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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.

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.

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.

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; 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.

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.

Data Paper; International audience; The Bellecombe pilot site - SIPIBEL - was created in 2010 in order to study the characterisation, treatability and impacts of hospital effluents in an urban wastewater treatment plant. This pilot site is composed of: i) the Alpes Leman hospital (CHAL), opened in February 2012, ii) the Bellecombe wastewater treatment plant, with two separate treatment lines allowing to fully separate the hospital wastewater and the urban wastewater, and iii) the Arve River as the receiving water body and a tributary of the Rhone River and the Geneva aquifer. The database includes in total 48 439 values measured on 961 samples (raw and treated hospital and urban wastewater, activated sludge in aeration tanks, dried sludge after dewatering, river and groundwater, and a few additional campaigns in aerobic and anaerobic sewers) with 44 455 physico-chemistry values (including 15 pharmaceuticals and 14 related transformation products, biocides compounds, metals, organic micropollutants), 2 193 bioassay values (ecotoxicity), 1 679 microbiology values (including microorganisms and antibioresistance indicators) and 112 hydrobiology values.

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; Pharmaceutical industry effluents are complex and highly variable in time. Assessing the efficiency of a pharmaceutical industry wastewater treatment plant (WWTP) and the resulting decrease in effluent toxicity and ecological risk is thus not straightforward. We set up an original in situ pilot directly connected to a pharmaceutical WWTP to monitor the chronic toxicity of successive effluents using natural periphytic biofilms. Their structural and functional responses to effluent exposure were assessed by combining (i) a molecular approach to characterize the bacterial and diatom diversity and (ii) functional measurements of photosynthetic and enzyme activities. Effluent contamination by pharmaceuticals strongly decreased after the quaternary treatment (activated carbon). Most of the structural biological characteristics improved with cumulative WWTP treatment (bacterial diversity, microbial genetic structure, and biological diatom index), showing community recovery along the treatment process. However, functional parameters did not show clear links with treatment steps, suggesting that microbial activities were not solely driven by pharmaceuticals produced during the experimental period. Operationally, this type of pilot system offers a useful tool for biomonitoring approaches and offers new approaches for industrial managers to assess the ecological risk of production effluents in receiving water.

International audience; Altitude lakes are weakly impacted by human activities. This makes them choice ecosystems to understand how biological communities are impacted by natural factors. This question was addressed to littoral benthic diatoms, a largely used ecological indicator. We wanted to know if benthic diatoms in lakes are more impacted by local varying factors (altitude, lake depth...) or regional varying factors (geology). The study area takes place in the Northern French Alps. Littoral benthic diatoms of 63 natural lakes situated between 1350 and 2700 m · a.s.l. were sampled. Two categories of communities were observed: one of deep and lower altitude lakes and one of higher altitude and shallower lakes. In each category, communities were characterized and were corresponding to particular lake types: lakes dominated by a particular geology, lakes with a water level fluctuation, turbid lakes,... Communities did not show a spatial structure. We observed that local factors were more important than regional factors. Indeed, the study area displayed a mixed geology even at a local level. On another hand, altitude a local varying factor determines freezing period a determining item of high-altitude lake functioning.

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.

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; Land-use imposes an important potential threat on the aquatic ecosystems of riverine habitats. In this study, DNA metabarcoding was used to assess the effect of land-use on diatom assemblages, with a special focus on cropland area as an integrative proxy for several direct-acting pressures. The so-called taxonomy-free approach was tested using exact sequence variants (ESVs) without pre-clustering and without assigning them to traditional taxonomy. Our hypothesis is that the taxonomy-free approach gives better interpretation of the effect of agricultural land use and it provides a more efficient index to indicate agricultural stress than the classical method with taxonomical clustering. Classical ordination techniques (PCA, NMDS) were performed to study the effects of cropland area proportion on diatom assemblages and a modified version of the Zelinka-Marvan equation for the index development. Results showed that (i) although ESVs provided better results when studying land-use effects on diatoms, taxonomic assignment after analysis was necessary to give ecological interpretations and that (ii) a better performing index could be developed by using the taxonomy-free approach. By using ESVs without taxonomic assignment, information on the ecology of sequences belonging to the same species and of unassigned sequences could be kept. New types of clustering methods are welcome in the future of biomonitoring where the delimitation of taxonomic units should be refined based on a higher emphasis on their ecology rather than on morphological or genetical criteria.

