Assembly processes and functional diversity of marine protists and their rare biosphere.

BACKGROUND: The mechanisms shaping the rare microbial biosphere and its role in ecosystems remain unclear. We developed an approach to study ecological patterns in the rare biosphere and use it on a vast collection of marine microbiomes, sampled in coastal ecosystems at a regional scale. We study the assembly processes, and the ecological strategies constituting the rare protistan biosphere. Using the phylogeny and morpho-trophic traits of these protists, we also explore their functional potential. RESULTS: Taxonomic community composition remained stable along rank abundance curves. Conditionally rare taxa, driven by selection processes, and transiently rare taxa, with stochastic distributions, were evidenced along the rank abundance curves of all size-fractions. Specific taxa within the divisions Sagenista, Picozoa, Telonemia, and Choanoflagellida were rare across time and space. The distribution of traits along rank abundance curves outlined a high functional redundancy between rare and abundant protists. Nevertheless, trophic traits illustrated an interplay between the trophic groups of different size-fractions. CONCLUSIONS: Our results suggest that rare and abundant protists are evolutionary closely related, most notably due to the high microdiversity found in the rare biosphere. We evidenced a succession of assembly processes and strategies of rarity along rank abundance curves that we hypothesize to be common to most microbiomes at the regional scale. Despite high functional redundancy in the rare protistan biosphere, permanently rare protists were evidenced, and they could play critical functions as bacterivores and decomposers from within the rare biosphere. Finally, changes in the composition of the rare protistan biosphere could be influenced by the trophic regime of aquatic ecosystems. Our work contributes to understanding the role of rare protists in microbiomes.

Apparent biogeographical trends in Alexandrium blooms for northern Europe: identifying links to climate change and effective adaptive actions.

The marine dinoflagellate Alexandrium Halim represents perhaps the most significant and intensively studied genus with respect to species diversity, life history strategies, toxigenicity, biogeographical distribution, and global magnitude and consequences harmful algal blooms (HABs). The socioeconomic impacts, environmental and human health risks, and mitigation strategies for toxigenic Alexandrium blooms have also been explored in recent years. Human adaptive actions based on future scenarios of bloom dynamics and shifts in biogeographical distribution under climate-change parameters remain under development and not yet implemented on a regional scale. In the CoCliME (Co-development of climate services for adaptation to changing marine ecosystems) project these issues were addressed with respect to past, current and anticipated future status of key HAB genera and expected benefits of enhanced monitoring. Data on the distribution and frequency of Alexandrium blooms related to paralytic shellfish toxin (PST) events from key CoCliME Case Study areas, comprising the North Sea and adjacent Kattegat-Skagerrak, Norwegian Sea, and Baltic Sea, and eastern North Atlantic marginal seas, were evaluated in a contemporary and historical context over the past several decades. The first evidence of possible biogeographical expansion of Alexandrium taxa into eastern Arctic gateways was provided from DNA barcoding signatures. Various key climate change indicators, such as salinity, temperature, and water-column stratification, relevant to Alexandrium bloom initiation and development were identified. The possible influence of changing variables on bloom dynamics, magnitude, frequency and spatial and temporal distribution were interpreted in the context of regional ocean climate models. These climate change impact indicators may play key roles in selecting for the occurrence and diversity of Alexandrium species within the broader microeukaryote communities. For example, shifts to higher temperature and lower salinity regimes predicted for the southern North Sea indicate the potential for increased Alexandrium blooms, currently absent from this area. Ecological and socioeconomic impacts of Alexandrium blooms and effects on fisheries and aquaculture resources and coastal ecosystem function are evaluated, and, where feasible, effective adaptation strategies are proposed herein as emerging climate services.

Strong population genomic structure of the toxic dinoflagellate Alexandrium minutum inferred from meta-transcriptome samples.

Despite theoretical expectations, marine microeukaryote population are often highly structured and the mechanisms behind such patterns remain to be elucidated. These organisms display huge census population sizes, yet genotyping usually requires clonal strains originating from single cells, hindering proper population sampling. Estimating allelic frequency directly from population wide samples, without any isolation step, offers an interesting alternative. Here, we validate the use of meta-transcriptome environmental samples to determine the population genetic structure of the dinoflagellate Alexandrium minutum. Strain and meta-transcriptome based results both indicated a strong genetic structure for A. minutum in Western Europe, to the level expected between cryptic species. The presence of numerous private alleles, and even fixed polymorphism, would indicate ancient divergence and absence of gene flow between populations. Single nucleotide polymorphisms (SNPs) displaying strong allele frequency differences were distributed throughout the genome, which might indicate pervasive selection from standing genetic variation (soft selective sweeps). However, a few genomic regions displayed extremely low diversity that could result from the fixation of adaptive de novo mutations (hard selective sweeps) within the populations.

