No lukewarm diatom communities-the response of freshwater benthic diatoms to phosphorus in streams as basis for a new phosphorus diatom index (PDISE)

In the present study, we developed a new Swedish phosphorus diatom index (PDISE) to improve the poor fit of existing indices to match the needs of water managers to detect and mitigate eutrophication. We took advantage of a large amount of data (820 Swedish stream sites) collected in recent years. During our work, we found an unexpected bimodal response of the diatom assemblages to phosphorus. The taxa clustered either into an assemblage with a low or with a high site-specific averaged TP optimum (a calculated value comprised of the diatom taxa-specific optima). We could not find a characteristic diatom assemblage for sites with intermediate site-specific averaged TP optima. To our knowledge, this bimodal community response has not been shown earlier. The PDISE correlated more strongly than the currently used TDI to changes in TP concentrations. Thus, the PDISE should replace the TDI in the Swedish standard method. The modeled TP optima (expressed as categories) were different compared to the TDI for most of the taxa included in the index, indicating that the realized niche for these morphotaxa was different between Sweden and the UK where the TDI was developed originally. With a r(2) of 0.68, the correlation of the PDISE to TP is among the highest reported for other diatom nutrient indices globally; thus, we believe that it might be worth to test it for other bioregions with similar geography and climate

Occurrence and mass development of Mougeotia Spp. (Zygnemataceae) in large, deep lakes

Over the last decades, mass developments by the filamentous conjugating green alga Mougeotia have been followed in three large peri-alpine lakes(Lake Geneva, Lake Garda, Lake Maggiore) and in the sub-tropical Lake Kinneret. The aim of this study is to highlight annual and interannual patterns of Mougeotia biomass in the studied lakes and select key environmental parameters that may favour and maintain its mass development. Our results confirm former studies that planktic Mougeotia favours meso-oligotrophic conditions and becomes dominant when annual mean total phosphorus concentrations in the epilimnion fall below 20 ?gl-1. This triggering factor has effect with interactions of other environmental circumstances such as the water column stability. Physiological and morphological features of the taxon make it a successful competitor under stratified conditions. Results also showed that in three out of the four studied lakes, the annual peak was higher when the annual population development started earlier. Focusing on Lake Geneva, depth and strength of the thermocline, as well as wind speed in the beginning of summer that can cause nutrient replenishment and mix the epilimnion are key factors in the blooming of the taxon

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

Assessing ecological status with diatoms DNA metabarcoding: Scaling-up on a WFD monitoring network (Mayotte island, France)

International audienceDiatoms are excellent ecological indicators of water quality because they are broadly distributed, they show high species diversity and they respond rapidly to human pressures. In Europe, the Water Framework Directive (WFD) gives the legal basis for the use of this indicator for water quality assessment and its management. Several quality indices, like the Specific Polluosensitivity Index (SPI), were developed to assess the ecological quality status of rivers based on diatom communities. It is based on morphological identifications and count of diatom species present in natural biofilms using a microscope. This methodology requires high taxonomic skills and several hours of analysis per sample as 400 individuals must be identified to species level. Since several years, a molecular approach based on DNA metabarcoding combined to High-Throughput Sequencing (HTS) is developed to characterize species assemblages in environmental samples which is potentially faster and cheaper. The ability of this approach to provide reliable diatom inventories has been demonstrated and its application to water quality assessment is currently being improved. Despite optimization of the DNA metabarcoding process with diatoms, few studies had yet extended it at the scale of a freshwater monitoring network and evaluated the reliability of its quality assessment compared to the classical morphological approach. In the present study we applied DNA metabarcoding to the river monitoring network of the tropical Island Mayotte. This island is a French departement since 2011 and the WFD has to be applied. This offered the opportunity to scale up the comparison of molecular and morphological approaches and their ability to produce comparable community inventories and water quality assessments. Benthic diatoms were sampled following WFD standards in 45 river sites in 2014 and 2015 (80 samples). All samples were submitted in parallel to the molecular and the morphological approaches. DNA metabarcoding was carried out using Genelute DNA extraction method, rbcL DNA barcode and PGM sequencing, while microscopic counts were carried out for the classical methodology. Diatom community structures in terms of molecular (OTUs) and of morphological (species) were significantly correlated. However, only 13% of the species was shared by both approaches, with qualitative and quantitative variation due to i) the incompleteness of the reference library (82% of morphological species are not represented in the database), ii) limits in taxonomic knowledge and iii) biases in the estimation of relative abundances linked to diatom cell biovolume. However, ecological quality status assessed with the molecular and morphological SPI values were congruent, and little affected by sequencing depth. DNA metabarcoding of diatom communities allowed a reliable estimation of the quality status for most of the rivers at the scale of the full biomonitoring network of Mayotte Island

