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My PhD project focuses on the uncultivable marine protist lineage of Radiolaria. In order to elucidate their life cycle and shed light onto the long speculated reproductive radiolarian cycle, the functional profile of single-cell transcriptomes of various radiolarian life forms is investigated for sex-related essential genes.The described genes will serve assexual cycle marker genesthat will allow the estimation of radiolarianlifespanand provide a better insight into theecological implicationsof each reproductive life stage. Overall, these results aim atexpanding the current knowledge over the life of Rhizaria, a lineage with few cultivable representatives.

Ribosomal RNA (rRNA) genes are known to be valuable markers for the barcoding of eukaryotic life and its phylogenetic classification at various taxonomic levels. The large-scale exploration of environmental microbial diversity through metabarcoding approaches has been focused mainly on the V4 and V9 regions of the 18S rRNA gene. The accurate interpretation of such environmental surveys is hampered by technical (e.g. PCR and sequencing errors) and biological biases (e.g. intra-genomic variability). Here we explored the intra-genomic diversity of Nassellaria and Spumellaria specimens (Radiolaria) by comparing Sanger sequencing with Illumina and Oxford Nanopore Technologies (MinION). Our analysis determined that intra-genomic variability of Nassellaria and Spumellaria is generally low, yet some Spumellaria specimens showed two different copies of the V4 with

Coastal marine habitats constitute hotspots of primary productivity. In temperate regions, this is due both to massive phytoplankton blooms and dense colonization by macroalgae that mostly store carbon as glycans, contributing substantially to local and global carbon sequestration. Because they control carbon and energy fluxes, algae-degrading microorganisms are crucial for coastal ecosystem functions. Environmental surveys revealed consistent seasonal dynamics of alga-associated bacterial assemblages, yet resolving what factors regulate thein situabundance, growth rate and ecological functions of individual taxa remains a challenge. Here, we specifically investigated the seasonal dynamics of abundance and activity for a well-known alga-degrading marine flavobacterial genus in a tidally-mixed coastal habitat of the Western English Channel. We show that members of the genusZobelliaare a stable, low-abundance component of healthy macroalgal microbiota and can also colonize particles in the water column. This genus undergoes recurring seasonal variations with higher abundances in winter, significantly associated to biotic and abiotic variables.Zobelliacan become a dominant part of bacterial communities on decaying macroalgae, showing a strong activity and high estimatedin situgrowth rates. These results provide insights into the seasonal dynamics and environmental constraints driving natural populations of alga-degrading bacteria that influence coastal carbon cycling.Originality-significance statementGlycan-degrading bacteria play a crucial role in marine habitats to remineralize organic carbon sequestered in algal biomass. Yet, resolving what factors regulate thein situabundance, growth rate and ecological functions of individual taxa remains a challenge. Here, we investigate the seasonal dynamics of abundance and activity of an environmentally relevant glycan-degrading bacterial genus in two constrasted compartments of the same coastal habitat, i.e. the surface of diverse macroalgae and the water column. These results provide insights into the recurring temporal patterns and environmental constraints driving natural populations of alga-degrading bacteria that influence ocean carbon cycling.

The smallest phytoplankton species are key actors in oceans biogeochemical cycling and their abundance and distribution are affected with global environmental changes. Among them, algae of the Pelagophyceae class encompass coastal species causative of harmful algal blooms while others are cosmopolitan and abundant. The lack of genomic reference in this lineage is a main limitation to study its ecological importance. Here, we analysed Pelagomonas calceolata relative abundance, ecological niche and potential for the adaptation in all oceans using a complete chromosome-scale assembled genome sequence. Our results show that P. calceolata is one of the most abundant eukaryotic species in the oceans with a relative abundance favoured by high temperature, low-light and iron-poor conditions. Climate change projections based on its relative abundance suggest an extension of the P. calceolata habitat toward the poles at the end of this century. Finally, we observed a specific gene repertoire and expression level variations potentially explaining its ecological success in low-iron and low-nitrate environments. Collectively, these findings reveal the ecological importance of P. calceolata and lay the foundation for a global scale analysis of the adaptation and acclimation strategies of this small phytoplankton in a changing environment.