Sediment archives reveal irreversible shifts in plankton communities after World War II and agricultural pollution.

To evaluate the stability and resilience1 of coastal ecosystem communities to perturbations that occurred during the Anthropocene,2 pre-industrial biodiversity baselines inferred from paleoarchives are needed.3,4 The study of ancient DNA (aDNA) from sediments (sedaDNA)5 has provided valuable information about past dynamics of microbial species6-8 and communities9-18 in relation to ecosystem variations. Shifts in planktonic protist communities might significantly affect marine ecosystems through cascading effects,19-21 and therefore the analysis of this compartment is essential for the assessment of ecosystem variations. Here, sediment cores collected from different sites of the Bay of Brest (northeast Atlantic, France) allowed ca. 1,400 years of retrospective analyses of the effects of human pollution on marine protists. Comparison of sedaDNA extractions and metabarcoding analyses with different barcode regions (V4 and V7 18S rDNA) revealed that protist assemblages in ancient sediments are mainly composed of species known to produce resting stages. Heavy-metal pollution traces in sediments were ascribed to the World War II period and coincided with community shifts within dinoflagellates and stramenopiles. After the war and especially from the 1980s to 1990s, protist genera shifts followed chronic contaminations of agricultural origin. Community composition reconstruction over time showed that there was no recovery to a Middle Ages baseline composition. This demonstrates the irreversibility of the observed shifts after the cumulative effect of war and agricultural pollutions. Developing a paleoecological approach, this study highlights how human contaminations irreversibly affect marine microbial compartments, which contributes to the debate on coastal ecosystem preservation and restoration.

Diversity and regional distribution of harmful algal events along the Atlantic margin of Europe.

The IOC-ICES-PICES Harmful Algal Event Database (HAEDAT) was used to describe the diversity and spatiotemporal distribution of harmful algal events along the Atlantic margin of Europe from 1987 - 2018. The majority of events recorded are caused by Diarrhetic Shellfish Toxins (DSTs). These events are recorded annually over a wide geographic area from southern Spain to northern Scotland and Iceland, and are responsible for annual closures of many shellfish harvesting areas. The dominant causative dinoflagellates, members of the morphospecies 'Dinophysis acuminata complex' and D. acuta, are common in the waters of the majority of countries affected. There are regional differences in the causative species associated with PST events; the coasts of Spain and Portugal with the dinoflagellates Alexandrium minutum and Gymnodinium catenatum, north west France/south west England/south Ireland with A. minutum, and Scotland/Faroe Islands/Iceland with A. catenella. This can influence the duration and spatial scale of PST events as well as the toxicity of shellfish. The diatom Pseudo-nitzschia australis is the most widespread Domoic Acid (DA) producer, with records coming from Spain, Portugal, France, Ireland and the UK. Amnesic Shellfish Toxins (ASTs) have caused prolonged closures for the scallop fishing industry due to the slow depuration rate of DA. Amendments to EU shellfish hygiene regulations introduced between 2002 and 2005 facilitated end-product testing and sale of adductor muscle. This reduced the impact of ASTs on the scallop fishing industry and thus the number of recorded HAEDAT events. Azaspiracids (AZAs) are the most recent toxin group responsible for events to be characterised in the ICES area. Events associated with AZAs have a discrete distribution with the majority recorded along the west coast of Ireland. Ciguatera Poisoning (CP) has been an emerging issue in the Canary Islands and Madeira since 2004. The majority of aquaculture and wild fish mortality events are associated with blooms of the dinoflagellate Karenia mikimotoi and raphidophyte Heterosigma akashiwo. Such fish killing events occur infrequently yet can cause significant mortalities. Interannual variability was observed in the annual number of HAEDAT areas with events associated with individual shellfish toxin groups. HABs represent a continued risk for the aquaculture industry along the Atlantic margin of Europe and should be accounted for when considering expansion of the industry or operational shifts to offshore areas.

Bacteria enhance the production of extracellular polymeric substances by the green dinoflagellate Lepidodinium chlorophorum.

