Co-occurrence, ecological profiles and geographical distribution based on unique molecular identifiers of the common freshwater diatoms Fragilaria and Ulnaria

Diatom taxonomy has evolved in recent years, with many new species described and new approaches such as molecular genetics showing the existence of cryptic diversity within currently accepted species. This cryptic diversity is not well understood even for common freshwater genera such as Fragilaria and Ulnaria. The purpose of our study was to define taxon-specific ecological profiles and geographical distributions for unique molecular identifiers (amplicon sequence variants, ASVs) linked to curated taxonomy for these genera. Our goal is to contribute to the development of ecological assessment methods, and to the understanding why we often observe so many diatom species co-occurring in a single sample. We filtered a large (770 samples) metabarcoding dataset with linked environmental data covering several countries in Europe for genetic variants (ASVs) assigned to currently accepted species of our target genera. We studied the geographical distribution of the ASVs, and tested for ASV-pair co-occurrence. We modelled ASV-specific preferences for pH, alkalinity, total nitrogen, total phosphorus and conductivity, and analysed their preference for lakes or streams as habitat. Our study confirmed that there seems to be no general geographical barrier for the distribution of freshwater benthic diatom ASVs in Europe, but that dispersal is not rapid enough to hide historical events. The Fragilaria and Ulnaria ASVs in our study showed considerable overlap in geographical distribution, habitat and ecological preferences. We found evidence that only large differences in preferences for the analysed water chemistry variables prevented the co-occurrence of ASVs at the same sites. Instead, Fragilaria and Ulnaria ASVs co-occurred frequently in samples. We found subtle differences in ecological preferences for some ASV pairs, which might in part explain the co-occurrence by the avoidance of direct competition. However, the great overlap in distribution and ecological preferences suggests that other factors not studied here were also responsible for the observed co-occurrences and high richness of ASVs found at many sites. To our knowledge, we are the first to use ASVs in combination with a curated taxonomy to understand co-occurrence, specific ecological profiles and large-scale geographical distribution for unique identifiers not biased by the quality of reference databases, clustering methods, or non-harmonized morphological identification. Thus, our results can now be used in subsequent projects to interpret ASV occurrences, e.g. for development of ecological assessment methods

Co-occurrence, ecological profiles and geographical distribution based on unique molecular identifiers of the common freshwater diatoms Fragilaria and Ulnaria

Diatom taxonomy has evolved in recent years, with many new species described and new approaches such as molecular genetics showing the existence of cryptic diversity within currently accepted species. This cryptic diversity is not well understood even for common freshwater genera such as Fragilaria and Ulnaria. The purpose of our study was to define taxon-specific ecological profiles and geographical distributions for unique molecular identifiers (amplicon sequence variants, ASVs) linked to curated taxonomy for these genera. Our goal is to contribute to the development of ecological assessment methods, and to the understanding why we often observe so many diatom species co-occurring in a single sample. We filtered a large (770 samples) metabarcoding dataset with linked environmental data covering several countries in Europe for genetic variants (ASVs) assigned to currently accepted species of our target genera. We studied the geographical distribution of the ASVs, and tested for ASV-pair co-occurrence. We modelled ASV-specific preferences for pH, alkalinity, total nitrogen, total phosphorus and conductivity, and analysed their preference for lakes or streams as habitat. Our study confirmed that there seems to be no general geographical barrier for the distribution of freshwater benthic diatom ASVs in Europe, but that dispersal is not rapid enough to hide historical events. The Fragilaria and Ulnaria ASVs in our study showed considerable overlap in geographical distribution, habitat and ecological preferences. We found evidence that only large differences in preferences for the analysed water chemistry variables prevented the co-occurrence of ASVs at the same sites. Instead, Fragilaria and Ulnaria ASVs co-occurred frequently in samples. We found subtle differences in ecological preferences for some ASV pairs, which might in part explain the co-occurrence by the avoidance of direct competition. However, the great overlap in distribution and ecological preferences suggests that other factors not studied here were also responsible for the observed co-occurrences and high richness of ASVs found at many sites. To our knowledge, we are the first to use ASVs in combination with a curated taxonomy to understand co-occurrence, specific ecological profiles and large-scale geographical distribution for unique identifiers not biased by the quality of reference databases, clustering methods, or non-harmonized morphological identification. Thus, our results can now be used in subsequent projects to interpret ASV occurrences, e.g. for development of ecological assessment methods

Recommendations for the preservation of environmental samples in diatom metabarcoding studies

Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa

Recommendations for the preservation of environmental samples in diatom metabarcoding studies

Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa

Boosting Institute of Chemistry, Technology and Metallurgy in water biomonitoring - BIOLAWEB

Project "Boosting Institute of Chemistry, Technology and Metallurgy in Water Biomonitoring" (BIOLAWEB) aims to strengthen the research and innovation capacity of the Institute of Chemistry, Technology and Metallurgy, National Institute of the Republic of Serbia, University of Belgrade (UB-ICTM) in biodiversity assessment and biomonitoring. UB-ICTM researchers made a noticeable contribution to the study of biodiversity, community ecology, and conservation of water bodies in South-Eastern Europe. However, a knowledge on index development and intercalibration following the EU standards for lakes and watercourse monitoring is still lacking in this geographic region. Similarly, there is a knowledge gap in DNA-based ecological status assessment in SEE.Abstract from ExcellMater Conference 2024 (ExcellMater Conference 2024: Innovative biomaterials for novel medical devices) published in Supplementary Issue

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems

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.info:eu-repo/semantics/publishedVersio