Coral reefs are of paramount importance in marine ecosystems, where they provide support for a large part of the biodiversity. Being quite sensitive to global changes, they are therefore the prime targets for biodiversity conservation policies. However, such conservation goals require accurate species identification, which are notoriously difficult to get in these highly morphologically variable organisms, rich in cryptic species. There is an acute need for easy-to-use and resolutive species diagnostic molecular markers. The present study builds on the huge sequencing effort developed during the TARA Pacific expedition to develop a genotyping strategy to assign coral samples to the correct species within two coral genera (PoritesandPocillopora). For this purpose, we developed a technique that we called “Divergent Fragment” based on the sequencing of a less than 2kb long diagnostic genomic fragment determined from the metagenomic data of a subset of the corals collected. This method has proven to be rapid, resolvable and cost-effective. Sequencing of PCR fragments nested along the species diagnostic fragment allowed us to assign 232 individuals of the genusPocilloporaand 247 individuals of the genusPoritesto previously identified independent genetic lineages (i.e. species). This genotyping method will allow to fully analyze the coral samples collected across the Pacific during the Tara Pacific expedition and opens technological perspectives in the field of population genomics-guided conservation.

Tropical coral reefs are among the worst affected ecosystems by climate change with predictions ranging between a 70-90% loss of reefs in the coming decades. Effective conservation strategies that maximize ecosystem resilience, and potential for recovery, must be informed by the accurate characterization of extant genetic diversity and population structure together with an understanding of the adaptive potential of keystone species. Here, we analyzed samples from theTaraPacific Expedition (2016 to 2018) that completed an 18,000 km longitudinal transect of the Pacific Ocean sampling three widespread corals –Pocillopora meandrina, Porites lobata, andMilleporacf.platyphylla– across 33 sites from 11 islands. Using deep metagenomic sequencing of 269 colonies in conjunction with morphological analyses and climate variability data we can show that the sampled transect encompasses multiple morphologically cryptic species that exhibit disparate biogeographic patterns, and most importantly, distinct evolutionary patterns, despite exposure to identical environmental regimes. Our findings demonstrate on a basin-scale that evolutionary trajectories are species-specific and complex, and can only in part be predicted from the environment. This highlights that conservation strategies must integrate multi-species investigations to consider the distinct genomic footprints shaped by selection as well as the genetic potential for adaptive change.

Nucleic acid based studies of marine biodiversity often focus on Kingdom-level diversity. Such approaches often largely miss diversity of less studied groups, likely to harbour many unknown lineages which are likely playing significant ecological roles. Among these elusive groups are Endomyxa (Rhizaria), a ubiquitous, but understudied lineage comprising parasites (e.g. Ascetosporea, Phytomyxea), free-living amoebae (e.g. Vampyrellida, Gromia, Filoreta), flagellates (e.g. Tremula, Aquavolon), and unknown environmental lineages. Using Endomyxa-biased primers targeting the hypervariable region V4 of the 18S rRNA gene, we explored the diversity of Endomyxa in marine samples from European coastal sites and compared it to that found in pan-eukaryote V4 libraries of the same samples. In total 458 endomyxan OTUs were identified, of which 38% were only detected by the specific primers. Most are distinct from published sequences, and include novel diversity within known clades, and putative novel lineages. The data revealed variations in endomyxan assemblages related to habitat (benthic vs. pelagic, sampling site), mode of nutrition (parasitic vs. free-living) and nucleic acid type (DNA vs. RNA). Overall, the vast majority of endomyxan diversity occurs in sediments (including Vampyrellida, Reticulosida, and the environmental “Novel Clade 12”) where they form diverse and active communities including many uncharacterised lineages.

