Assessing the relevance of DNA metabarcoding compared to morphological identification for lake phytoplankton monitoring.

Phytoplankton is a key biological group used to assess the ecological status of lakes. The classical monitoring approach relies on microscopic identification and counting of phytoplankton species, which is time-consuming and requires high taxonomic expertise. High-throughput sequencing, combined with metabarcoding, has recently demonstrated its potential as an alternative approach for plankton surveys. Several studies have confirmed the relevance of the diatom metabarcoding approach to calculate biotic indices based on species ecology. However, phytoplankton communities have not yet benefited from such validation. Here, by comparing the results obtained with the two methods (molecular and microscopic counting), we evaluated the relevance of metabarcoding approach for phytoplankton monitoring by considering different metrics: alpha diversity, taxonomic composition, community structure and a phytoplankton biotic index used to assess the trophic level of lakes. For this purpose, 55 samples were collected in four large alpine lakes (Aiguebelette, Annecy, Bourget, Geneva) during the year 2021. For each sample, a metabarcoding analysis based on two genetic markers (16S and 23S rRNA) was performed, in addition to the microscopic count. Regarding the trophic level of lakes, significant differences were found between index values obtained with the two approaches. The main hypothesis to explain these differences comes from the incompleteness, particularly at the species level, of the barcode reference library for the two genetic markers. It is therefore necessary to complete reference libraries for using such species-based biotic indices with metabarcoding data. Besides this, species richness and diversity were higher in the molecular inventories than in the microscopic ones. Moreover, despite differences in taxonomic composition of the floristic lists obtained by the two approaches, their community structures were similar. These results support the possibility of using metabarcoding for phytoplankton monitoring but in a different way. We suggest exploring alternative approaches to index development, such as a taxonomy-free approach.

Evidence of Bacterial Community Coalescence between Freshwater and Discharged tpm-Harboring Bacterial Taxa from Hospital and Domestic Wastewater Treatment Plants among Epilithic Biofilms.

The ability of WWTP outflow bacteria at colonizing rock surfaces and contributing to the formation of river epilithic biofilms was investigated. Bacterial community structures of biofilms (b-) developing on rocks exposed to treated wastewaters (TWW) of a hospital (HTWW) and a domestic (DTWW) clarifier, and to surface waters of the stream located at 10 m, 500 m, and 8 km from the WWTP outlet, were compared. Biofilm bacterial contents were analyzed by cultural approaches and a tpm-based DNA metabarcoding analytical scheme. Co-occurrence distribution pattern analyses between bacterial datasets and eighteen monitored pharmaceuticals were performed. Higher concentrations of iohexol, ranitidine, levofloxacin, and roxithromycin were observed in the b-HTWW while atenolol, diclofenac, propranolol, and trimethoprim were higher in the b-DTWW. MPN growth assays showed recurrent occurrences of Pseudomonas aeruginosa and Aeromonas caviae among these biofilms. An enrichment of multi-resistant P. aeruginosa cells was observed in the hospital sewer line. P. aeruginosa MPN values were negatively correlated to roxithromycin concentrations. The tpm DNA metabarcoding analyses confirmed these trends and allowed an additional tracking of more than 90 species from 24 genera. Among the recorded 3082 tpm ASV (amplicon sequence variants), 41% were allocated to the Pseudomonas. Significant differences through ANOSIM and DESeq2 statistical tests were observed between ASV recovered from b-HTWW, b-DTWW, and epilithic river biofilms. More than 500 ASV were found restricted to a single sewer line such as those allocated to Aeromonas popoffii and Stenotrophomonas humi being strictly found in the b-HTWW file. Several significant correlations between tpm ASV counts per species and pharmaceutical concentrations in biofilms were recorded such as those of Lamprocystis purpurea being positively correlated with trimethoprim concentrations. A tpm source tracking analysis showed the b-DTWW and b-HTWW tpm ASV to have contributed, respectively, at up to 35% and 2.5% of the epilithic river biofilm tpm-taxa recovered downstream from the WWTP outlet. Higher contributions of TWW taxa among epilithic biofilms were recorded closer to the WWTP outlet. These analyses demonstrated a coalescence of WWTP sewer communities with river freshwater taxa among epilithic biofilms developing downstream of a WWTP outlet.

