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The genus Operculina, a large symbiont-bearing benthic foraminifer, is characterized by high morphological variability showing thick involute to intermediate semi-involute to flat evolute tests. Different morphotypes are either considered as ecophenotypes or distinct species. In order to test the hypothesis of ecophenotypes versus different species, a single cell high throughput sequencing approach was applied to assess the interspecific diversity of Operculina. This results in two groups of ribotypes, one corresponding to Operculina ammonoides/Operculina discoidalis, the other containing Operculina complanata/Operculina elegans. These groups can also be separated morphologically. Therefore, O. complanata and O. elegans represent a single species and the latter can be regarded as a junior synonym of O. complanata. Operculina ammonoides and O. discoidalis also form a single species, which makes the latter a junior synonym of O. ammonoides. Because generic differences in Operculina species are manifested in morphology and molecular genetics, the genus Neoassilina with the designated species Neoassilina ammonoides is installed. Additional analysis of ribosomal SSU rDNA data of eight recent nummulitid genera confirms the obtained high troughput sequencing results and further shows that Palaeonummulites venosus builds a clade with O. complanata that branches at the base of other Nummulitidae containing Planostegina, Planoperculina, Cycloclypeus, Heterostegina, Operculinella and Neoassilina.

Since 2010, considerable efforts have been undertaken to monitor the environmental status of European marine waters and ensuring the development of methodological standards for the evaluation of this status. However, the current routine biomonitoring implicates time-consuming and costly manual sorting and morphological identification of benthic macrofauna. Environmental DNA (eDNA) metabarcoding represents an alternative to the traditional monitoring method with very promising results. Here, we tested it further by performing eDNA metabarcoding of benthic eukaryotic communities in the vicinity of two offshore oil and gas platforms in the North Sea. Three different genetic markers (18S V1V2, 18S V9 and COI) were used to assess the environmental pressures induced by the platforms. All markers showed patterns of alpha and beta diversity consistent with morphology-based macrofauna analyses. In particular, the communities' structure inferred from metabarcoding and morphological data significantly changed along distance gradients from the platforms. The impact of the operational discharges was also detected by the variation of biotic index values, AMBI index showing the best correlation between morphological and eDNA data sets. Finally, the sediment physicochemical parameters were used to build a local de novo pressure index that served as benchmark to test the potential of a taxonomy-free approach. Our study demonstrates that metabarcoding approach outperforms morphology-based approach and can be used as a cost and time-saving alternative solution to the traditional morphology-based monitoring in order to monitor more efficiently the impact of industrial activities on marine biodiversity.

The last decade brought a spectacular development of so-called environmental (e)DNA studies. In general, "environmental DNA" is defined as DNA isolated from environmental samples, in contrast to genomic DNA that is extracted directly from specimens. However, the variety of different sources of eDNA and the range of taxonomic groups that are targeted by eDNA studies is large, which has led to some discussion about the breadth of the eDNA concept. In particular, there is a recent trend to restrict the use of the term "eDNA" to the DNA of macro-organisms, which are not physically present in environmental samples. In this paper, we argue that such a distinction may not be ideal, because the eDNA signal can come from organisms across the whole tree of life. Consequently, we advocate that the term "eDNA" should be used in its generic sense, as originally defined, encompassing the DNA of all organisms present in environmental samples, including microbial, meiofaunal and macrobial taxa. We first suggest specifying the environmental origin of the DNA sample, such as water eDNA, sediment eDNA or soil eDNA. A second specification would then define the taxonomic group targeted through polymerase chain reaction amplification, such as fish eDNA, invertebrate eDNA and bacterial eDNA. This terminology does also not require assumptions about the specific state of the DNA sampled (intracellular or extracellular). We hope that such terminology will help better define the scope of eDNA studies, especially for environmental managers, who use them as reference in routine biomonitoring and bioassessment.