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

Revisiting global biogeography of freshwater diatoms: new insights from molecular data

The high-dispersal rates of microorganisms have driven to the expectation of their cosmopolitan geographic distribution. However, recent studies demonstrate that microorganisms instead show particular biogeography. Despite the existence of cosmopolitan species, geographically limited microbial groups have been found in aquatic and terrestrial environments worldwide.Diatoms are long time used model to study the biogeography of microorganisms. They are unicellular eukaryotic microalgae that contribute significantly to the aquatic primary production and have huge taxonomic diversity and marked species-specific ecological preferences. Several authors considered that diatoms have no limits in dispersion and are ubiquitously present. On the other hand, recent studies have shown that endemism exists for several genera, and species may have low dispersal capacity. However, all these studies are based on data obtained by microscopy and therefore suffer from the many well-identified biases associated with the optical identification of microorganisms at large scale.Metabarcoding technologies provide an access to taxonomic precision with a higher resolution compared to microscopy and open therefore the possibility of analyzing microbial diversity at genetic level. Recent bioinformatics tools allow reliable and standardized comparison of large datasets originating from distant geographic regions, overcoming issues related to biases in species identification.In this study we used metabarcoding data to revisit central questions in freshwater diatom biogeography. We assembled a large dataset of samples of benthic diatoms collected from rivers in seven different geographic regions. These regions cover the subpolar (Fennoscandia), temperate (France Mainland) and tropical (West Africa, French Guyana, New Caledonia, Tahiti island and Mayotte island) climate zones. The selected geographic regions can also be classified into four continental areas (Fennoscandia, France Mainland, West Africa, and French Guyana) and three islands (New Caledonia, Tahiti and Mayotte).We analyzed diatom alpha, beta and gamma diversity patterns in this dataset to address two main questions: 1) the presence of a latitudinal gradient in diatom diversity and 2) the cosmopolitanism of diatoms.Similarly to results previously reported by Soininen et al. 2016, our data showed a decrease in diatom richness with a decrease in latitude. However, testing the effect of land type (island vs. mainland) showed that this factor explains the actual variability of richness along the climatic gradient and the effect of latitude is not significant. Differences in community structure between regions and climate zones were significant. In multivariate analysis, tropical samples did not overlap with any of the other climate zones, suggesting the specificity of these communities

Strategy for Successful Integration of eDNA-based Methods in Aquatic Monitoring

Recent developments in the use of environmental DNA are opening up new horizons for the assessment of the quality of aquatic environments. These rapid and cost-effective methods, in very swift progress, will potentially offer the opportunity to identify all the taxa present in an environmental sample (water or biota) by the use of complementary markers. The produced inventories can then be used for the assessment of biodiversity and ecological quality. However, the inclusion of these new DNA-based methods in monitoring practices is not straightforward and requires harmonised actions in the coming years at national and international levels.In order to foresee and stimulate such a harmonised implementation, the European network DNAqua-Net (COST Action CA15219) brought together some of its members, experts of ECOSTAT and other environmental biomonitoring stakeholders from different European countries. Through workshops, bringing together 51 participants in 7 sub-groups in April 2020, an implementation roadmap was designed. The coordinated actions to be taken in the different countries, and the possible collaborations and steps to be taken at the EU level were identified.This presentation will give an overview of all discussions (Lefrançois et al. 2020) reflecting the diversity of situations in Europe, as well as common views. We will highlight important actions required for a successful implementation of DNA-based biomonitoring of aquatic ecosystems to the horizon of 2030

Aquatic biofilms can act as natural environmental DNA samplers

Diatoms, macroinvertebrates and fish communities are widely used for the assessment of the ecological status of rivers and lakes. Metabarcoding studies of these communities are usually performed from “bulk” samples in the case of diatoms and macroinvertebrates; and from water samples in the case of fish.Recent studies, suggest that aquatic biofilms can physically act as environmental catchers of environmental DNA (eDNA) (e.g. Mariani et al. 2019). Thus, we propose an alternative metabarcoding approach to study macroinvertebrates and fishes directly from this matrix.The capacity of aquatic biofilms to catch macroinvertebrate eDNA was tested from a previous study in Mayotte Island were both biofilm samples and macroinvertebrate morphological inventories were available at same river sites (Rivera et al. 2021). 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. Our results showed that both methods provided comparable structures and diversities for macroinvertebrate communities when using unassigned OTUs suggesting that macroinvertebrate DNA is present in biofilms and representative of the communities. However, after taxonomic assignment of OTUs, diversity and richness were no longer correlated. Indeed, many constraints were observed as the need for: a) more specific primers to avoid co-amplification of untargeted taxa inhabiting biofilms, b) primers targeting shorter barcodes to sequence more easily degraded eDNA that may be captured in biofilms, and c) a reference database well adapted to our tropical study sites. Finally, even if the results of this first study were encouraging, we wanted to test the biofilm approach on organisms that do not inhabit this environmental matrix in order to be able to distinguish between intra or extra-cellular DNA.Based on these observations, a second study looking for a fish eDNA signal in aquatic biofilms was performed. Environmental biofilm and water samples were collected in parallel at littoral sites at Lake Geneva. DNA was extracted from these samples, and fish communities were targeted using a standard 12S barcode. The molecular inventories derived from the biofilm and the water samples were compared. Both methods provide comparable floristic lists, providing a novel approach for ecological studies related to fish phenology using eDNA in biofilms.Our results open the door to the study of diatoms, macroinvertebrates and fish communities through metabarcoding from a single matrix reducing sampling efforts and costs