International audience; Benthic diatoms inhabiting littoral zones are regarded as promising bioindicators in lakes. Some pre-standard protocols were proposed and suggested to collect a single site per lake to assess its overall quality. In large lakes, such as Lake Geneva, a spatial heterogeneity of diatom assemblages was suspected, because of differing human pressures. Thirty-five sites were sampled along the shoreline in locations showing various population densities and crossed by rivers flowing into the lake. Different assemblages were found; one corresponded to more oligotrophic areas and was mostly situated on the shoreline next to low population densities; it was mostly composed of low-profile diatoms. A second one corresponded to more eutrophic areas and was mostly situated near densely inhabited areas; it was composed of many high-profile diatoms. A third one was in close vicinity to river inlets, with high abundances of motile diatoms. This last assemblage was spatially restricted to the river plume. After testing several diatom indices, one showed better capacities to assess nutrient level (EPI-L). If we avoid areas near river inlets, the ecological quality assessed with diatom indices ranged from moderate to high ecological status depending on the vicinity situated more or less near densely populated areas.

International audience; Climatic extreme events such as droughts (unpredictable), dry periods (predictable) or even flush floods, threaten freshwater ecosystems worldwide. The filtering mechanisms of these events and their strength on communities, however, can be different among regions. While time-for-adaptation theory defines whether or not water scarcity can be considered as disturbance, the stress-dominance theory predicts an increase in importance of environmental filtering and a decrease in the role of biotic interactions in communities with increasing environmental stress. Here, we tested whether environmental filtering (leading to trait convergence) or limiting similarity (leading to trait divergence) is the main assembly rule shaping the structure and trait composition of benthic diatom assemblages in Mediterranean (Portuguese) and continental (Hungarian) temporary and perennial streams. We assumed that the trait composition of diatom assemblages in the two stream types would be less different in the Mediterranean than in the continental region (addressed to time-for-adaptation theory). We also hypothesized that trait composition would be shaped by environmental filtering in the Hungarian streams while by biotic interactions in Portuguese streams (addressed to stress-dominance theory). Our results supported our first hypothesis since traits, which associated primarily to temporary streams were found only in the continental region. Our findings, however, only partially proved the stress-dominance hypothesis. In the continental region, where drying up of streams were induced by unpredictable droughts, biotic interactions were the main assembly rules shaping community structure. In contrast, environmental filtering was nearly as important as limiting similarity in structuring trait composition in the Mediterranean region during the predictable dry phase with no superficial flow. These analyses also highlighted that drought events (both predictable and unpredictable ones) have a complex and strong influence on benthic diatom assemblages resulting even in irreversible changes in trait composition and thereby in ecosystem functioning.

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.

Increasing release of pharmaceuticals and personal care products (PPCPs) into aquatic ecosystems is a growing environmental concern. Triclosan and fluoxetine are two widely used PPCPs and frequently detected in aquatic ecosystems. In this study, the sensitivities of 7 algal species from 4 genera to triclosan, fluoxetine and their mixture were evaluated. The results showed that the inhibitory effect on algal growth (EC50-96h) of triclosan varied with 50 times differences among the 7 algal species. Chlorella ellipsoidea was the least susceptible species and Dunaliella parva was the most sensitive species to triclosan. The inhibitory effect of fluoxetine was less variable than triclosan. Slightly higher toxicity of fluoxetine than triclosan was shown in the 7 tested algal species. No consistent pattern of the effects from mixture of triclosan and fluoxetine was observed among the 7 algal species and among the 4 genera. Additive effects of the mixture occured in 4 species and antagonistic effects in the other 3 species but no synergistic effect was detected. The algal species might show some sign of phylogenetic response to triclosan, as evidenced by the wide range of differences in their sensitivity at the genus level. This study provides important data which could be beneficial for biomonitoring programs on the ecological risk (algal species diversity) of these two chemicals.

