219). Even though we cannot claim that the organisation of DNAQUA2021 was a piece of cake, it is simply wonderful to see the great interest in this event. With 1,498 registered participants from 79 nations, 204 contributed talks and posters for only two and a half days, the conference shows how timely and relevant research on DNA-based aquatic bioassessment and monitoring is. As the managing team of DNAqua-Net, we could have hardly imagined the impact of DNAqua-Net back in 2015, when we wrote the proposal (Leese et al. 2016). Yet, the more we are now delighted and thankful to see the success. Together with many experts from many different countries, taxonomists, ecologists, geneticists and bioinformaticians, we have made significant methodological progress. Above all, we have succeeded in connecting biomonitoring experts all across Europe and beyond. With more than 100 scientific publications from DNAqua-Net's five working groups, the research impact of the network is obvious. Furthermore, with "Metabarcoding and Metagenomics" (MBMG), we have established an international journal for basic and applied aspects of genetic bioassessment and monitoring. However, in many ways, the impact of DNAqua-Net goes far beyond the mere scientific progress. Capacity building e.g. via barcoding projects have been initiated in many countries, validation studies were co-designed by researchers and stakeholders from the applied sector and launched - even across several countries as for example the SCANDNAnet project shows. DNAqua-Net has supported over 50 research exchanges that fostered close cooperation among the institutions and countries. Also, DNAqua-Net accompanied the fourth Joint Danube Survey (JDS4) and conducted the (e)DNA-based surveys for fish, benthic invertebrates, phytobenthos and the sediment community. Last but not least, we have developed many essential pieces of an applied concept for future implementation of DNA-based methods together with various stakeholders at national and international level. Here, of particular importance was the establishment of a working group within the European Standardisation Organisation CEN on DNA and eDNA-based methods (CEN/TC230/WG28). We are particularly grateful also to our colleagues from 'beyond Europe' that have supported us, participated in workshops, discussions and training schools, invited us to their national meetings on DNA and eDNA-based biomonitoring on five continents. The implementation of (e)DNA-based methods into bioassessment and monitoring programs of our rivers, lakes, oceans and the groundwater, will be particularly successful if we sustainably stay connected across countries, generations, cultures and disciplines (Fig. 1). Many of the findings from basic to applied research will be presented at DNAQUA2021. We are particularly pleased that so many early career researchers present their findings. Please take the chance and discuss with them (but not only with them) about their findings. With "Spatial.Chat" we offer you a nice and intuitive environment that allows for some 'real' conference spirit even in these COVID-19 virtual meeting times. Now enjoy two and a half days packed with fascinating insights from (e)DNA-based aquatic biomonitoring. Take the chance, foster and extend your collaborations. We hope to see and discuss with you over the next days at DNAQUA2021 and beyond. THANK YOU! Florian, Agnès, Charly & Alex (Fig. 2)

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)

Environmental conditions, such as nutrient concentrations, salinity, elevation etc., shape diatom assemblages of periphytic biofilms. These assemblages respond rapidly to environmental changes, a fact which makes diatoms valuable bioindicators. Hence, freshwater biomonitoring programmes currently use diatom indices (e.g. EU Water Framework Directive - WFD). To date, microscopy-based assessments require high taxonomic expertise for diatom identification at the species level. High-throughput technologies now provide cost-effective identification approaches that are promising, complementary or alternative tools for bioassessment. The suitability of the metabarcoding method is evaluated for the first time in the Cyprus streams WFD monitoring network, an eastern Mediterranean country with many endemic species and results are compared to the results acquired from the morphotaxonomic analysis. Morphotaxonomic identification was conducted microscopically, using the most updated taxonomic concepts, literature and online resources. At the same time, DNA metabarcoding involved the use of the rbcL 312 bp barcode, high-throughput sequencing and bioinformatic analysis. The ecological status was calculated using the IPS Index. Results show a positive correlation between morpho-taxonomic and molecular IPS scores. Discrepancies between the two methodologies are related to the limitations of both techniques. This study confirmed that Fistulifera saprophila can have a crucial role in key differences observed, as it negatively influences IPS scores and microscopy methods frequently overlook it. Importantly, gaps in the DNA barcoding reference databases lead to a positive overestimation in IPS scores. Overall, we conclude that DNA metabarcoding offsets the morphotaxonomic methodology for the ecological quality assessment of freshwaters.