During the last decade, considerable efforts have been undertaken to achieve a “good ecological status” of European coastal waters and ensuring the development of methodological standards for the evaluation of this status. However, the current routine biomonitoring implicates time-consuming and costly manual sorting and morphological identification of benthic macrofauna. In our study, we tested the performance of environmental DNA metabarcoding targeting microbial communities and meiofauna as an alternative to traditional macrofauna-based monitoring. We focused on environmental impact assessment of offshore oil and gas industry. We used three genetic markers (18S V1V2, 18S V9 and COI) to assess the environmental pressures induced by the platforms. All markers showed patterns of alpha and beta diversity consistent with morphology-based macrofauna analyses, significantly changing along distance gradients from the platforms. The impact of the operational discharges was also detected by the variation of biotic indices values, AMBI index showing the best correlation between morphological and eDNA datasets. Finally, the sediment physicochemical parameters were used to build a local de novo pressure index that served as benchmark to test the potential of a taxonomy-free approach. Our study demonstrates that metabarcoding approach outperforms morphology-based approach and can be used as a cost and time-saving alternative solution to the traditional morphology-based monitoring in order to monitor more efficiently the impact of industrial activities on marine biodiversity.

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems. Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring.

In a recent paper, “Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring,” Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra‐organismal DNA from macro‐organisms. We agree with this view. However, we are concerned that their proposed two‐level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra‐organismal) from which ecological interpretations are derived.

Since the 1960 s, there has been a rapid expansion of drilling activities in the central and northern Adriatic Sea to meet the increasing global energy demand. The discharges of organic and inorganic pollutants, as well as the alteration of the sediment substrate, are among the main impacts associated with these activities. In the present study, we evaluate the response of benthic foraminifera to the activities of three gas platforms in the northwestern Adriatic Sea, with a special focus on the Armida A platform for which extensive geochemical data (organic matter, trace elements, polycyclic aromatic hydrocarbons, other hydrocarbons, and volatile organic compounds) are available. The response to disturbance is assessed by analyzing the foraminiferal diversity using the traditional morphology-based approach and by 18S rDNA-based metabarcoding. The two methods give congruent results, showing relatively lower foraminiferal diversity and higher dominance values at stations closer to the platforms (

Newts are amphibians commonly present in small ponds or garden pools in urban areas. They are protected in many countries and their presence is monitored through visual observation and/or trapping. However, newts are not easy to spot as they are small, elusive and often hidden at the bottom of water bodies. In recent years, environmental DNA (eDNA) has become a popular tool for detecting newts, with a focus on individual species using qPCR assays. Here, we assess the effectiveness of eDNA metabarcoding compared to conventional visual surveys of newt diversity in 45 ponds within urban areas of Geneva canton, Switzerland. We designed newt-specific mitochondrial 16S rRNA primers, which assign the majority of amplicons to newts, and were able to detect four species known to be present in the region, including the invasive subspecies Lissotriton vulgaris meridionalis, native to the Italian peninsula, that has been introduced in the Geneva area recently. The obtained eDNA results were congruent overall with conventional surveys, confirming the morphological observations in the majority of cases (67%). In 25% of cases, a species was only detected genetically, while in 8% of cases, the observations were not supported by eDNA metabarcoding. Our study confirms the usefulness of eDNA metabarcoding as a tool for the effective and non-invasive monitoring of newt community and suggests its broader use for the survey of newt diversity in urban area at larger scales.

International audience; 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.

International audience; A decade after environmental scientists integrated high-throughput sequencing technologies in their toolbox, the genomics-based monitoring of anthropogenic impacts on the biodiversity and functioning of ecosystems is yet to be implemented by regulatory frameworks. Despite the broadly acknowledged potential of environmental genomics to this end, technical limitations and conceptual issues still stand in the way of its broad application by end-users. In addition, the multiplicity of potential implementation strategies may contribute to a perception that the routine application of this methodology is premature or "in development", hence restraining regulators from binding these tools into legal frameworks. Here, we review recent implementations of environmental genomics-based methods, applied to the biomonitoring of ecosystems. By taking a general overview, without narrowing our perspective to particular habitats or groups of organisms, this paper aims to compare, review and discuss the strengths and limitations of four general implementation strategies of environmental genomics for monitoring: (A) Taxonomy-based analyses focused on identification of known bioindicators or described taxa; (B) De novo bioindicator analyses; (C) Structural community metrics including inferred ecological networks; and (D) Functional community metrics (metagenomics or metatranscriptomics). We emphasise the utility of the three latter strategies to integrate meiofauna and microorganisms that are not traditionally utilised in biomonitoring because of difficult taxonomic identification. Finally, we propose a roadmap for the implementation of environmental genomics into routine monitoring programs that leverage recent analytical advancements, while pointing out current limitations and future research needs.

