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.

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.

Ripe for reassessment: A synthesis of available molecular data for the speciose diatom family Bacillariaceae.

The Bacillariaceae is a very species-rich family of raphid diatoms and includes the large and taxonomically difficult genus Nitzschia, whose species are often small-celled and finely structured and have few discrete morphological characters visible in the light microscope. The classification of Nitzschia is still mostly based on one developed in the second half of the 19th century by Grunow, who separated the genus into a series of sections largely on cell shape and symmetry, the position of the raphe, transverse extension of the fibulae, and folding of the valve. We assembled and analysed single-gene and concatenated alignments of nSSU, nLSU, rbcL, psbC and cox1 to test Grunow's and subsequent classifications and to examine selected morphological characters for their potential to help define monophyletic groups. The maximum likelihood trees were equivocal as to monophyly of the family itself but showed good support for each of eight main clades of Bacillariaceae, three of which corresponded more or less to existing genera (Hantzschia, Cylindrotheca and Bacillaria). The other five main clades and some subclades comprised groups of Nitzschia species or assemblies of Nitzschia species with other genera (Pseudo-nitzschia, Fragilariopsis, Neodenticula, Tryblionella, Psammodictyon). Relationships between most of the eight main clades were not resolved robustly but all analyses recovered Nitzschia as non-monophyletic. The Grunowian classification of Nitzschia into sections was not supported, though in some respects (e.g. treatment of sigmoid species) it is better than subsequent reclassifications. Several of the main clades and subclades are cryptic (lacking morphological synapomorphies) and homoplasy is common in both light microscopical and ultrastructural characters (to the extent that organisms initially assigned to the same species sometimes prove to belong to a different main clade). Nevertheless, some characters, including the structure of the raphe canal and girdle, seem to be sufficiently conservative evolutionarily to give a provisional estimate of relationships if molecular data are unavailable. No new formal classifications are proposed but various options are explored and research needs identified.

Connecting the morphological and molecular species concepts to facilitate species identification within the genus Fragilaria (Bacillariophyta).

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 rbcL 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 rbcL, 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.

Can we predict diatoms herbicide sensitivities with phylogeny? Influence of intraspecific and interspecific variability.

Diatoms are used as indicators of freshwater ecosystems integrity. Developing diatom-based tools to assess impact of herbicide pollution is expected by water managers. But, defining sensitivities of all species to multiple herbicides would be unattainable. The existence of a phylogenetic signal of herbicide sensitivity was shown among diatoms and should enable prediction of new species sensitivity. However, diatoms present a cryptic diversity that may lead to variation in their sensitivity to herbicides that would need to be taken into account. Using bioassays, the sensitivity to four herbicides (Atrazine, Terbutryn, Diuron, Isoproturon) was evaluated for 11 freshwater diatom taxa and intraspecific variability was assessed for two of them (Nitzschia palea and Achnanthidium spp.). Intraspecific variability of herbicide sensitivity was always smaller than interspecific variability, but intraspecific variability was more important in N. palea than in Achnanthidium spp. Indeed, one species showed no intraspecific phylogenetic signal (N. palea) whereas the other did (Achnanthidium spp.). On one hand, species boundaries are not set properly for Achnanthidium spp. which encompass several taxa. On the other hand, there is a higher phenotypic plasticity for N. palea. Finally, a phylogenetic signal of herbicide sensitivity was measured at the interspecific level, opening up prospects for setting up reliable biomonitoring tools based on sensitivity prediction, insofar as species boundaries are correctly defined.

Phylogenetic signal in diatom ecology: perspectives for aquatic ecosystems biomonitoring.

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.

Linking diatom sensitivity to herbicides to phylogeny: a step forward for biomonitoring?

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 II 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.

Seasonal shift in the sensitivity of a natural benthic microalgal community to a herbicide mixture: impact on the protective level of thresholds derived from species sensitivity distributions.

Seasonal changes in the structure and composition of a benthic microalgal community may lead to different responses to herbicide contamination during different seasons. Consequently, the thresholds derived from risk assessment tools such as species sensitivity distributions (SSDs) must allow for these changes. We built a single-substance SSD for each of four herbicides (atrazine, terbutryn, diuron and isoproturon), which was specific to the sensitivity of the benthic diatoms found in Lake Geneva, in order to derive protective thresholds for a mixture of these four herbicides using the concentration addition model. We then investigated (1) the structural parameters of a Lake Geneva benthic microalgal community during two contrasting seasons (summer 2012 and winter 2013), (2) the response of these communities to a herbicide mixture, and (3) the protective levels of the thresholds derived. The winter community was characterized by having greater biomass, diatom species richness, and diversity metrics, and lower non-diatom species richness than the summer community. The differences in the diatom communities composition in these seasons appeared to be primarily driven by the environmental nitrate concentrations and the temperature. Moreover, the species in the winter community were more resistant to herbicides than those found in the summer community. Consequently, the protective threshold for this herbicide mixture obtained in this study was in fact protective for the winter community, but not for the summer community based on their structural parameters. Thus, the protective level against herbicides of the threshold for the benthic microalgal community should take into account changes in the environmental physico-chemical conditions that strongly influence the structure and composition of the community. The fact that the succession of species over time (i.e., over the seasons) is difficult to predict introduces uncertainties into the estimation of protective thresholds and questions their applicability year round.

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.

