Revisiting global diversity and biogeography of freshwater diatoms: New insights from molecular data

The distribution of microorganisms has long been assumed to be cosmopolitan and primarily controlled by the environment, but recent studies suggest that microbes may also exhibit strong biogeographical patterns driven by dispersal limitation. Past attempts to study the global biogeography of freshwater diatoms have always encountered the great difficulty of collecting taxonomically harmonized large-scale data. However, developments in molecular techniques and DNA metabarcoding provide a unique opportunity to overcome these limitations and to disclose diatom biodiversity at an unprecedented scale and resolution. Here, we assembled DNA metabarcoding data of freshwater benthic diatom communities sampled in seven geographic regions across the world to investigate how diatom diversity varies along latitude and to assess the proportion of genetic variants of these microorganisms which are exclusive or shared across regions. We observed significant differences in assemblages among climate zones and found that genetic richness is not affected by latitude, but by an island effect. The genetic resolution directly impacts the proportion of variants shared across regions; however, the majority of taxa remained specific to a single geographic region. Freshwater diatoms disperse over long distances and across oceans but at rates that allow the appearance of local genetic variants and the regionalization of assemblages. Future work should focus on putting these diversity dynamics into a temporal context, an approach that should be possible by bringing together new sequencing techniques and phylogeography

Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization

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 185-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. (C) 2020 The Authors. Published by Elsevier B.V

New molecular methods to assess biodiversity. Potentials and pitfalls of DNA metabarcoding: a workshop report

This report presents the outcome of the joint work of PhD students and senior researchers working with DNA-based biodiversity assessment approaches with the goal to facilitate others the access to definitions and explanations about novel DNA-based methods. The work was performed during a PhD course (SLU PNS0169) at the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden. The course was co-organized by the EU COST research network DNAqua-Net and the SLU Research Schools Focus on Soils and Water (FoSW) and Ecology - basics and applications. DNAqua-Net (COST Action CA15219, 2016-2020) is a network connecting researchers, water managers, politicians and other stakeholders with the aim to develop new genetic tools for bioassessment of aquatic ecosystems in Europe and beyond. The PhD course offered a comprehensive overview of the paradigm shift from traditional morphology-based species identification to novel identification approaches based on molecular markers. We covered the use of molecular tools in both basic research and applied use with a focus on aquatic ecosystem assessment, from species collection to the use of diversity in environmental legislation. The focus of the course was on DNA (meta)barcoding and aquatic organisms. The knowledge gained was shared with the general public by creating Wikipedia pages and through this collaborative Open Access publication, co-authored by all course participants

New methods for improving water management - Exploring the role of diatoms in ecosystems

Diatoms are photosynthetic microalgae which are well known to react quickly to environmental changes and are widely used as bioindicators within Water Framework Directive. However, diatom identification by light microscopy is time-consuming and error-prone, creating a need for alternative, faster and more reproducible methods for characterising diatom communities. This thesis describes the development and application of two such methods: a molecular approach using metabarcoding and a trait-based approach using combinations of different diatom morphological traits. Both approaches were shown to have potential for use in environmental assessment, as they revealed the response of diatoms to environmental change and enabled better representation of the diatom community. However, results from the two approaches were not directly comparable to results obtained by conventional microscopy and diatom DNA and trait data should not be used with tools calibrated for morphotaxa data. Discrepancies in molecular output data between laboratories revealed a strong need for standardisation and best-practice guidelines. Nutrient indices developed for each novel approach performed well and showed potential for use in assessment of total phosphorus levels in Fennoscandia freshwater. The molecular-based index showed differences in performance for stream and lake samples. The trait-based index performed equally well for both types of site, but current limitation of this index is that selection of meaningful traits must be done for each new gradient. Further work is needed on diatom traits and their response to environmental changes, to obtain reproducible and comparable molecular outputs and to continue the development of new indices calibrated to diatom DNA and trait data

Same same, but different: The response of diatoms to environmental gradients in Fennoscandian streams and lakes – barcodes, traits and microscope data compared

We developed and compared the performance of freshwater benthic diatom indices calculated from (i) traditional morphological species identification, (ii) Amplicon Sequence Variants (ASVs) obtained via DNA metabarcoding, and (iii) morphological traits to indicate eutrophication in rivers and lakes in Fennoscandia. Based on the results, we provided recommendations for the future routine use of diatom bioassessment tools in environmental monitoring and assessment. Our results show that ASVs are the most promising candidates to be used in environmental assessment. Indices based on ASVs correlated better with TotP concentrations than morphological taxa data, whereas the trait indices correlated least. We could see by studying the taxonomic assignments of the ASVs that traditional morphotaxa were divided up into several ASVs with different ecological profiles, which explained part of the better index performance and also encourages further studies on diatom diversity and ecological preferences. In general, ASV- and morphotaxon-specific optima differed slightly between streams and lakes, but were significantly correlated with each other. This means that it should be possible to develop a common index that is applicable in both streams and lakes, but boundary values with respect to TotP might need to be set separately for them. More knowledge on diatom traits is required to enable their use for environmental assessment