Diatom DNA metabarcoding to assess the effect of natural radioactivity in mineral springs on ASV of benthic diatom communities.

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.

Diatom endemism and taxonomic turnover: Assessment in high-altitude alpine lakes covering a large geographical range.

Diatoms are widely used as ecological indicators and show various degrees of endemism. Many studies that support the idea of endemic species integrate several climate zones, a variety of ecosystem types, and often focus on a global scale. Here, we investigated whether endemism could be detected when considering a homogeneous type of ecosystem in a single climate zone. We sampled stone biofilms at 40-50 cm depth in high-altitude lakes in the Alpine climate zone. A total of 149 samples were obtained from the French and Georgian mountains, two areas separated by ∼3000 km. Using Amplicon Sequence Variants derived from DNA metabarcoding, we assessed taxonomic turnover and Zeta-diversity (a measure of endemism). We ran haplotype networks and phylogenetic tests to measure geographical signal in the phylogenies of dominant taxa. The French and Georgian communities shared 51 % of species. Species that were not shared across both regions were mostly rare, and often not characteristic of lakes but of neighboring habitats instead. In contrast, at the sub-species level, 87 % of the genotypes showed restricted distributions. Whereas endemism was the rule at sub-species level, most species were shared across both French and Georgian lakes, suggesting that geographic barriers strongly limited dispersal at the sub-species level but not species level. Dominant species hosted higher levels of sub-specific diversity than rare species. In contrast to global-scale studies, we did not find any significant geographical structuring in the phylogeny of the investigated species. This could indicate ongoing dispersal at a frequency fast enough to prevent allopatric divergence, yet slow enough to prevent sharing most haplotypes between France and Georgia. These results have implications for biomonitoring: depending on the taxonomic level chosen, robust generic tools (species level) or tools dedicated to a region able to discriminate fine pressures differences (sub-species level) may be developed.

Environmental filtering and mass effect are two important processes driving lake benthic diatoms: Results of a DNA metabarcoding study in a large lake.

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.

Passive sampling of environmental DNA in aquatic environments using 3D-printed hydroxyapatite samplers.

The study of environmental DNA (eDNA) released by aquatic organisms in their habitat offers a fast, noninvasive and sensitive approach to monitor their presence. Common eDNA sampling methods such as water filtration and DNA precipitation are time-consuming, require difficult-to-handle equipment and partially integrate eDNA signals. To overcome these limitations, we created the first proof of concept of a passive, 3D-printed and easy-to-use eDNA sampler. We designed the samplers from hydroxyapatite (HAp samplers), a natural mineral with a high DNA adsorption capacity. The porous structure and shape of the samplers were designed to optimize DNA adsorption and facilitate their handling in the laboratory and in the field. Here we show that HAp samplers can efficiently collect genomic DNA in controlled set-ups, but can also collect animal eDNA under controlled and natural conditions with yields similar to conventional methods. However, we also observed large variations in the amount of DNA collected even under controlled conditions. A better understanding of the DNA-hydroxyapatite interactions on the surface of the samplers is now necessary to optimize eDNA adsorption and to allow the development of a reliable, easy-to-use and reusable eDNA sampling tool.

SIPIBEL observatory: Data on usual pollutants (solids, organic matter, nutrients, ions) and micropollutants (pharmaceuticals, surfactants, metals), biological and ecotoxicity indicators in hospital and urban wastewater, in treated effluent and sludge from wastewater treatment plant, and in surface and groundwater.

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 Léman 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 Rhône 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.

Fish eDNA metabarcoding from aquatic biofilm samples: Methodological aspects.

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.

Herbicide-Induced Shifts in the Periphyton Community Composition Indirectly Affect Feeding Activity and Physiology of the Gastropod Grazer Physella acuta.

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 µg/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.

A pilot experiment to assess the efficiency of pharmaceutical plant wastewater treatment and the decreasing effluent toxicity to periphytic biofilms.

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.