Aquatic oligochaete communities are valuable indicators of the biological quality of sediments in streams and lakes, but identification of specimens to the species level based on morphological features requires solid expertise in taxonomy and is possible only for a fraction of specimens present in a sample. The identification of aquatic oligochaetes using DNA barcodes would facilitate their use in biomonitoring and allow a wider use of this taxonomic group for ecological diagnoses. Previous approaches based on DNA metabarcoding of samples composed of total sediments or pools of specimens have been proposed for assessing the biological quality of ecosystems, but such methods do not provide precise information on species abundance, which limits the value of resulting ecological diagnoses. Here, we tested how a DNA barcoding approach based on high-throughput sequencing of sorted and genetically tagged specimens performed to assess oligochaete species diversity and abundance and the biological quality of sediments in streams and lakes. We applied both molecular and morphological approaches at 13 sites in Swiss streams and at 7 sites in Lake Geneva. We genetically identified 33 or 66 specimens per site. For both approaches, we used the same index calculations. We found that the ecological diagnoses derived from the genetic approach matched well with those of the morphological approach and that the genetic identification of only 33 specimens per site provided enough ecological information for correctly estimating the biological quality of sediments in streams and lakes.

The environmental DNA (eDNA) metabarcoding represents a new promising tool for biomonitoring and environmental impact assessment. One of the main advantages of eDNA metabarcoding, compared to the traditional morphotaxonomy-based methods, is to provide a more holistic biodiversity information that includes inconspicuous morphologically non-identifiable taxa. Here, we use eDNA metabarcoding to survey marine biodiversity in the vicinity of the three offshore gas platforms in North Adriatic Sea (Italy). We isolated eDNA from 576 water and sediment samples collected at 32 sampling sites situated along four axes at increasing distances from the gas platforms. We obtained about 46 million eDNA sequences for 5 markers from nuclear 18S V1V2, 18S V4, 18S 37F and mitochondrial 16S and COI genes that cover a wide diversity of benthic and planktonic eukaryotes. Our results showed some impact of platform activities on benthic and pelagic communities at very close distance (50 m), while communities for intermediate (125 m, 250 m, 500 m) and reference (1000 m, 2000 m) sites did not show any particular biodiversity changes that could be related to platforms activities. The most significant community change along the distance gradient was obtained with the 18S V1V2 marker targeting benthic eukaryotes, even though other markers showed similar trends, but to a lesser extent. These results were congruent with the AMBI index inferred from the eDNA sequences assigned to benthic macrofauna. We finally explored the relation between various physicochemical parameters, including hydrocarbons, on benthic community in the case of one of the platforms. Our results showed that these communities were not significantly impacted by most of hydrocarbons, but rather by macro-elements and sediment texture.

Aquatic oligochaetes are important bioindicators of sediment quality in watercourses and lakes, but their morphological identification to the species level is challenging and sometimes impossible. The use of DNA barcoding and metabarcoding could greatly facilitate the identifications of specimens and improve ecological diagnoses based on oligochaete communities. The aim of this study was to test how well metabarcoding approaches based on high throughout sequencing (HTS) could perform in assessing oligochaete species diversity and the biological quality of sediments. We analysed oligochaete communities at several sites in Swiss rivers, comparing morphological data to metabarcoding of mixed specimen, sieved sediment and total sediment samples. We amplified the cytochrome c oxidase (COI) marker using universal primers and/or specific primers to metazoans. Our results showed that metabarcoding analysis of mixed specimens allowed a more representative assessment of the biodiversity than the metabarcoding of sieved or total sediments. Although community structures obtained with the morphological and metabarcoding analyses were different in term of presence/absence of species and species abundances, the ecological diagnoses based on these two approaches largely agreed.