Same same, but different : The response of diatoms to environmental gradients in Fennoscandian streams and lakes – barcodes, traits and microscope data compared

Highlights • Amplicon Sequence Variants (ASVs) are a promising unit for diatom nutrient indices. • Diatom indices based on morphology, ASVs and traits were related to TotP concentrations. • Correlations of indices to TotP was best for ASVs, followed by morphology and traits.We developed and compared the performance of freshwater benthic diatom indices calculated from (i) traditional morphological species identification, (ii) Amplicon Sequence Variants (ASVs) obtained via DNA metabarcoding, and (iii) morphological traits to indicate eutrophication in rivers and lakes in Fennoscandia. Based on the results, we provided recommendations for the future routine use of diatom bioassessment tools in environmental monitoring and assessment. Our results show that ASVs are the most promising candidates to be used in environmental assessment. Indices based on ASVs correlated better with TotP concentrations than morphological taxa data, whereas the trait indices correlated least. We could see by studying the taxonomic assignments of the ASVs that traditional morphotaxa were divided up into several ASVs with different ecological profiles, which explained part of the better index performance and also encourages further studies on diatom diversity and ecological preferences. In general, ASV- and morphotaxon-specific optima differed slightly between streams and lakes, but were significantly correlated with each other. This means that it should be possible to develop a common index that is applicable in both streams and lakes, but boundary values with respect to TotP might need to be set separately for them. More knowledge on diatom traits is required to enable their use for environmental assessment

Molecular versus morphological data for benthic diatoms biomonitoring in Northern Europe freshwater and consequences for ecological status

International audienceDiatoms are known to be efficient bioindicators for water quality assessment because of their rapid response to environmental pressures and their omnipresence in water bodies. The identification of benthic diatoms communities in the biofilm, coupled with quality indices such as the Indice de polluosensibilité spécifique (IPS) can be used for biomonitoring purposes in freshwater. However, the morphological identification and counting of diatoms species under the microscope is time-consuming and requires extensive expertise to deal with a constantly evolving taxonomy. In response, a molecular-based and potentially more cost-effective method has been developed, coupling high-throughput sequencing and DNA metabarcoding. The method has already been tested for water quality assessment with diatoms in Central Europe. In this study, we applied both the traditional and molecular methods on 180 biofilms samples from Northern Europe (rivers and lakes of Fennoscandia and Iceland). The DNA metabarcoding data were obtained on two different DNA markers, the 18S-V4 and rbcL barcodes, with the NucleoSpin Soil kit for DNA extraction and sequenced on an Ion Torrent PGM platform. We assessed the ability of the molecular method to produce species inventories, IPS scores and ecological status class comparable to the ones generated by the traditional morphology-based approach. The two methods generated correlated but significantly different IPS scores and ecological status assessment. The observed deviations are explained by presence/absence and abundance discrepancies in the species inventories, mainly due to the incompleteness of the barcodes reference databases, primer bias and strictness of the bioinformatic pipeline. Abundance discrepancies are less common than presence/absence discrepancies but have a greater effect on the ecological assessment. Missing species in the reference databases are mostly acidophilic benthic diatoms species, typical of the low pH waters of Northern Europe. The two different DNA markers also generated significantly different ecological status assessments. The use of the 18S-V4 marker generates more species inventories discrepancies, but achieves an ecological assessment more similar to the traditional morphology-based method. Further development of the metabarcoding method is needed for its use in environmental assessment. For its application in Northern Europe, completion and curation of reference databases are necessary, as well as evaluation of the currently available bioinformatics pipelines. New indices, fitted for environmental biomonitoring, should also be developed directly from molecular data

Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization

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 185-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

Revisiting global diversity and biogeography of freshwater diatoms: New insights from molecular data

International audienceAbstract The distribution of microorganisms has long been assumed to be cosmopolitan and primarily controlled by the environment, but recent studies suggest that microbes may also exhibit strong biogeographical patterns driven by dispersal limitation. Past attempts to study the global biogeography of freshwater diatoms have always encountered the great difficulty of collecting taxonomically harmonized large‐scale data. However, developments in molecular techniques and DNA metabarcoding provide a unique opportunity to overcome these limitations and to disclose diatom biodiversity at an unprecedented scale and resolution. Here, we assembled DNA metabarcoding data of freshwater benthic diatom communities sampled in seven geographic regions across the world to investigate how diatom diversity varies along latitude and to assess the proportion of genetic variants of these microorganisms which are exclusive or shared across regions. We observed significant differences in assemblages among climate zones and found that genetic richness is not affected by latitude, but by an island effect. The genetic resolution directly impacts the proportion of variants shared across regions; however, the majority of taxa remained specific to a single geographic region. Freshwater diatoms disperse over long distances and across oceans but at rates that allow the appearance of local genetic variants and the regionalization of assemblages. Future work should focus on putting these diversity dynamics into a temporal context, an approach that should be possible by bringing together new sequencing techniques and phylogeography