Diatoms are among the most successful marine eukaryotic phytoplankton groups. Their diversity has been investigated in the world’s oceans through expeditions and observations carried out from the equator to the poles. Traditionally, diatom species have been distinguished based on morphological characters of their frustules, but high-throughput sequencing offers new, high-resolution data that can be used to re-examine spatial and/or temporal patterns of diversity. Here we investigated diatoms’ genetic diversity using metabarcoding (18S V4 rRNA gene) obtained along the years 2011 to 2013 at two coastal time series stations (SOMLIT-Astan and LTER-MareChiara) installed respectively off Roscoff in the Western English Channel and in the Gulf of Naples in the Mediterranean Sea. Diatom species pools detected were different, which fitted with previous observations and with our expectations, since these stations are installed in two contrasted pelagic habitats (permanently-mixed versus stratified in summer). However, this analysis also shows a pool of common ASVs among which some are persistent and dominant in both sites. The observed synchronous variations in relative read abundances of shared ASVs assigned to Chaetoceros socialis, C. tenuissimus, Cyclotella, Mediolabrus comicus and Leptocylindrus aporus at the two geographically distant sites could indicate that internal controls of growth rate and sexual reproduction, rather that external environmental parameters are at work.

Endogenous viral elements (EVEs) offer insight into the evolutionary histories and hosts of contemporary viruses. This study leveraged DNA metagenomics and genomics to detect and infer the host of a non-retroviral dinoflagellate-infecting +ssRNA virus (dinoRNAV) common in coral reefs. As part of the Tara Pacific Expedition, this study surveyed 269 newly sequenced cnidarians and their resident symbiotic dinoflagellates (Symbiodiniaceae), associated metabarcodes, and publicly available metagenomes, revealing 178 dinoRNAV EVEs, predominantly among hydrocoral-dinoflagellate metagenomes. Putative associations between Symbiodiniaceae and dinoRNAV EVEs were corroborated by the characterization of dinoRNAV-like sequences in 17 of 18 scaffold-scale and one chromosome-scale dinoflagellate genome assembly, flanked by characteristically cellular sequences and in proximity to retroelements, suggesting potential mechanisms of integration. EVEs were not detected in dinoflagellate-free (aposymbiotic) cnidarian genome assemblies, including stony corals, hydrocorals, jellyfish, or seawater. The pervasive nature of dinoRNAV EVEs within dinoflagellate genomes (especially Symbiodinium), as well as their inconsistent within-genome distribution and fragmented nature, suggest ancestral or recurrent integration of this virus with variable conservation. Broadly, these findings illustrate how +ssRNA viruses may obscure their genomes as members of nested symbioses, with implications for host evolution, exaptation, and immunity in the context of reef health and disease.

Coccolithophores have global ecological and biogeochemical significance as the most important calcifying marine phytoplankton group. The structure and selection of prokaryotic communities associated with the most abundant coccolithophore and bloom-forming species, Emiliania huxleyi, are still poorly known. In this study, we assessed the diversity of bacterial communities associated with an E. huxleyi bloom in the Celtic Sea, exposed axenic E. huxleyi cultures to prokaryotic communities derived from bloom and non-bloom conditions and followed the dynamics of their microbiome composition over one year. Bloom-associated prokaryotic communities were dominated by SAR11, Marine group II Euryarchaeota, Rhodobacterales and contained substantial proportions of known indicators of phytoplankton bloom demises such as Flavobacteriaceae and Pseudoalteromonadaceae. Taxonomic richness of replicated co-cultures resulting from natural communities with axenic E. huxleyi rapidly shifted and then stabilized over time, presumably by ecological selection favoring more beneficial populations. Recruited microbiomes from the environment were consistently dependent on the composition of the initial bacterioplankton community. Phycosphere-associated communities derived from the E. huxleyi bloom depth were highly similar to one another, suggesting deterministic processes, whereas cultures from non-bloom conditions show an effect of both deterministic processes and stochasticity. Overall, this work sheds new light on the importance of the initial inoculum composition in microbiome recruitment and elucidates the temporal dynamics of its composition and long-term stability.