International audience; The effectiveness of environmental protection measures is based on the early identification and diagnosis of anthropogenic pressures. Similarly, restoration actions require precise monitoring of changes in the ecological quality of ecosystems, in order to highlight their effectiveness. Monitoring the ecological quality relies on bioindicators, which are organisms revealing the pressures exerted on the environment through the composition of their communities. Their implementation, based on the morphological identification of species, is expensive because it requires time and experts in taxonomy. Recent genomic tools should provide access to reliable and high-throughput environmental monitoring by directly inferring the composition of bioindicators' communities from their DNA (metabarcoding). The French-Swiss program SYNAQUA (INTERREG France-Switzerland 2017-2019) proposes to use and validate the tools of environmental genomic for biomonitoring and aims ultimately at their implementation in the regulatory bio-surveillance. SYNAQUA will test the metabarcoding approach focusing on two bioindicators, diatoms, and aquatic oligochaetes, which are used in freshwater biomonitoring in France and Switzerland. To go towards the renewal of current biomonitoring practices, SYNAQUA will (1) bring together different actors: scientists, environmental managers, consulting firms, and biotechnological companies, (2) apply this approach on a large scale to demonstrate its relevance, (3) propose robust and reliable tools, and (4) raise public awareness and train the various actors likely to use these new tools. Biomonitoring approaches based on such environmental genomic tools should address the European need for reliable, higher-throughput monitoring to improve the protection of aquatic environments under multiple pressures, guide their restoration , and follow their evolution.

Genomics is fast becoming a routine tool in medical diagnostics and cutting-edge biotechnologies. Yet, its use for environmental biomonitoring is still considered a futuristic ideal. Until now, environmental genomics was mainly used as a replacement of the burdensome morphological identification, to screen known morphologically distinguishable bioindicator taxa. While prokaryotic and eukaryotic microbial diversity is of key importance in ecosystem functioning, its implementation in biomonitoring programs is still largely unappreciated, mainly because of difficulties in identifying microbes and limited knowledge of their ecological functions. Here, we argue that the combination of massive environmental genomics microbial data with machine learning algorithms can be extremely powerful for biomonitoring programs and pave the way to fill important gaps in our understanding of microbial ecology.

Most foraminifera inhabit marine habitats, but some species of monothalamids have been described from freshwater environments, mainly from Swiss water bodies over 100 years ago. Recent environmental DNA surveys revealed the presence of four major phylogenetic clades of freshwater foraminifera. However, until now only one of them (clade 2) has been associated to a morphologically described taxon-the family Reticulomyxidae. Here, we present morphological and molecular data for the genera representing the three remaining clades. We describe two new agglutinated freshwater genera from China and the Netherlands, Lacogromia and Limnogromia, which represent clades 3 and 4, respectively. We also report the first ribosomal DNA sequences of the genus Lieberkuehnia, which place this genus within clade 1. Our study provides the first morphotaxonomic documentation of molecular clades of freshwater foraminifera, showing that the environmental DNA sequences correspond to the agglutinated monothalamous species, morphologically similar to those described 100 years ago.

Monothalamous (single-chambered) foraminifera have long been considered as the "poor cousins" of multichambered species, which calcareous and agglutinated tests dominate in the fossil record. This view is currently changing with environmental DNA surveys showing that the monothalamids may be as diverse as hard-shelled foraminifera. Yet, the majority of numerous molecular lineages revealed by eDNA studies remain anonymous. Here, we describe a new monothalamous species and genus isolated from the sample of sea grass collected in Gulf of Eilat (Red Sea). This new species, named Leannia veloxifera, is characterized by a tiny ovoid theca (about 50-100 μm) composed of thin organic wall, with two opposite apertures. The examined individuals are multinucleated and show very active reticulopodial movement. Phylogenetic analyses of SSU rDNA, actin, and beta-tubulin (ß-tubulin) show that the species represents a novel lineage branching separately from other monothalamous foraminifera. Interestingly, the SSU rDNA sequence of the new species is very similar to an environmental foraminiferal sequence from Bahamas, suggesting that the novel lineage may represent a group of shallow-water tropical allogromiids, poorly studied until now.