Water covers over 70% of our planet's surface and it is a key resource for the survival of all organisms, among them humans. Unfortunately, water resources face increasing pressures due to the exponential expansion of and resource exploitation by human beings. The consequences of this on water ecosystems represent hallmarks of the Anthropocene such as chemical pollution, warming, scarcity of clean drinking water, ocean acidification and a dramatic loss of biodiversity. As a consequence, the direct and indirect benefits humanity obtained from these ecosystems as cheap services, such as clean water, biomass production, climate regulation and matter fluxes, are increasingly at risk. Therefore, it is of utmost importance to assess the ecological state of aquatic ecosystems and to protect and manage them in a sustainable way. In order to assess the ecological status of a given water body, aquatic biodiversity data are collected by morphological identification of bioindicator species and a comparison of site-specific species lists to those of fairly natural reference water bodies. Quantifying the differences between the lists guides subsequent management actions. Examples of European standard bioasseessments (so far morphologically-based) are Marine Strategy Framework Directive (2008/56/EC) and the Water Framework Directive (2000/60/EC). While the implementation of biomonitoring programs is already a great success, there is room for improvement. In the field of molecular genetics, revolutionary high-throughput DNA-based analyses have been developed. These can be applied to assess taxon lists of hundreds to many thousands at once and greatly improve speed and accuracy of assessments. However, while these novel genetic tools have attracted a lot of interest, they are not implemented in any of the regular legal biomonitoring programs. In order to change this, the European Co-Operation in Science and Technology (COST) program's Action CA15219 'DNAqua-Net' was launched in November 2016 (Leese et al. 2016). The Action aims to gather existing knowledge and complement those standard procedures by developing and implementing these novel genomic DNA-based approaches for biomonitoring and bioassessment. The Action is comprised of five working groups (WGs): WG1: DNA Barcode References; WG2: Biotic Indices & Metrics; WG3: Lab & Field Protocols; WG4: Data Analysis & Storage and WG5: Implementation Strategies & Legal Issues. However, central to this is the standardisation of the various protocols, methods and biotic indices and integration of DNA-based datasets (e.g. resulting from DNA metabarcoding, mito- and metagenomics) with existing data standards. Here, the TDWG community will be of central importance and its participation in DNAqua-Net is highly desirable. Moreover, the innovative open access journal Metabarcoding & Metagenomics (MBMG) has been recently initiated to promote open science and enhance data exchange in this field as well as to connect the diverse actors and communities (Leese et al. 2017).

Hospital effluents cause growing interest, as they may be an important contamination source of micro- and macropollutants to WWTP influents. These effluents are usually characterised by higher concentrations and greater diversity of pollutants compared to urban wastewater. However, in certain cases, hospital effluents may represent only a small fraction of the total WWTP influent. Several recent studies report that their contribution to WWTP influents is limited and they are only one of the important sources of micropollutants in the environment. Nevertheless, specific micropollutants may exhibit relatively high hospital contribution, which may cause environmental risks. Several other important sources of micropollutants (chronic medication, nursing home, outpatients, cattle, etc.) are released in urban wastewaters. These sources should not be neglected, because they represent an important load that may impact aquatic environments.

International audience; Phylogeny has not yet been fully accepted in the field of ecotoxicology, despite studies demonstrating its potential for developing environmental biomonitoring tools, as it can provide an a priori assessment of the sensitivity of several indicator organisms. We therefore investigated the relationship between phylogeny and sensitivity to herbicides in freshwater diatom species. This study was performed on four photosystem 11 inhibitor herbicides (atrazine, terbutryn, diuron, and isoproturon) and 14 diatom species representative of Lake Geneva biofilm diversity. Using recent statistical tools provided by phylogenetics, we observed a strong phylogenetic signal for diatom sensitivity to herbicides. There was a major division in sensitivity to herbicides within the phylogenetic tree. The most sensitive species were mainly centrics and araphid diatoms (in this study, Thalassiosirales and Fragilariales), whereas the most resistant species were mainly pennates (in this study, Cymbellales, Naviculales, and Bacillariales). However, there was considerable variability in diatom sensitivity within the raphid clade, which could be explained by differences in trophic preferences (autotrophy or heterotrophy). These traits appeared to be complementary in explaining the differences in sensitivity observed at a refined phylogenetic level. Using phylogeny together with complementary traits, as trophic preferences, may help to predict the sensitivity of communities with a view to protecting their ecosystem.

