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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, 238 U 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 238 U, suggesting its high tolerance to this particular radionuclide. This diatom species may therefore represent a bio-indicator of high natural levels of uranium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ecological assessment of lakes and rivers using benthic diatom assemblages currently requires considerable taxonomic expertise to identify species using light microscopy. This traditional approach is also time-consuming. Diatom metabarcoding is a promising alternative and there is increasing interest in using this approach for routine assessment. However, until now, analysis protocols for diatom metabarcoding have been developed and optimised by research groups working in isolation. The diversity of existing bioinformatics methods highlights the need for an assessment of the performance and comparability of results of different methods. The aim of this study was to test the correspondence of outputs from six bioinformatics pipelines currently in use for diatom metabarcoding in different European countries. Raw sequence data from 29 biofilm samples were treated by each of the bioinformatics pipelines, five of them using the same curated reference database. The outputs of the pipelines were compared in terms of sequence unit assemblages, taxonomic assignment, biotic index score and ecological assessment outcomes. The three last components were also compared to outputs from traditional light microscopy, which is currently accepted for ecological assessment of phytobenthos, as required by the Water Framework Directive. We also tested the performance of the pipelines on the two DNA markers (rbcL and 18S-V4) that are currently used by the working groups participating in this study. The sequence unit assemblages produced by different pipelines showed significant differences in terms of assigned and unassigned read numbers and sequence unit numbers. When comparing the taxonomic assignments at genus and species level, correspondence of the taxonomic assemblages between pipelines was weak. Most discrepancies were linked to differential detection or quantification of taxa, despite the use of the same reference database. Subsequent calculation of biotic index scores also showed significant differences between approaches, which were reflected in the final ecological assessment. Use of the rbcL marker always resulted in better correlation among molecular datasets and also in results closer to these generated using traditional microscopy. This study shows that decisions made in pipeline design have implications for the dataset's structure and the taxonomic assemblage, which in turn may affect biotic index calculation and ecological assessment. There is a need to define best-practice bioinformatics parameters in order to ensure the best representation of diatom assemblages. Only the use of similar parameters will ensure the compatibility of data from different working groups. The future of diatom metabarcoding for ecological assessment may also lie in the development of new metrics using, for example, presence/absence instead of relative abundance data., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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: R 2  ≈ 0.7, interception ≈ 0.8) and sensitive to global anthropogenic disturbance (Rs 2  > 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs., (Copyright © 2018 Elsevier B.V. All rights reserved.)