Coral reef science is a fast-growing field propelled by the need to better understand coral health and resilience to devise strategies to slow reef loss resulting from environmental stresses. Key to coral resilience are the symbiotic interactions established within a complex holobiont, i.e. the multipartite assemblages comprising the host coral organism, endosymbiotic dinoflagellates, bacteria, archaea, fungi, and viruses. Tara Pacific is an ambitious project built upon the experience of previous Tara Oceans expeditions, and leveraging state-of-the-art sequencing technologies and analyses to dissect the biodiversity and biocomplexity of the coral holobiont screened across most archipelagos spread throughout the entire Pacific Ocean. Here we detail the Tara Pacific workflow for multi-omics data generation, from sample handling to nucleotide sequence data generation and deposition. This unique multidimensional framework also includes a large amount of concomitant metadata collected side-by-side that provide new assessments of coral reef biodiversity including micro-biodiversity and shape future investigations of coral reef dynamics and their fate in the Anthropocene.

International audience; Major seasonal community reorganizations and associated biomass variations are landmarks of plankton ecology. However, the processes of plankton community turnover rates have not been fully elucidated so far. Here, we analyse patterns of planktonic protist community succession in temperate latitudes, based on quantitative taxonomic data from both microscopy counts (cells >10 mu m) and ribosomal DNA metabarcoding (size fraction >3 mu m, 18S rRNA gene) from plankton samples collected bimonthly over 8 years (2009-2016) at the SOMLIT-Astan station (Roscoff, Western English Channel). Based on morphology, diatoms were clearly the dominating group all year round and over the study period. Metabarcoding uncovered a wider diversity spectrum and revealed the prevalence of Dinophyceae and diatoms but also of Cryptophyta, Chlorophyta, Cercozoa, Syndiniales and Ciliophora in terms of read counts and or richness. The use of morphological and molecular analyses in combination allowed improving the taxonomic resolution and to identify the sequence of the dominant species and OTUs (18S V4 rDNA-derived taxa) that drive annual plankton successions. We detected that some of these dominant OTUs were benthic as a result of the intense tidal mixing typical of the French coasts in the English Channel. Our analysis of the temporal structure of community changes point to a strong seasonality and resilience. The temporal structure of environmental variables (especially Photosynthetic Active Radiation, temperature and macronutrients) and temporal structures generated by species life cycles and or species interactions, are key drivers of the observed cyclic annual plankton turnover.

This protocol has been developped for simultaneous nucleic acid (RNA and DNA) extraction from environmental water samples collected by filtration on polycarbonate 47mm and 142mm filters. It has been developped during BioMarks project (2009-2013) and has been used in many projects studying plankton diversity (TARA-OCEANS 2009-2013, TARA-PACIFIC 2016-2018, TONGA 2019), and plankton monitoring projects (MOOSE-GE 2017- ongoing). This protocol is applied in routine in our lab and also in CEA Genoscope.

Sea spray aerosol (SSA) formation have a major role in the climate system, but measurements at a global-scale of this micro-scale process are highly challenging. We measured high-resolution temporal patterns of SSA number concentration over the Atlantic Ocean, Caribbean Sea, and the Pacific Ocean covering over 42,000 km. We discovered a ubiquitous 24-hour rhythm to the SSA number concentration, with concentrations increasing after sunrise, remaining higher during the day, and returning to predawn values after sunset. The presence of dominating continental aerosol transport can mask the SSA cycle. We did not find significant links between the diel cycle of SSA number concentration and diel variations of surface winds, atmospheric physical properties, radiation, pollution, nor oceanic physical properties. However, the daily mean sea surface temperature positively correlated with the magnitude of the day-to-nighttime increase in SSA concentration. Parallel diel patterns in particle sizes were also detected in near-surface waters attributed to variations in the size of particles smaller than ~1 µm. These variations may point to microbial day-to-night modulation of bubble-bursting dynamics as a possible cause of the SSA cycle., Sea spray aerosol (SSA) are an important way through which oceans can influence the atmosphere’s radiative properties. Here, the authors present measurements taken over a 42,000 km ship cruise in the Atlantic and Pacific Ocean and show that SSA number concentrations vary over a 24-hour cycle, possibly linked to surface water bubble-bursting dynamics.