Diatom DNA Metabarcoding for Biomonitoring: Strategies to Avoid Major Taxonomical and Bioinformatical Biases Limiting Molecular Indices Capacities

Recent years provided intense progression in the implementation of molecular techniques in a wide variety of research fields in ecology. Biomonitoring and bioassessment can greatly benefit from DNA metabarcoding and High-Throughput Sequencing (HTS) methods that potentially provide reliable, high quantity and quality standardized data in a cost- and time-efficient way. However, DNA metabarcoding has its drawbacks, introducing biases at all the steps of the process, particularly during bioinformatics treatments used to prepare HTS data for ecological analyses. The high diversity of bioinformatics methods (e.g., OTU clustering, chimera detection, taxonomic assignment) and parameters (e.g., percentage similarity threshold used to define OTUs) make inter-studies comparison difficult, limiting the development of standardized and easy-accessible bioassessment procedures for routine freshwater monitoring. In order to study and overcome these drawbacks, we constructed four de novo indices to assess river ecological status based on the same biological samples of diatoms analyzed with morphological and molecular methods. The biological inventories produced are (i) morphospecies identified by microscopy, (ii) OTUs provided via metabarcoding and hierarchical clustering of sequences using a 95% similarity threshold, (iii) individual sequence units (ISUs) via metabarcoding and only minimal bioinformatical quality filtering, and (iv) exact sequence variants (ESVs) using DADA2 denoising algorithm. The indices based on molecular data operated directly with ecological values estimated for OTUs/ISUs/ESVs. Our study used an approach of bypassing taxonomic assignment, so bias related to unclassified sequences missing from reference libraries could be handled and no information on ecology of sequences is lost. Additionally, we showed that the indices based on ISUs and ESVs were equivalent, outperforming the OTU-based one in terms of predictive power and accuracy by revealing the hidden ecological information of sequences that are otherwise clustered in the same OTU (intra-species/intra-population variability). Furthermore, ISUs, ESVs, and morphospecies indices provided similar estimation of site ecological status, validating that ISUs with limited bioinformatics treatments may be used for DNA freshwater monitoring. Our study is a proof of concept where taxonomy- and clustering-free approach is presented, that we believe is a step forward a standardized and comparable DNA bioassessment, complementary to morphological methods

Spatial- and niche segregation of DCM-forming cyanobacteria in Lake Stechlin (Germany)

At low trophic state, stable stratified water columns may provide favorable conditions for adapted phytoplankton species to form deep chlorophyll maxima (DCM). Such maxima occur regularly in Lake Stechlin, mainly contributed by the cyanobacterial species Cyanobium sp. and occasionally by Planktothrix rubescens. In the early twenty-first century, a rapid invasion by nostocalean cyanobacteria occurred in the lake and a number of Dolichospermum species together with Aphanizomenon flos-aquae appeared. As revealed by both microscopic and fluorimetric methods, during the summer stratification of 2013, a multispecific DCM was formed by Cyanobium, Planktothrix rubescens, and A. flos-aquae, however with spatial segregation. Planktothrix occurred in the upper hypolimnion, Aphanizomenon and Cyanobium dominated in the metalimnetic layer. Coexistence of these three cyanoprokaryota is possibly the consequence of different environmental factors limiting them (light, availability of N and P). This study represents a rare case when spatial niche segregation of phytoplankton species occurs in close to equilibrium conditions. DCM formed by Aphanizomenon and Cyanobium was detected by the fluoroprobe; Planktothrix with its different pigment compositions remained largely hidden. Our results indicate the necessity of parallel microscopic investigations and the need of careful calibration when fluorimetric methods are used for detecting cyanobacterial populations

Taxonomy- or trait-based ecological assessment for tropical rivers? Case study on benthic diatoms in Mayotte island (France, Indian Ocean)

Diatom-based ecological quality assessment methods have been implemented and used regularly in the Water Framework Directive. These indices use the species' abundance profiles along a specific environmental gradient, which they aim to assess. However, this approach has several problematic issues including the unstable and fast-changing diatom taxonomy. The use of traits can be a solution if their responses to the environmental pressure are well-defined. Here, we developed taxonomy-based and trait-based diatom assemblage indices to assess the ecological status of riverine sites on a tropical island. The two indices are based on two sub-indices that measure the diatom assemblage response to a nutrient and organic matter/turbidity gradient. Both taxonomy- and trait-based indices correlated significantly with the selected environmental gradients of the test database, which was not used during index development. We showed that traits could be used for quality assessment of the Mayotte rivers and require much less effort than taxonomy-based indices. There were differences between the two types of indices, which are discussed in this paper. As a perspective for further studies, tests of trait-based indices among different eco-regions would be challenging