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; Intermittent rivers and ephemeral streams (IRES) – waterways in which flow ceases periodically or that dry completely – are found worldwide, and their frequency and extent are expected to increase in the future in response to global climate change and growing anthropogenic demand for fresh water. Repeated wet–dry cycles generate highly dynamic settings within river networks composed of aquatic and terrestrial habitats, which act as evolutionary triggers for aquatic and terrestrial biota. Drying also alters functions and processes within river networks, with consequences for ecosystem services. Despite the emergence of promising conceptual and methodological developments, our understanding of the occurrence and diversity of organisms in these ecosystems is limited primarily due to their coupled aquatic–terrestrial characteristics. Novel genomic tools based on high-throughput sequencing have the potential to tackle unanswered questions of pivotal importance to predict future change in IRES. Here, we outline why genomic tools are needed to assess these dynamic ecosystems from the population to the metacommunity scale, and their potential role in bridging ecological–evolutionary dynamics.

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.

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.

International audience; Since the early 20's antibiotics have been massively produced and consumed for the benefit of bothhuman and animal health. Nevertheless, antibiotics have also reached the aquatic environmentthrough diffuse sources (e.g. veterinary treatment, contaminated manure application...) and throughwastewater. Consequently, antibiotics concentrations between the ng/L and μg/L range are regularlydetected in surface water and those molecules have also been found in sediments and aquatic biota.The ubiquitous presence of antibiotics exerts a selective pressure on microbial communities leading tothe acquisition and dissemination of antibioresistance in the environment. While both antibiotics andantibioresistance have been found in different aquatic compartments, more investigation is requiredto better understand their distribution and to identify hot spots of accumulation.In this context, we investigated the repartition of antibiotics and antibioresistance in different aquaticcompartments on 4 stations belonging to regional observatories and presenting contrasting levels ofpharmaceuticals: 2 on the Arve river belonging to Sipibel observatory and 2 on Lake Geneva belongingto the Observatory on Lakes. On the Arve river, the 2 stations were located up- and down- stream thedischarge place of a wastewater treatment plant (WWTP) collecting both urban and hospitalwastewaters. On Lake Geneva, one station was located in a relatively pristine area while the secondwas close to the discharge of an urban WWTP. To better identify a potential temporal dynamic ofantibiotics and antibioresistances over seasons, 6 samplings were conducted during 1.5 year. On eachsampling campaign, the following parameters were determined: (i) antibiotics levels in water,sediments and periphyton; (ii) antibioresistance in periphyton and sediments using various techniques:detection of resistance genes, integrons quantification, detection of tolerance acquisition via a PICT(Pollution Induced Community Tolerance) approach; (iii) antibiotics biodegradation potential ofmicrobial community from sediments (by radiorespirometric measurement); (iv) diversity of bacteriaand diatoms in periphyton and sediments; (v) physico-chemical parameters and (vi) metalliccontamination in sediments.While microbial resistance to antibiotics is commonly assessed by quantifying resistance genes orisolating antibiotic resistant bacteria, antibiotic resistance can also be estimated by measuring theacquisition of antibiotics tolerance at community level, following a PICT approach. In our study,periphytic microbial communities from the Arve river were found to have a higher tolerance to thetested antibiotics (ciprofloxacin, ofloxacin, sulfamethazine and erythromycin) than communities fromLake Geneva, in agreement with the expected levels of contamination. In addition, in some cases, ahigher tolerance was also found at stations close to WWTP effluents than in upstream/protectedstations. For example, periphytic microbial communities collected downstream the WWTP on the Arveriver were generally found to have a higher tolerance to ciprofloxacin than the upstream communities.Comparing whole community tolerance to other classical indicators of antibioresistance and toantibiotics levels in the aquatic ecosystems allows us to better understand the interconnectionbetween pharmaceutical exposure, in situ tolerance and genetic potential for antibioresistance.