Major seasonal community reorganizations and associated biomass variations are landmarks of plankton ecology. However, the processes determining marine species and community turnover rates have not been fully elucidated so far. Here, we analyse patterns of planktonic protist community succession in temperate latitudes, based on quantitative taxonomic data from both microscopy counts and ribosomal DNA metabarcoding from plankton samples collected biweekly over 8 years (2009-2016) at the SOMLIT-Astan station (Roscoff, Western English Channel). Considering the temporal structure of community dynamics (creating temporal correlation), we elucidated the recurrent seasonal pattern of the dominant species and OTUs (rDNA-derived taxa) that drive annual plankton successions. The use of morphological and molecular analyses in combination allowed us to assess absolute species abundance while improving taxonomic resolution, and revealed a greater diversity. Overall, our results underpinned a protist community characterised by a seasonal structure, which is supported by the dominant OTUs. We detected that some were partly benthic as a result of the intense tidal mixing typical of the French coasts in the English Channel. While the occurrence of these microorganisms is driven by the physical and biogeochemical conditions of the environment, internal community processes, such as the complex network of biotic interactions, also play a key role in shaping protist communities.

This DNA extraction protocol allows to get both eukaryotic and prokaryotic DNA from microalgae strains, so the microbiome diversity can be studied in cultures by using this protocol.

Nowadays metabarcoding approaches allow to explore the diversity of different communities using next-generation sequencing (NGS). Here we describe the 16S-V4V5 DNA amplification method applied for prokaryote metabarcoding analyses using Illumina Miseq technology. This protocol has been used in many projects studying prokaryotic diversity (TARA-OCEANS 2009-2013, TARA-PACIFIC 2016-2018), and prokaryote monitoring projects (ROSCOFF ASTAN 2009-ongoing). We developed the flowchart for 2 different sequencing platforms : Fasteris-Gene Support SA (Plan-Les-Ouates, Swiss) and GeT-PlaGE (Toulouse, France).

Nowadays metabarcoding approaches allow to explore the diversity of different communities using next-generation sequencing (NGS). Here we describe the 18S-V4 DNA amplification method applied for eukaryote metabarcoding analyses using Illumina Miseq technology. This protocol has been used in many projects studying eukaryotic diversity (TARA-OCEANS 2009-2013, TARA-PACIFIC 2016-2018), and eukaryote monitoring projects (MOOSE-GE 2017-..., ROSCOFF ASTAN 2009-ongoing). We developed the flowchart for 2 different sequencing platforms : Fasteris-Gene Support SA (Plan-Les-Ouates, Swiss) and GeT-PlaGE (Toulouse, France).

SummaryThe smallest phytoplankton species are key actors in oceans biogeochemical cycling and their abundance and distribution are affected with global environmental changes. Picoalgae (cells Pelagomonas calceolata. We identified unusual large low-GC and gene-rich regions potentially representing centromeres. These particular genomic structures could be explained by the absence of genes from a recombination pathway involving double Holiday Junctions. We identified a large repertoire of genes involved in inorganic nitrogen sensing and uptake and several genes replacing iron-requiring proteins potentially explaining P. calceolata ecological success in oligotrophic waters. Finally, based on this high-quality assembly, we evaluated P. calceolata relative abundance in all oceans using environmental Tara Oceans datasets. Our results suggest that P. calceolata is one of the most abundant eukaryotic species in the oceans with a relative abundance favoured by high temperature and iron-poor conditions. Climate change projections based on its relative abundance suggest an extension of the P. calceolata habitat toward the poles at the end of this century. Collectively, these findings reveal the ecological importance of P. calceolata and lay the foundation for a global scale analysis of the adaptation and acclimation strategies of picoalgae in a changing environment.