International audience; Benthic diatom communities are used in the ecological assessment of Portuguese rivers through the calculation of an autecological index, the IPS (Indice de Polluosensibilite Specifique), officially adopted for Portugal. This index requires a high level of taxonomic expertise for morphological identification of individuals at species level. Advances in genomics, such as the DNA metabarcoding combined with high-throughput sequencing techniques offer a promising alternative to this classical approach, decreasing expertise requirement and therefore reducing time and costs. The aim of this study was to test the potential of DNA metabarcoding of diatoms in the bioassessment of Portuguese rivers, by comparing the classifications of the IPS obtained with morphological and molecular approaches. A total of 88 samples were collected in four river types and a quality gradient in central Portugal in the spring of 2017, following the Water Framework Directive (WFD) standards. The morphological approach comprised taxonomic identification of at least 400 valves, under the light microscope. Molecular approach included eDNA extraction followed by PCR amplification and DNA high-throughput sequencing (Illumina MiSeq) using a diatom-specific 312 bp rbcL DNA barcode. Sequences were analysed with Mothur software, producing Operational Taxonomic Units (OTUs) that were taxonomically assigned using the R-Syst::diatom reference library. We also tested the effect of a correction factor (CF) based on biovolume for the quantification of molecular data. When comparing the number of species identified, morphological, molecular without and with CF inventories revealed 306, 125 and 111 species, respectively. Molecular data showed 67% of unassigned OTUs. These results were mainly due to the incompleteness of the reference library, which currently represents the major difficulty in taxonomic assignment of DNA sequences in metabarcoding. Regarding IPS values, the results indicated a good correlation between morphological and molecular methods, particularly when applying the CF. Furthermore, Principal Coordinates analysis plot based on species abundances revealed a quality gradient in all 3 methodologies (morphological, molecular without and with CF). These results support the hypothesis that DNA metabarcoding is a valid approach for ecological quality assessment of Portuguese rivers. Yet, there is still work to be done on this new methodology to enable a smooth transition between the traditional and this new approach without misspend accumulated knowledge from the last decades on water quality assessment.

International audience; Benthic diatoms evolved in a biofilm structure, at the interface between water and substrata. Biofilms can adsorb toxicants, such as herbicides, but little is known about the exposure of biofilm organisms, such as benthic diatoms, to these adsorbed herbicides. We assessed the sensitivity of 11 benthic diatoms species to 6 herbicides under both planktonic and benthic conditions using single-species bioassays. The concentration that reduced the growth rate of the population by 10% (EC10) and 50% (EC50), respectively, varied depending on the species, the herbicides, and the growth forms involved. As a general trend, the more hydrophobic the herbicide, the more species were found to be sensitive under benthic growth conditions. Statistical differences (alpha < 5%) were observed between the sensitivities under planktonic and benthic growth conditions for many hydrophobic herbicides. A protective effect of the biofilm against herbicides was observed, and this tended to decrease (at both the EC10 and EC50 levels) with increasing hydrophobicity. The biofilm matrix appeared to control exposure to herbicides, and consequently their toxicity towards benthic diatoms. For metolachlor, terbutryn and irgarol, benthic thresholds derived from species sensitivity distributions were more protective than planktonic thresholds. For hydrophobic herbicides, deriving sensitivity thresholds from data obtained under benthic growth seems to offer a promising alternative. (C) 2013 Elsevier B.V. All rights reserved.

International audience; Diatoms include a great diversity of taxa and are recognized as powerful bioindicators in rivers. However using diatoms for monitoring programs is costly and time consuming because most of the methodologies necessitate species-level identification. This raises the question of the optimal trade-off between taxonomic resolution and bioassessment quality. Phylogenetic tools may form the bases of new, more efficient approaches for biomonitoring if relationships between ecology and phylogeny can be demonstrated. We estimated the ecological optima of 127 diatom species for 19 environmental parameters using count data from 2119 diatom communities sampled during eight years in eastern France. Using uni- and multivariate analyses, we explored the relationships between freshwater diatom phylogeny and ecology (i.e., the phylogenetic signal). We found a significant phylogenetic signal for many of the ecological optima that were tested, but the strength of the signal varied significantly from one trait to another. Multivariate analysis also showed that the multidimensional ecological niche of diatoms can be strongly related to phylogeny. The presence of clades containing species that exhibit homogeneous ecology suggests that phylogenetic information can be useful for aquatic biomonitoring. This study highlights the presence of significant patterns of ecological optima for freshwater diatoms in relation to their phylogeny. These results suggest the presence of a signal above the species level, which is encouraging for the development of simplified methods for biomonitoring survey.