High biomasses of the marine dinoflagellate Lepidodinium chlorophorum cause green seawater discolorations along Southern Brittany (NE Atlantic, France). The viscosity associated to these phenomena has been related to problems in oyster cultivation. The harmful effect of L. chlorophorum might originate from the secretion of Extracellular Polymeric Substances (EPS). To understand whether the EPS are produced by L. chlorophorum or its associated bacteria, or if they are a product of their interaction, batch cultures were performed under non-axenic and pseudo-axenic conditions for three strains. Maximum dinoflagellate cell abundances were observed in pseudo-axenic cultures. The non-sinking fraction of polymers (Soluble Extracellular Polymers, SEP), mainly composed of proteins and the exopolysaccharide sulphated galactan, slightly increased in pseudo-axenic cultures. The amount of Transparent Exopolymer Particles (TEP) per cell increased under non-axenic conditions. Despite the high concentrations of Particulate Organic Carbon (POC) measured, viscosity did not vary. These results suggest that the L. chlorophorum-bacteria interaction could have a detrimental consequence on the dinoflagellate, translating in a negative effect on L. chlorophorum growth, as well as EPS overproduction by the dinoflagellate, at concentrations that should not affect seawater viscosity.

Variable inter and intraspecies alkaline phosphatase activity within single cells of revived dinoflagellates.

Adaptation of cell populations to environmental changes is mediated by phenotypic variability at the single-cell level. Enzyme activity is a key factor in cell phenotype and the expression of the alkaline phosphatase activity (APA) is a fundamental phytoplankton strategy for maintaining growth under phosphate-limited conditions. Our aim was to compare the APA among cells and species revived from sediments of the Bay of Brest (Brittany, France), corresponding to a pre-eutrophication period (1940's) and a beginning of a post-eutrophication period (1990's) during which phosphate concentrations have undergone substantial variations. Both toxic marine dinoflagellate Alexandrium minutum and the non-toxic dinoflagellate Scrippsiella acuminata were revived from ancient sediments. Using microfluidics, we measured the kinetics of APA at the single-cell level. Our results indicate that all S. acuminata strains had significantly higher APA than A. minutum strains. For both species, the APA in the 1990's decade was significantly lower than in the 1940's. For the first time, our results reveal both inter and intraspecific variabilities of dinoflagellate APA and suggest that, at a half-century timescale, two different species of dinoflagellate may have undergone similar adaptative evolution to face environmental changes and acquire ecological advantages.

Phytoplankton taxonomic and functional diversity patterns across a coastal tidal front.

Oceanic physics at fine scale; e.g. eddies, fronts, filaments; are notoriously difficult to sample. However, an increasing number of theoretical approaches hypothesize that these processes affect phytoplankton diversity which have cascading effects on regional ecosystems. In 2015, we targeted the Iroise Sea (France) and evidenced the setting up of the Ushant tidal front from the beginning of spring to late summer. Seawater samples were taken during three sampling cruises and DNA-barcoding allowed us to investigate patterns of eukaryotic phytoplankton diversity across this front. First focusing on patterns of taxonomic richness, we evidenced that the front harbored a hotspot of eukaryotic phytoplankton diversity sustained throughout summer. We then detail the ecological processes leading to the formation of this hotspot by studying shifts in community composition across the Iroise Sea. Physical mixing mingled the communities surrounding the front, allowing the formation of a local ecotone, but it was cycles of disturbances and nutrient inputs over the front that allowed a decrease in competitive exclusion, which maintained a higher diversity of rare phytoplankton taxa. These processes did not select a specific ecological strategy as inferred by a trait approach coupled to our taxonomic approach. Instead the front favored higher richness within widespread strategies, resulting in functional redundancy. We detail how fine-scale ocean physics affect phytoplankton diversity and suppose that this interplay is a major control on regional ecosystems.

Current distribution and potential expansion of the harmful benthic dinoflagellate Ostreopsis cf. siamensis towards the warming waters of the Bay of Biscay, North-East Atlantic.

In a future scenario of increasing temperatures in North-Atlantic waters, the risk associated with the expansion of the harmful, benthic dinoflagellate Ostreopsis cf. siamensis has to be evaluated and monitored. Microscopy observations and spatio-temporal surveys of environmental DNA (eDNA) were associated with Lagrangian particle dispersal simulations to: (i) establish the current colonization of the species in the Bay of Biscay, (ii) assess the spatial connectivity among sampling zones that explain this distribution, and (iii) identify the sentinel zones to monitor future expansion. Throughout a sampling campaign carried out in August to September 2018, microscope analysis showed that the species develops in the south-east of the bay where optimal temperatures foster blooms. Quantitative PCR analyses revealed its presence across almost the whole bay to the western English Channel. An eDNA time-series collected on plastic samplers showed that the species occurs in the bay from April to September. Due to the water circulation, colonization of the whole bay from the southern blooming zones is explained by inter-site connectivity. Key areas in the middle of the bay permit continuous dispersal connectivity towards the north. These key areas are proposed as sentinel zones to monitor O. cf. siamensis invasions towards the presumably warming water of the North-East Atlantic.