Ribosomal DNA (rDNA) genes are known to be valuable markers for the barcoding of eukaryotic life and its phylogenetic classification at various taxonomic levels. The large scale exploration of environmental microbial diversity through metabarcoding approaches have been focused mainly on the hypervariable regions V4 and V9 of the 18S rDNA gene. Yet, the accurate interpretation of such environmental surveys is hampered by technical (e.g., PCR and sequencing errors) and biological biases (e.g., intra-genomic variability). Here we explored the intra-genomic diversity of Nassellaria and Spumellaria specimens (Radiolaria) by comparing Sanger sequencing with two different high-throughput sequencing platforms: Illumina and Oxford Nanopore Technologies (MinION). Our analysis determined that intra-genomic variability of Nassellaria and Spumellaria is generally low, yet in some Spumellaria specimens we found two different copies of the V4 with a similarity lower than 97%. From the different sequencing methods, Illumina showed the highest number of contaminations (i.e., environmental DNA, cross-contamination, tag-jumping), revealed by its high sequencing depth; and Minion showed the highest sequencing rate error (~14%). Yet the long reads produced by MinION (~2900 bp) allowed accurate phylogenetic reconstruction studies. These results, highlight the requirement for a careful interpretation of Illumina based metabarcoding studies, in particular regarding low abundant amplicons, and open future perspectives towards full environmental rDNA metabarcoding surveys.

The study of protistan functional diversity is crucial to understand the dynamics of oceanic ecological processes. We combined the metabarcoding data of various coastal ecosystems and a newly developed trait-based approach to study the link between taxonomic and functional diversity across marine protistan communities of different size-classes. Environmental DNA was extracted and the V4 18S rDNA genomic region was amplified and sequenced. In parallel, we tried to annotate the operational taxonomic units (OTUs) from our metabarcoding dataset to 30 biological traits using published and accessible information on protists. We then developed a method to study trait correlations across protists (i.e. trade-offs) in order to build the best functional groups. Based on the annotated OTUs and our functional groups, we demonstrated that the functional diversity of marine protist communities varied in parallel with their taxonomic diversity. The coupling between functional and taxonomic diversity was conserved across different protist size classes. However, the smallest size-fraction was characterized by wider taxonomic and functional groups diversity, corroborating the idea that nanoplankton and picoplankton are part of a more stable ecological background on which larger protists and metazoans might develop.

Ocean-atmosphere interactions such as sea spray aerosol (SSA) formation have a major role in the climate system, but a global-scale assessment of this micro-scale process is highly challenging. We measured high-resolution temporal patterns of SSA number concentration over the Atlantic Ocean, Caribbean Sea, and the Pacific Ocean covering 42,000 km of open ocean waters. We discovered a ubiquitous 24-hour rhythm to the number concentration, clearly seen for particle diameters > ~ 0.58 µm, with spikes at dawn and drops at dusk throughout the Pacific Ocean and Caribbean Sea, showing more than doubling of the SSA number concentration during the day than at night. No correlation with surface winds, atmospheric radiation, pollution nor oceanic physical properties were found. Instead, parallel diel patterns in particle sizes detected in near-surface waters, attributed to variations in the size of particles smaller than ~ 1 µm, point to microbial day-to-night modulation of bubble-bursting dynamics as the cause of the SSA cycle.

Spumellaria (Radiolaria, Rhizaria) are holoplanktonic amoeboid protists, ubiquitous and abundant in the global ocean. Their silicified skeleton preserves very well in sediments displaying an excellent fossil record, from the early middle Cambrian (ca. 509-521 Ma), extremely valuable for paleo-environmental reconstruction studies. Spumellaria are tedious to maintain in laboratory conditions preventing an accurate perception of their extant diversity and ecology in today’s oceans, most of which being inferred from sediment records. This study represents an integrated and comprehensive classification of Spumellaria based on the combination of ribosomal taxonomic marker genes (rDNA) and morphological characteristics. In contrast to established taxonomic knowledge, we demonstrate that symmetry of the skeleton takes more importance than internal structures at high taxonomic rank classification. Such reconsideration allows gathering different morphologies with concentric structure and a spherical or radial symmetry believed to belong to other Radiolaria orders from the fossil record, as for some Entactinaria families. Results obtained in this study suggest the existence of new Spumellaria diversity at early diverging positions, in which a non-bearing skeleton organism lives within shelled ones. Using fossil calibrated molecular clock we estimated the origin of Spumellaria in the middle Cambrian (ca. 515 Ma), in agreement with the appearance of the first radiolarian representatives in the fossil record. The morpho-molecular and evolutionary framework established herein allows a direct connection between living specimens and fossil morphologies from the Cambrian, bringing both a standpoint for future molecular environmental surveys and a better understanding for paleo-environmental reconstruction studies.