The objective of this protocol is to provide a reliable and replicable method for the sampling of lake micro-phytobenthos and associated microbes in biofilms, to be used in both downstream DNA analysis and algal microscopic counts. The field protocol is optimized for routine sampling and is in agreement with CEN guidance (NF EN 13946) and CEN technical report (CEN/TR 17245) for the analysis of benthic diatoms from rivers and lakes. The application proposed here in the context of EcoAlpsWater aims at comparing DNA inventories to traditional inventories (microscopy). The sampling strategy has been designed to assess localized hotspots pollutions/perturbations impacts on the shoreline, taking into account shoreline’s heterogeneity. This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium have contributed to the optimization of this protocol. The use of microalgae to assess ecological quality of lakes is traditionally based on phytoplankton that allow to evaluate the trophic status. A single depth-integrated water sample from one sampling site above the deepest point of the lake is chosen, and samplings are carried out in the euphotic zone at different seasons. Though not always included as biological quality indicator in lake monitoring, phytobenthos has relevance for lake ecological quality assessment and might be used as a complementary indicator beside phytoplankton in the future (e.g. DeNicola & Kelly 2013). Phytobenthic communities of the lake’s shorelines are generally heterogeneous. The reason for this heterogeneity can be due to many factors, including substrate characteristics as well as anthropogenic factors such as eutrophication, the presence of pollution hotspots, like arrivals of polluted rivers in the lakes or arrivals of wastewaters through drains (e.g. Crossetti et al. 2013, Rimet et al. 2015/2016/2018, Cicek & Yamuc, 2017). But it can also be caused by the nature of the neighboring terrestrial habitats and land use (for instance closed forest vs open fields, or pasture vs steep rock falls). The objective of this protocol is to provide a reliable and replicable method for the sampling of lake micro-phytobenthos and associated microbes biofilms to be used in both downstream DNA analysis and algal microscopic counts. Examples of applications to assess localized hotspots pollutions impact on the shoreline, taking into account shoreline’s heterogeneity, will be provided. The field protocol is based on routine methods used for biofilms sampling and is in agreement with : - CEN 2014. Water quality - NF EN 13946 - Guidance for the routine sampling and preparation of benthic diatoms from rivers and lakes. Afnor, 1-23. - CEN 2018. Water quality - CEN/TR 17245 - Technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for metabarcoding analyses. CEN, 1-8.

The objective of this protocol is to provide a reliable and replicable method for the sampling of river micro-phytobenthos and associated microbes in biofilms, to be used in both downstream DNA analysis and algal microscopic counts. The filed protocol is optimized for routine sampling and is in agreement with CEN guidance (NF EN 13946) and CEN technical report (CEN/TR 17245) for the analysis of benthic diatoms from rivers and lakes. The application proposed here in the context of EcoAlpsWater aims in comparing DNA inventories to traditional inventories (microscopy). This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium (involving 12 partners : http://www.alpine-space.eu/projects/eco-alpswater/en/home) have contributed to the optimization of this protocol. Phytobenthos-based ecological assessment of running waters is part of the EU Water Framework Directive 2000/60/EC (European Commission) allowing to evaluate the level of nutrients and organic matter pollution in rivers. According to the countries, phytobenthos assessment includes all groups of algae or is based only on diatoms communities. Other periphytic organisms such as fungi, bacteria or microbial eukaryotes as cilates or planktonic (metaphytic) algae are not considered in this assessment method. Further the group of charophytes is covered by the macrophyte ecology method assessment. The recent development of DNA metabarcoding has the potential to complement the traditional biological monitoring based on the direct observation of the organisms, both by reducing sample-processing cost and time and by offering the opportunity to consider a larger taxonomic diversity (e.g. bacteria, heterotrophic micro-eukaryotes …). The protocol described here is dedicated to the sampling of biofilms in rivers both for microscopic enumeration of the phytobenthic community and for downstream DNA analyses of microbial assemblages. This field protocol is based on routine methods used for biofilms sampling and is in agreement with: - CEN, 2014. Water quality - NF EN 13946 - Guidance for the routine sampling and preparation of benthic diatoms from rivers and lakes. Afnor, 1-23. - CEN 2018. Water quality - CEN/TR 17245 - Technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for metabarcoding analyses. CEN, 1-8.

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.

Lakes are exposed to anthropogenic pollution including the release of allochthonous bacteria into their waters. Antibiotic resistance genes (ARGs) stabilize in bacterial communities of temperate lakes, and these environments act as long-term reservoirs of ARGs. Still, it is not clear if the stabilization of the ARGs is caused by a periodical introduction, or by other factors regulated by dynamics within the water column. Here we observed the dynamics of the tetracycline resistance gene (tetA) and of the class 1 integron integrase gene intI1 a proxy of anthropogenic pollution in the water column and in the sediments of subalpine Lake Maggiore, together with several chemical, physical and microbiological variables. Both genes resulted more abundant within the bacterial community of the sediment compared to the water column and the water-sediment interface. Only at the inset of thermal stratification they reached quantifiable abundances in all the water layers, too. Moreover, the bacterial communities of the water-sediment interface were more similar to deep waters than to the sediments. These results suggest that the vertical distribution of tetA and intI1 is mainly due to the deposition of bacteria from the surface water to the sediment, while their resuspension from the sediment is less important.