Morphological and phylogenetic data do not support the split of Alexandrium into four genera.

A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: "The polyphyly [sic] of Alexandrium is solved with the split into four genera". However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted.

Revival of Ancient Marine Dinoflagellates Using Molecular Biostimulation.

The biological processes involved in the preservation, viability, and revival of long-term dormant dinoflagellate cysts buried in sediments remain unknown. Based on studies of plant seed physiology, we tested whether the revival of ancient cysts preserved in century-old sediments from the Bay of Brest (France) could be stimulated by melatonin and gibberellic acid, two molecules commonly used in seed priming. Dinoflagellates were revived from sediments dated to approximately 150 years ago (156 ± 27, 32 cm depth), extending the known record age of cyst viability previously established as around one century. A culture suspension of sediments mixed with melatonin and gibberellic acid solutions as biostimulants exhibited germination of 11 dinoflagellate taxa that could not be revived under controlled culture conditions. The biostimulants revived some dinoflagellates from century-old sediments, including the potentially toxic species Alexandrium minutum. The biostimulants showed positive effects on germination on even more ancient cysts, showing dose-dependent effects on the germination of Scrippsiella acuminata. Concentrations of 1, 10, and 100 µM melatonin and gibberellic acid promoted germination. In contrast, 1,000 µM solutions, particularly for melatonin, drastically decreased germination, suggesting a potential noxious effect of high doses of these molecules on dinoflagellate revival. Our findings suggest that melatonin and gibberellic acid are involved in the stimulation of germination of dinoflagellate cysts. These biostimulants can be used to germinate long-term stored dinoflagellate cysts, which may promote studies of ancient strains in the resurrection ecology research field.

Species specific gene expression dynamics during harmful algal blooms.

Harmful algal blooms are caused by specific members of microbial communities. Understanding the dynamics of these events requires comparing the strategies developed by the problematic species to cope with environmental fluctuations to the ones developed by the other members of the community. During three consecutive years, the meta-transcriptome of micro-eukaryote communities was sequenced during blooms of the toxic dinoflagellate Alexandrium minutum. The dataset was analyzed to investigate species specific gene expression dynamics. Major shifts in gene expression were explained by the succession of different species within the community. Although expression patterns were strongly correlated with fluctuation of the abiotic environment, and more specifically with nutrient concentration, transcripts specifically involved in nutrient uptake and metabolism did not display extensive changes in gene expression. Compared to the other members of the community, A. minutum displayed a very specific expression pattern, with lower expression of photosynthesis transcripts and central metabolism genes (TCA cycle, glucose metabolism, glycolysis…) and contrasting expression pattern of ion transporters across environmental conditions. These results suggest the importance of mixotrophy, cell motility and cell-to-cell interactions during A. minutum blooms.

Cryptic species in the parasitic Amoebophrya species complex revealed by a polyphasic approach.

As critical primary producers and recyclers of organic matter, the diversity of marine protists has been extensively explored by high-throughput barcode sequencing. However, classification of short metabarcoding sequences into traditional taxonomic units is not trivial, especially for lineages mainly known by their genetic fingerprints. This is the case for the widespread Amoebophrya ceratii species complex, parasites of their dinoflagellate congeners. We used genetic and phenotypic characters, applied to 119 Amoebophrya individuals sampled from the same geographic area, to construct practical guidelines for species delineation that could be applied in DNA/RNA based diversity analyses. Based on the internal transcribed spacer (ITS) regions, ITS2 compensatory base changes (CBC) and genome k-mer comparisons, we unambiguously defined eight cryptic species among closely related ribotypes that differed by less than 97% sequence identity in their SSU rDNA. We then followed the genetic signatures of these parasitic species during a three-year survey of Alexandrium minutum blooms. We showed that these cryptic Amoebophrya species co-occurred and shared the same ecological niche. We also observed a maximal ecological fitness for parasites having narrow to intermediate host ranges, reflecting a high cost for infecting a broader host range. This study suggests that a complete taxonomic revision of these parasitic dinoflagellates is long overdue to understand their diversity and ecological role in the marine plankton.

Coupling between taxonomic and functional diversity in protistan coastal communities.

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.

Novel Widespread Marine Oomycetes Parasitising Diatoms, Including the Toxic Genus Pseudo-nitzschia: Genetic, Morphological, and Ecological Characterisation.