Morphology and phylogeny of Nitzschianandorii sp. nov. (Bacillariophyceae), a new small-celled lanceolate species from a post-mining reservoir.

Post-mining reservoirs are distinguished by characteristic environmental conditions where specific diatom communities can be observed. Reservoirs created as a part of the reclamation plan after human mining activities are marked by unique chemical and physical water parameters. In the course of research on the diatoms from Bogdalów reservoir, we examined the taxonomic and morphological diversity of Nitzschia taxa from the section Lanceolatae occurring in a post-mining lignite reservoir. Our study describes a new species of Nitzschia from a post-mining reservoir, Nitzschianandorii Olszynski, Zakrzewski & Zelazna-Wieczorek, sp. nov. Morphometry and morphology analyses of new species were performed with light and scanning electron microscopy. Chloroplast morphology analysis was conducted with differential interference contrast microscopy and confocal laser scanning microscopy. Molecular data from SSU 18S, rbcL and psbC sequences were obtained from cultures of this taxon. Differential diagnosis of Nitzschianandorii Olszynski, Zakrzewski & Zelazna-Wieczorek, sp. nov. with co-occurring taxa: N.lacuum and N.alpinobacillum was performed using morphological traits and nMDS analysis of the valves' morphometry.

Combined Earth observations reveal the sequence of conditions leading to a large algal bloom in Lake Geneva.

Freshwater algae exhibit complex dynamics, particularly in meso-oligotrophic lakes with sudden and dramatic increases in algal biomass following long periods of low background concentration. While the fundamental prerequisites for algal blooms, namely light and nutrient availability, are well-known, their specific causation involves an intricate chain of conditions. Here we examine a recent massive Uroglena bloom in Lake Geneva (Switzerland/France). We show that a certain sequence of meteorological conditions triggered this specific algal bloom event: heavy rainfall promoting excessive organic matter and nutrients loading, followed by wind-induced coastal upwelling, and a prolonged period of warm, calm weather. The combination of satellite remote sensing, in-situ measurements, ad-hoc biogeochemical analyses, and three-dimensional modeling proved invaluable in unraveling the complex dynamics of algal blooms highlighting the substantial role of littoral-pelagic connectivities in large low-nutrient lakes. These findings underscore the advantages of state-of-the-art multidisciplinary approaches for an improved understanding of dynamic systems as a whole.

The effect of temperature and a herbicide mixture on freshwater periphytic algae.

Temperature is a strong driver of biofilm formation and of the dynamics of microalgae in freshwater. Moreover, exposure to herbicides is a well-known stressor of periphytic communities in anthropized aquatic environments. We tested these two environmental factors on periphytic communities that had been sampled from the littoral zone of Lake Geneva and acclimatized in the lab for 3 weeks at 18, 21, 24 and 28 °C. After this acclimation period, differences in the composition of the diatom community and decreases in cell density were observed corresponding to the temperature gradient. These acclimated communities were then exposed to 23 and 140 nM of a mixture composed of equitoxic quantities of atrazine, terbutryn, diuron and isoproturon. The periphytic community was more sensitive to the herbicide mixture at 18 °C than at higher temperatures, suggesting that higher temperature reduced its toxicity. Small and pioneer diatom species known to be promoted by contamination also appeared to benefit from higher temperatures. Temperature therefore appears to condition the herbicide sensitivity of periphytic communities.

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.

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.

Pioneer marine biofilms on artificial surfaces including antifouling coatings immersed in two contrasting French Mediterranean coast sites.

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.

Risk of herbicide mixtures as a key parameter to explain phytoplankton fluctuation in a great lake: the case of Lake Geneva, Switzerland.

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 co-variables. 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.

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.

DNA sequence and taxonomic gap analyses to quantify the coverage of aquatic cyanobacteria and eukaryotic microalgae in reference databases: Results of a survey in the Alpine region.

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

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.

DNA metabarcoding reveals differences in distribution patterns and ecological preferences among genetic variants within some key freshwater diatom species.

Our study evaluates differences in the distribution and ecology of genetic variants within several ecologically important diatom species that are also key for Water Framework Directive monitoring of European rivers: Fistulifera saprophila (FSAP), Achnanthidium minutissimum (ADMI), Nitzschia inconspicua (NINC) and Nitzschia soratensis (NSTS). We used DADA2 to infer amplicon sequence variants (ASVs) of a short rbcL barcode in 531 environmental samples from biomonitoring campaigns in Catalonia and France. ASVs within each species showed different distribution patterns. Threshold Indicator Taxa ANalysis revealed three ecological groupings of ASVs in both ADMI and FSAP. Two of these in each species were separated by opposite responses to calcium and conductivity. Boosted regression trees additionally showed that both variables greatly influenced the occurrence of these groupings. A third grouping in FSAP was characterized by a negative response to total organic carbon and hence was better represented in waters with higher ecological status than the other FSAP ASVs, contrasting with what is generally assumed for the species. In the two Nitzschia species, our analyses confirmed earlier studies: NINC preferred higher levels of calcium and conductivity. Our findings suggest that the broad ecological tolerance of some diatom species results from overlapping preferences among genetic variants, which individually show much more restricted preferences and distributions. This work shows the importance of studying the ecological preferences of genetic variants within species complexes, now possible with DNA metabarcoding. The results will help reveal and understand biogeographical distributions and facilitate the development of more accurate biological indexes for biomonitoring programmes.