Marine aerosols play a significant role in the global radiative budget, in clouds’ processes, and in the chemistry of the marine atmosphere. There is a critical need to better understand their production mechanisms, composition, chemical properties, and the contribution of ocean-derived biogenic matter to their mass and number concentration. Here we present an overview of a new dataset of in situ measurements of marine aerosols conducted over the 2.5-yr Tara Pacific Expedition over 110,000 km across the Atlantic and Pacific Oceans. Preliminary results are presented here to describe the new dataset that will be built using this novel set of measurements. It will characterize marine aerosols properties in detail and will open a new window to study the marine aerosol link to the water properties and environmental conditions.

Cell Lysis can be difficult to conduct for envirronmental water samples collected on big filters, specially when big water volumes (20-100L) have been filtered on polycarbanate 142mm-diameter filters. Here we developped a mecanic lyis protocol in liquid nitrogen conditions to get preserved DNA and RNA. This protocol was developped for combined RNA and DNA extraction from environmental water samples for Biomarks (2009-2013) and TARA OCEANS projects (2009-ongoing).

Radiolaria are protists which can't be cultivated. These microoganisms have to be isolated by single-cell for genetic identification and it can be difficult to get clean DNA. Here we optimized a DNA extraction protocol from protist single-cell. It works very well on single-cell Radiolaria, Foraminifers, but also Ciliates, Dinoflagelletes, Diatoms.

Radiolaria are protists which can't be cultivated. These microoganisms have to be isolated by single-cell for genetic identification and it can be difficult to get clean DNA. Here we optimized a DNA extraction protocol from protist single-cell. It works very well on single-cell Radiolaria, Foraminifers, but also Ciliates, Dinoflagelletes, Diatoms.

Spumellaria (Radiolaria, Rhizaria) are holoplanktonic amoeboid protists, ubiquitous and abundant in the global ocean. Their silicified skeleton preserves very well in sediments, displaying an excellent fossil record extremely valuable for paleo-environmental reconstruction studies, from where most of their extant diversity and ecology have been inferred. This study represents a comprehensive classification of Spumellaria based on the combination of ribosomal taxonomic marker genes (rDNA) and morphological characteristics. In contrast to established taxonomic knowledge, we demonstrate that symmetry of the skeleton takes more importance than internal structures at high classification ranks. Such reconsideration allows gathering different morphologies with concentric structure and spherical or radial symmetry believed to belong to other Radiolaria orders from the fossil record, as for some Entactinaria families. Our calibrated molecular clock dates the origin of Spumellaria in the middle Cambrian (ca. 515 Ma), among the first radiolarian representatives in the fossil record. This study allows a direct connection between living specimens and extinct morphologies from the Cambrian, bringing both a standpoint for future molecular environmental surveys and a better understanding for paleo-environmental reconstruction analysis. (C) 2021 The Authors. Published by Elsevier GmbH, This work was supported by the IMPEKAB ANR 15-CE02-0011 grant and the Brittany Region ARED C16 1520A01, the Japan Society for Promotion of Science KAKENHI Grant No. K16K0-74750 for N. Suzuki and "the Cooperative Research Project with the Japan Science and Technology Agency (JST) and Centre National de la Recherche Scientifique (CNRS, France) "Morphomolecular Diversity Assessment of Ecologically, Evolutionary, and Geo-logically Relevant Marine Plankton (Radiolaria) ". We are grateful to the CNRS-Sorbonne University ABiMS bioinformatics platform (http://abims.sbroscoff.fr) for providing computational resources. The authors are grateful to the MOOSE observation national network (funded by CNRS-INSU and Research Infrastructure ILICO) which sustain the annual ship-based hydrographic sections in the northwestern Mediterranean Sea (MOOSEGE) , as well as John Dolan for hosting us multiple times at the Laboratoire d'Oceanographie of Villefranche sur Mer. We are greatly thankful to Cedric Berney for the phylogenetic advice and the valuable help on the interpretation of the "symbiotic" clade, as well as Vasily Zlatogursky for his contributions and feed-back on the heliozoan-like organism.