International audience; Mixture risk assessment predictions have rarely been confronted with biological changes observed in the environment. In this study, long-term monitoring of a European great lake, Lake Geneva, provides the opportunity to assess to what extent the predicted toxicity of herbicide mixtures explains the changes in the composition of the phytoplankton community next to other classical limnology parameters such as nutrients. To reach this goal, the gradient of the mixture toxicity of 14 herbicides regularly detected in the lake was calculated using concentration addition and response addition models. A temporal gradient of toxicity was observed which decreased from 2004 to 2009. Redundancy analysis and partial redundancy analysis showed that this gradient explains a significant portion of the variation in phytoplankton community composition with and without having removed the effect of all other covariables. Moreover, species that are significantly influenced, positively or negatively, by the decrease of toxicity in the lake over time are highlighted. It can be concluded that the herbicide mixture toxicity is one of the key parameters to explain phytoplankton changes in Lake Geneva.

1. The development of periphytic algae and bacteria is controlled by a combination of interacting biotic processes and abiotic factors. Distinguishing between the selection pressure resulting from pollution and that of natural environmental factors is therefore one of the most critical aspects of assessing the impact of pollutants on the diversity and function of benthic microbial communities in natural ecosystems. 2. We studied how current velocity and season affect the ability of river biofilms to cope with complex chemical pollution. We compared the diversity, structure and production of periphytic algae and bacteria from four sampling zones with differing chemical water quality levels and different flow velocities over the course of two seasons (summer and winter). 3. The three factors tested all influenced biofilm development, but this depended on the biological variable being measured. Bacterial and algal densities were highly dependent on season and chemical water quality. Algal density was lower in summer than in winter, but bacterial density and production increased from upstream (reference) to downstream (polluted), and this increase was more marked in winter. The impact of chemical water quality was also dependent on the season. 4. An interaction between current velocity and pollution was also detected. During the summer, there was no difference in bacterial density or production between the upstream and downstream segments in the fast current zones, whereas both variables were higher downstream in the slow current zones. Such interactions between environmental factors and the impact of water quality on biofilms must be taken into account in assessments of the effects of chemicals on biofilm community structure and functioning in rivers.

International audience; The European Union’s Water Framework Directive has set a target of achieving good ecological status for all aquatic environments in Europe by 2015. In order to determine the quality of aquatic environments, biological indicators such as diatoms are often used. However, biotic diatom indices can be difficult and time consuming to use because of complexity of species determination. We investigated whether the biological traits of diatoms in rivers (life-forms, size classes and ecological guilds) could be used to assess organic pollution and trophic level. We worked on a data set comprising 315 diatom species, determined at 328 river stations of south-east France and a variety of parameters. The abundances of some biological traits differed significantly between the different organic pollution and trophic levels, particularly stalked diatoms, and the motile and low-profile guilds.

International audience; The effects of chemical and physical factors on periphyton structure, diversity and functioning were investigated in an outdoor mesocosm experiment. Stream biofilms were subjected to a pesticide mix (diuron and azoxystrobin) under two different hydraulic regimes. The hydraulic regimes differed by spatial variations of flow conditions (turbulent with high variations vs. laminar with low variations). The effects of the hydraulic regime and pesticides were assessed at the level of the periphytic communities. We focused on the change in the biodiversity of these communities under the two hydraulic regimes, and on the role of these biodiversity changes in case of pesticide contamination. Changes in structural (biomass, cell density), diversity (community composition assessed by PCR-DGGE and microscopic analysis) and functional (bacterial and algal production, sensitivity to the herbicide) parameters were monitored throughout a 2-month experiment. The results showed that exposure to pesticides affected the phytobenthic community targeted by the herbicide, impacting on both its growth dynamics and its primary production. Conversely, the impact of the flow regime was greater than that of pesticides on the non-target bacterial community with higher bacterial density and production in laminar mesocosms (uniform regime). An interaction between flow and pollution effects was also observed. Communities that developed in turbulent mesocosms (heterogeneous regime) were more diversified, as a result of increased microhabitat heterogeneity due to high spatial variations. However, this higher biodiversity did not increase the ability of these biofilms to tolerate pesticides, as expected. On the contrary, the sensitivity of these communities to pesticide contamination was, in fact, increased.