Cyprus' diatom diversity and the association of environmental and anthropogenic influences for ecological assessment of rivers using DNA metabarcoding.

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.

Exploring the capacity of aquatic biofilms to act as environmental DNA samplers: Test on macroinvertebrate communities in rivers.

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.

Monitoring the ecological status of rivers with diatom eDNA metabarcoding: A comparison of taxonomic markers and analytical approaches for the inference of a molecular diatom index.

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

Ecosystems monitoring powered by environmental genomics: A review of current strategies with an implementation roadmap.

A decade after environmental scientists integrated high-throughput sequencing technologies in their toolbox, the genomics-based monitoring of anthropogenic impacts on the biodiversity and functioning of ecosystems is yet to be implemented by regulatory frameworks. Despite the broadly acknowledged potential of environmental genomics to this end, technical limitations and conceptual issues still stand in the way of its broad application by end-users. In addition, the multiplicity of potential implementation strategies may contribute to a perception that the routine application of this methodology is premature or "in development", hence restraining regulators from binding these tools into legal frameworks. Here, we review recent implementations of environmental genomics-based methods, applied to the biomonitoring of ecosystems. By taking a general overview, without narrowing our perspective to particular habitats or groups of organisms, this paper aims to compare, review and discuss the strengths and limitations of four general implementation strategies of environmental genomics for monitoring: (a) Taxonomy-based analyses focused on identification of known bioindicators or described taxa; (b) De novo bioindicator analyses; (c) Structural community metrics including inferred ecological networks; and (d) Functional community metrics (metagenomics or metatranscriptomics). We emphasise the utility of the three latter strategies to integrate meiofauna and microorganisms that are not traditionally utilised in biomonitoring because of difficult taxonomic identification. Finally, we propose a roadmap for the implementation of environmental genomics into routine monitoring programmes that leverage recent analytical advancements, while pointing out current limitations and future research needs.

Environmental filtering and limiting similarity as main forces driving diatom community structure in Mediterranean and continental temporary and perennial streams.

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.

Evaluation and sensitivity analysis of diatom DNA metabarcoding for WFD bioassessment of Mediterranean rivers.

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

A taxonomy-free approach based on machine learning to assess the quality of rivers with diatoms.

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.

Benthic diatom community dynamics in Mediterranean intermittent streams: Effects of water availability and their potential as indicators of dry-phase ecological status.

The study of intermittent rivers is a critical and timely issue due to their worldwide increase, triggered by several causes including climate change. The need to understand the response of intermittent river biota to water intermittency led us to conduct this study using benthic diatoms collected in southern Portugal. Benthic diatoms were explored in terms of assemblages, diversity indices, the Specific Pollution Sensitivity (SPI) Index, functional metrics (i.e. ecological guilds and life-forms) and conservation status. We verified that changes in water physico-chemical characteristics were highly controlled by flow intermittency, which in turn is directly linked to meteorological variables (air temperature and precipitation). Changes in diatom assemblages reflect the aquatic regime of sites, changes in aquatic states through time and mesohabitats (dry biofilm, samples collected in pools or under flowing conditions). Species richness, on the other hand, did not reflect these differences, whilst Shannon diversity and Pielou's Evenness indices only reflected mesohabitat differences. The SPI distinguished sampling periods, and mesohabitats. The relative abundance of ecological guilds changed with aquatic states, with the low-profile guild dominating in eurheic and arheic conditions (except during Summer), being replaced by motile taxa in summer arheic conditions, reflecting increases in nutrient and siltation. The hypothesis that benthic diatom assemblages in dry biofilm can be used as an indicator of ecological status during the dry-phase was validated, since no differences between the Ecological Quality Ratio determined in dry biofilm collected in Summer 2017 and the previous Spring 2017 in flowing water. A method is proposed for diatom sampling in dry biofilm, contributing to an integrated ecological status evaluation, which considers the dry-phase and enhances the reach of biomonitoring programs.

The vertical distribution of tetA and intI1 in a deep lake is rather due to sedimentation than to resuspension.

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.

Diat.barcode, an open-access curated barcode library for diatoms.

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 .