A new monothalamous (single-chambered) soft-walled foraminiferal species, Arnoldiellina fluorescens gen. et sp. nov., was isolated from samples collected in the Gulf of Eilat, Israel. The species is characterized by a small elongate organic theca with a single aperture of allogromiids. It is characterized by the emission of green autofluorescence (GAF) that has so far not been reported from foraminifera. Phylogenetic analysis of a fragment of the 18S rDNA indicates that the species is related to a group of monothalamous foraminiferans classified as clade I. Although the morphology of the new species is very different compared to the other members of this clade, a specific helix in 18S rRNA secondary structure strongly supports this position.

Current biodiversity assessment and biomonitoring are largely based on the morphological identification of selected bioindicator taxa. Recently, several attempts have been made to use eDNA metabarcoding as an alternative tool. However, until now, most applied metabarcoding studies have been based on the taxonomic assignment of sequences that provides reference to morphospecies ecology. Usually, only a small portion of metabarcoding data can be used due to a limited reference database and a lack of phylogenetic resolution. Here, we investigate the possibility to overcome these limitations using a taxonomy-free approach that allows the computing of a molecular index directly from eDNA data without any reference to morphotaxonomy. As a case study, we use the benthic diatoms index, commonly used for monitoring the biological quality of rivers and streams. We analysed 87 epilithic samples from Swiss rivers, the ecological status of which was established based on the microscopic identification of diatom species. We compared the diatom index derived from eDNA data obtained with or without taxonomic assignment. Our taxonomy-free approach yields promising results by providing a correct assessment for 77% of examined sites. The main advantage of this method is that almost 95% of OTUs could be used for index calculation, compared to 35% in the case of the taxonomic assignment approach. Its main limitations are under-sampling and the need to calibrate the index based on the microscopic assessment of diatoms communities. However, once calibrated, the taxonomy-free molecular index can be easily standardized and applied in routine biomonitoring, as a complementary tool allowing fast and cost-effective assessment of the biological quality of watercourses.

International audience; Diatoms are widely used as bioindicators for the assessment of water quality in rivers and streams. Classically, the diatom biotic indices are based on the relative abundance of morphologically identified species weighted by their autoecological value. Obtaining such indices is time-consuming, costly, and requires excellent taxonomic expertise, which is not always available. Here we tested the possibility to overcome these limitations using a next-generation sequencing (NGS) approach to identify and quantify diatoms found in environmental DNA and RNA samples. We analyzed 27 river sites in the Geneva area (Switzerland), in order to compare the values of the Swiss Diatom Index (DI-CH) computed either by microscopic quantification of diatom species or directly from NGS data. Despite gaps in the reference database and variations in relative abundance of analyzed species, the diatom index shows a significant correlation between morphological and molecular data indicating similar biological quality status for the majority of sites. This proof-of-concept study demonstrates the potential of the NGS approach for identification and quantification of diatoms in environmental samples, opening new avenues toward the routine application of genetic tools for bioassessment and biomonitoring of aquatic ecosystems.

The Mediterranean Sea is considered as one of the hotspots of marine bioinvasions, largely due to the influx of tropical species migrating through the Suez Canal, so-called Lessepsian migrants. Several cases of Lessepsian migration have been documented recently, however, little is known about the ecological characteristics of the migrating species and their aptitude to colonize the new areas. This study focused on Red Sea soritids, larger symbiont-bearing benthic foraminifera (LBF) that are indicative of tropical and subtropical environments and were recently found in the Israeli coast of the Eastern Mediterranean. We combined molecular phylogenetic analyses of soritids and their algal symbionts as well as network analysis of Sorites orbiculus Forskål to compare populations from the Gulf of Elat (northern Red Sea) and from a known hotspot in Shikmona (northern Israel) that consists of a single population of S. orbiculus. Our phylogenetic analyses show that all specimens found in Shikmona are genetically identical to a population of S. orbiculus living on a similar shallow water pebbles habitat in the Gulf of Elat. Our analyses also show that the symbionts found in Shikmona and Elat soritids belong to the Symbiodinium clade F5, which is common in the Red Sea and also present in the Indian Ocean and Caribbean Sea. Our study therefore provides the first genetic and ecological evidences that indicate that modern population of soritids found on the Mediterranean coast of Israel is probably Lessepsian, and is less likely the descendant of a native ancient Mediterranean species.