Recovery of bacterial and eukaryotic communities in biofilms naturally grown on stones was studied for 9 weeks after transferring them from a pesticide polluted downstream site of the river Morcille (Beaujolais, France) to a non-contaminated upstream site. Site-specific periphyton present on stones at both the down- and the upstream sampling site were collected to analyze the site-specific colonization. Throughout the experiment, structural and functional parameters were analyzed for the periphyton transferred and for the site-specific up- and downstream periphyton. Comparison between these three communities allowed quantifying recovery of the transferred one. Denaturing Gradient Gel Electrophoresis of PCR-amplified 16S and 18S rRNA gene fragments were used to assess prokaryotic and eukaryotic microbial community composition, respectively. Microscopy counts allowed characterizing the diatom taxa abundances. The sensitivity of the microalgal communities towards diuron and copper was investigated at the laboratory by short-term photosynthesis inhibition assays. The functional reaction of the bacterial communities towards copper was assessed by short-term respiration inhibition assays. The structure of transferred eukaryotic, bacterial and diatom communities was more similar to the structure of the downstream communities than to upstream ones even after 9 weeks acclimatization in particular for the bacterial community. In the same way, the community tolerance towards diuron and copper, as estimated by the EC50 values, was intermediate for the transferred biofilms compared to the local up- or downstream biofilm, even after 9 weeks of acclimatization. These results strongly suggest slow recovery, likely to be linked to long lasting exposure of pesticides and in particular copper adsorbed to the biofilm matrices and to the toughness for pioneer microorganisms to invade mature biofilms.

International audience; Developing new biological indicators for monitoring toxic substances is a major environmental challenge. Intensive agricultural areas are generally pesticide-dependent and generate water pollution due to transfer of pesticide residues through spray-drift, run-off and leaching. The ecological effects of these pollutants in aquatic ecosystems are broad-ranging owing to the variety of substances present (herbicides, fungicides, insecticides, etc.). Biofilms (or periphyton) are considered to be early warning systems for contamination detection and their ability to reveal effects of pollutants led researchers to propose a variety of methods to detect and assess the impact of pesticides. The present article sought to provide new insights into the ecological significance of biofilm microbial communities and to discuss their bioindication potential for water quality and land use by reporting on 4 years of research performed on the French Ardières-Morcille experimental watershed (AMEW). Various biological indicators have been applied during several surveys on AMEW, allowing the characterisation of (i) the structure and diversity of biofilm communities [community level finger printing (CLFP) such as PCRDGGE and pigment classes], (ii) functions associated with biofilm[community level physiological profiles (CLPP) such as extracellular enzymes, pesticides biodegradation or carbon sources biodegradation] and (iii) biofilm tolerance assessment (pollution-induced community tolerance, PICT) of the main contaminant in the AMEW (copper and diuron). Approaches based on CLFPs and PICT were consistent with each other and indicated the upstream-downstream impact due to the increasing land use by vineyards and the adaptation of algal and bacterial communities to the pollution gradient. CLPPs gave a contrasted bioindication because some parameters (most of the tested extracellular enzymes activities) did not detect a pollution gradient. Such CLPPs, CLFPs and PICT methods applied to biofilm could constitute the basis for a relevant in situ assessment both for chemical effects and aquatic ecosystem resilience.

International audience; DNA barcoding, being developed for biomonitoring, requires a database of reference sequences and knowledge of how much sequences can deviate before they are assigned to separate species. The molecular hunt for hidden species also raises the question of species definitions. We examined whether there are objective criteria for sequence-based species delimitation in diatoms, using Nitzschia palea, an important monophyletic indicator species already known to contain cryptic diversity. Strains from a wide geographical range were sequenced for 28S rRNA, COI and rbcL. Homogeneity indices and the Chao index failed to objectively select a precise number of species existing in N. palea as well as an evolutionary method based on coalescence theory. COI always gave higher diversity estimations than 28S rRNA or rbcL. Mating data did not provide a precise calibration of molecular species thresholds. Rarefaction curves indicated that further MOTUs would be detected with more isolates than we sampled (81 clones, 42 localities). Although some genotypes had intercontinental distributions, there was a positive relationship between genetic and geographical distance, suggesting even higher richness than we assessed, given that many regions were not sampled. Overall, no objective criteria were found for species separation; instead barcoding will need a consensual approach to molecular species limits.