The impact of OTU sequence similarity threshold on diatom-based bioassessment: A case study of the rivers of Mayotte (France, Indian Ocean)

International audienceExtensive studies on the taxonomic resolution required for bioassessment purposes have determined that resolution above species level (genus, family) is sufficient for their use as indicators of relevant environmental pressures. The high‐throughput se - quencing (HTS) and meta‐barcoding methods now used for bioassessment tradition - ally employ an arbitrary sequence similarity threshold (SST) around 95% or 97% to cluster sequences into operational taxonomic units, which is considered descriptive of species‐level resolution. In this study, we analyzed the effect of the SST on the resulting diatom‐based ecological quality index, which is based on OTU abundance distribution along a defined environmental gradient, ideally avoiding taxonomic as - signments that could result in high rates of unclassified OTUs and biased final values. A total of 90 biofilm samples were collected in 2014 and 2015 from 51 stream sites on Mayotte Island in parallel with measures of relevant physical and chemical param - eters. HTS sequencing was performed on the biofilms using the rbcL region as the genetic marker and diatom‐specific primers. Hierarchical clustering was used to group sequences into OTUs using 20 experimental SST levels (80%–99%). An OTU‐ based quality index (Idx OTU ) was developed based on a weighted average equation using the abundance profiles of the OTUs. The developed Idx OTU revealed significant correlations between the Idx OTU values and the reference pressure gradient, which reached maximal performance using an SST of 90% (well above species level delimi - tation). We observed an interesting and important trade‐off with the power to dis - criminate between sampling sites and index stability that will greatly inform future applications of the index. Taken together, the results from this study detail a thor - oughly optimized and validated approach to generating robust, reproducible, and complete indexes that will greatly facilitate effective and efficient environmental monitorin

Avoiding quantification bias in metabarcoding: Application of a cell biovolume correction factor in diatom molecular biomonitoring

International audience1. In recent years, remarkable progress has been made in developing environmental DNA metabarcoding. However, its ability to quantify species relative abundance remains uncertain, limiting its application for biomonitoring. In diatoms, although the rbcL gene appears to be a suitable barcode for diatoms, providing relevant qualitative data to describe taxonomic composition, improvement of species quantifica-tion is still required. 2. Here, we hypothesized that rbcL copy number is correlated with diatom cell biovol-ume (as previously described for the 18S gene) and that a correction factor (CF) based on cell biovolume should be applied to improve taxa quantification. We carried out a laboratory experiment using pure cultures of eight diatom species with contrasted cell biovolumes in order to (1) verify the relationship between rbcL copy numbers (estimated by qPCR) and diatom cell biovolumes and (2) define a potential CF. In order to evaluate CF efficiency, five mock communities were created by mixing different amounts of DNA from the eight species, and were sequenced using HTS and targeting the same rbcL barcode. 3. As expected, the correction of DNA reads proportions by the CF improved the congruence between morphological and molecular inventories. Final validation of the CF was obtained on environmental samples (metabarcoding data from 80 ben-thic biofilms) for which the application of CF allowed differences between molecular and morphological water quality indices to be reduced by 47%. 4. Overall, our results highlight the usefulness of applying a CF factor, which is effective in reducing over-estimation of high biovolume species, correcting quantitative biases in diatom metabarcoding studies and improving final water quality assessment. K E Y W O R D S benthic diatom, biovolume correction factor, freshwater ecosystems, gene copy number variation, quantitative metabarcodin

The potential of High-Throughput Sequencing (HTS) of natural samples as a source of primary taxonomic information for reference libraries of diatom barcodes

International audienceDiatoms are used routinely to assess pollution level in rivers and lakes. Current methods are based on identification by light microscopy, which is laborious. An alternative is to identify species based on short DNA fragments and High-Throughput Sequencing (HTS). However a potential limitation is the incomplete coverage of species in reference barcode libraries. Usually these libraries are compiled by isolating cells, before culturing and sequencing them, which is tedious and often unsuccessful. Here we propose the use of rbcL sequences from environmental samples analysed by HTS. We set several criteria to ensure good sequence quality and correspondence with the target species observed in microscopy: the sequence needed to be abundant in the sample, and with no insertions nor deletions or stop codon, phylogenetic neighbour taxa had to correspond to neighbour taxonomic taxa expected from morphological observations. Four species from tropical rivers are given as examples, including one that is new to science