24 páginas, 2 figuras, 1 tabla., The protection, preservation and restoration of aquatic ecosystems and their functions are of global importance. For European states it became legally binding mainly through the EUWater Framework Directive (WFD). In order to assess the ecological status of a given water body, aquatic biodiversity data are obtained and compared to a reference water body. The quantified mismatch obtained determines the extent of potential management actions. The current approach to biodiversity assessment is based on morpho-taxonomy. This approach has many drawbacks such as being time consuming, limited in temporal and spatial resolution, and error-prone due to the varying individual taxonomic expertise of the analysts. Novel genomic tools can overcome many of the aforementioned problems and could complement or even replace traditional bioassessment. Yet, a plethora of approaches are independently developed in different institutions, thereby hampering any concerted routine application. The goal of this Action is to nucleate a group of researchers across disciplines with the task to identify gold-standard genomic tools and novel ecogenomic indices for routine application in biodiversity assessments of European fresh- and marine water bodies. Furthermore, DNAqua-Net will provide a platform for training of the next generation of European researchers preparing them for the new technologies. Jointly with water managers, politicians, and other stakeholders, the group will develop a conceptual framework for the standard application of eco-genomic tools as part of legally binding assessments.

Marine biofilm communities that developed on artificial substrata were investigated using molecular and microscopic approaches. Polystyrene, Teflon® and four antifouling (AF) paints were immersed for 2 weeks at two contrasting sites near Toulon on the French Mediterranean coast (Toulon military harbour and the natural protected area of Porquerolles Island). Biofilms comprising bacteria and diatoms were detected on all the coatings. The population structure as well as the densities of the microorganisms differed in terms of both sites and coatings. Lower fouling densities were observed at Porquerolles Island compared to Toulon harbour. All bacterial communities (analysed by PCR-DGGE) showed related structure, controlled both by the sites and the type of substrata. Pioneer microalgal communities were dominated by the same two diatom species, viz. Licmophora gracilis and Cylindrotheca closterium, at both sites, irrespective of the substrata involved. However, the density of diatoms followed the same trend at both sites with a significant effect of all the AF coatings compared to Teflon and polystyrene.

Over the past 15 years, significant research efforts have been channeled into assessing the effects of organic herbicides on freshwater phototrophic microbial communities. The results of this research are reviewed herein. The main conclusions we have reached after performing this review can be summarized into five points: · Most relevant assessments have dealt with the effects of triazine and phenylurea herbicides. Herbicides from these chemical classes are often considered to be model compounds when photosystem-II inhibitors are studied. · Until the early 2000s, the vast majority of investigations conducted to evaluate herbicide effects on phototropic microbes were performed in microcosms or mesocosms. In such studies, herbicides were usually applied alone, and often at concentrations much higher than those detected in the environment. More recently, the trend has been toward more realistic and relevant studies, in which lower herbicide concentrations were considered, and compound mixtures or successive treatments were tested. Increasingly, in situ studies are being designed to directly evaluate microbial community responses, following chemical exposures in contaminated aquatic environments. · Several biological end points are used to evaluate how organisms in the phototrophic microbial community respond to herbicide exposure. These end points allow the detection of quantitative changes, such as chl a concentrations, total cell counts or periphytic biomass, qualitative changes such as community structure to algal diversity, or functional changes such as photosynthesis and respiration, among others. They may give different and complementary information concerning the responses of microbial communities. · PICT approaches, which have generally combined functional and structural measurements, may prove to be valuable for assessing both an immediate impact, and for factoring in the contamination history of an ecosystem at the community level. · Finally, any relevant assessment of pesticide effects should incorporate a detailed environmental characterization that would include abiotic parameters (light, flow speed, nutrient content), or biotic parameters (diversity and structure of biofilms), because these control the bioavailability of pesticides, and thereby the exposure of microbial communities. To improve the value of ecotoxicological risk assessments, future research is needed in two key areas: first, more information on the effects of pollutants at the community level must be obtained (new tools and new end points), and second, more effort must be directed to reinforce the ecological relevance of toxicological investigations.

International audience; Pollution of aquatic ecosystems by pesticide contamination is a major environmental concern. Numerous authors have addressed the frequent occurrence of chronic or acute herbicide contamination of freshwater ecosystems in both agricultural and urban areas of the world (Devault et al. 2007; Gilliom 2007; Schuler and Rand 2008; Woudneh et al. 2009). The physiological characteristics of photosynthetic microorganisms make them attractive as targets for herbicides in aquatic ecosystems. Since these primary producers form the basis of trophic structure in many aquatic environments, herbicides may threaten the entire equilibrium of the ecosystems they contaminate.

The potential of microbenthic algal assemblages to recover after diuron exposure was investigated. Microbenthic algal assemblages (periphyton) were grown on glass slides in correspondence to a diuron-polluted and a diuron-free sampling site of a river. After 5 weeks of colonization, the impacted periphyton was transferred by translocating the colonized glass slides to the unpolluted site. To monitor the changes in functional and structural parameters and to assess recovery, both the transferred and the local reference periphyton were sampled at the day of transfer (t0) and 1, 3, and 5 weeks after the transfer (t1, t3, t5). Structural transitions of eukaryotic communities were characterized by denaturing gradient gel electrophoresis (DGGE) and functional ones were characterized by short-term incubation toxicity tests with diuron. As shown by Bray–Curtis similarity values based on DGGE band patterns, almost total structural recovery of the transferred periphyton took place 5 weeks after transfer. For the transferred periphyton, previous diuron exposure at the contaminated site induced the development of diuron tolerance, which decreased after 1 week and became very similar to the tolerance of the nonimpacted community after 5 weeks. It is concluded that 5-week-old eukaryotic periphyton communities are capable of restoring their structural and functional attributes after 5 weeks within a noncontaminated environment.

An experimental study was undertaken to highlight the potential ecotoxicological impact of the herbicide diuron on biofilms during flooding events in a small river (Morcille) in the Beaujolais vineyard area (France). We investigated the responses of chronically contaminated biofilms exposed to short-term pulses (3 h) of diuron. Biofilms were grown in indoor microcosms that were either non-contaminated or exposed to low-level chronic contamination, and not exposed, or exposed to single or double pulses of two environmental concentrations (7 and 14 μg L−1) of diuron. Exposure to pollution and its impact on biofilms were assessed by measuring pesticide concentrations in biofilms, biomass parameters (chl a, AFDW), community structure (using 18S and 16S rDNA gene analysis by DGGE, and HPLC pigment analysis to target eukaryotes, bacteria and photoautotrophs, respectively) and by performing a physiological test. Control biofilms displayed very low diuron concentrations, whereas the herbicide was found in the contaminated biofilms. Nevertheless, diuron concentrations were not higher in the pulsed biofilms than in the non-pulsed ones. AFDW and chl a in vivo fluorescence increased in both microcosms during the experiment and biomass was higher in chronically exposed biofilms than in control ones. The impact on biomass was higher for the control double-pulsed biofilms than for the non-pulsed ones. Carbon incorporation by the chronically exposed biofilms was greater during the first 28 days of growth than during the first 28 days of growth in the control biofilms. Both single and double pulses inhibited carbon incorporation of all biofilm communities, especially of the control ones. Short-term inhibition of photosynthesis was never significantly different in exposed and non-exposed biofilms. Few differences in the pigment structure were found between chronically exposed and control biofilms, but pulses impacted on the pigment structure of all biofilm communities. Bacterial structural differences were observed between single-pulsed and non-pulsed biofilms, but not between double-pulsed and non-pulsed biofilms. The different pulses affected the eukaryotic community structures of the control biofilms, but not of the chronically exposed ones. Unlike the bacterial communities, the control eukaryotic communities were structurally different from the chronically exposed ones. This preliminary experimental study indicates that exposure to environmental concentrations of diuron and other agricultural contaminants and further exposure to diuron can have measurable effects on small river biofilm communities. The effects of a pulsed acute exposure to diuron on biofilms depended on whether the biofilms had previously been exposed to the same stressors or not.

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