Parasites are key drivers of phytoplankton bloom dynamics and related aquatic ecosystem processes. Yet, the dearth of morphological and molecular information hinders the assessment of their diversity and ecological role. Using single-cell techniques, we characterise morphologically and molecularly, intracellular parasitoids infecting four potentially toxin-producing Pseudo-nitzschia and one Melosira species on the North Atlantic coast. These sequences define two, morphologically indistinguishable clades within the phylum Oomycota, related to the genera of algal parasites Anisolpidium and Olpidiopsis and the diatom parasitoid species Miracula helgolandica. Our morphological data are insufficient to attribute either clade to the still unsequenced genus Ectrogella; hence it is proposed to name the clades OOM_1 and OOM_2. A screening of global databases of the barcode regions V4 and V9 of the 18S rDNA demonstrate the presence of these parasitoids beyond the North Atlantic coastal region. During a biweekly metabarcoding survey (Concarneau Bay, France), reads associated with one sequenced parasitoid coincided with the decline of Cerataulina pelagica bloom, whilst the other parasitoids co-occurred at low abundance with Pseudo-nitzschia. Our data highlight a complex and unexplored diversity of the oomycete parasitoids of diatoms and calls for the investigation of their phenology, evolution, and potential contribution in controlling their host spatial-temporal dynamics.

Analysis of the genomic basis of functional diversity in dinoflagellates using a transcriptome-based sequence similarity network.

Dinoflagellates are one of the most abundant and functionally diverse groups of eukaryotes. Despite an overall scarcity of genomic information for dinoflagellates, constantly emerging high-throughput sequencing resources can be used to characterize and compare these organisms. We assembled de novo and processed 46 dinoflagellate transcriptomes and used a sequence similarity network (SSN) to compare the underlying genomic basis of functional features within the group. This approach constitutes the most comprehensive picture to date of the genomic potential of dinoflagellates. A core-predicted proteome composed of 252 connected components (CCs) of putative conserved protein domains (pCDs) was identified. Of these, 206 were novel and 16 lacked any functional annotation in public databases. Integration of functional information in our network analyses allowed investigation of pCDs specifically associated with functional traits. With respect to toxicity, sequences homologous to those of proteins found in species with toxicity potential (e.g., sxtA4 and sxtG) were not specific to known toxin-producing species. Although not fully specific to symbiosis, the most represented functions associated with proteins involved in the symbiotic trait were related to membrane processes and ion transport. Overall, our SSN approach led to identification of 45,207 and 90,794 specific and constitutive pCDs of, respectively, the toxic and symbiotic species represented in our analyses. Of these, 56% and 57%, respectively (i.e., 25,393 and 52,193 pCDs), completely lacked annotation in public databases. This stresses the extent of our lack of knowledge, while emphasizing the potential of SSNs to identify candidate pCDs for further functional genomic characterization.

Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetoceros neogracilis species complex.

Seventy-five diatom strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM), as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, Attheya, Chaetoceros, Cylindrotheca, Eucampia, Nitzschia, Porosira, Pseudo-nitzschia, Shionodiscus, Thalassiosira, and Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS-1 and ITS-2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi-compensatory base changes (HCBCs) in paired positions of the ITS-2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co-occur in summer phytoplankton communities in the Beaufort Sea. C. neogracilis generally occurred as single cells but also formed short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis, but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic diatom taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.

Evolutionary processes and cellular functions underlying divergence in Alexandrium minutum.

Understanding divergence in the highly dispersive and seemingly homogeneous pelagic environment for organisms living as free drifters in the water column remains a challenge. Here, we analysed the transcriptome-wide mRNA sequences, as well as the morphology of 18 strains of Alexandrium minutum, a dinoflagellate responsible for harmful algal blooms worldwide, to investigate the functional bases of a divergence event. Analysis of the joint site frequency spectrum (JSFS) pointed towards an ancestral divergence in complete isolations followed by a secondary contact resulting in gene flow between the two diverging groups, but heterogeneous across sites. The sites displaying fixed SNPs were associated with a highly restricted gene flow and a strong overrepresentation of nonsynonymous polymorphism, suggesting the importance of selective pressures as drivers of the divergence. The most divergent transcripts were homologs to genes involved in calcium/potassium fluxes across the membrane, calcium transduction signal and saxitoxin production. The implication of these results in terms of ecological divergence and build-up of reproductive isolation is discussed. Dinoflagellates are especially difficult to study in the field at the ecological level due to their small size and the dynamic nature of their natural environment, but also at the genomic level due to their huge and complex genome and the absence of closely related model organism. This study illustrates the possibility to identify the traits of primary importance in ecology and evolution starting from high-throughput sequencing data, even for such organisms.

Benthic protists: the under-charted majority.

Marine protist diversity inventories have largely focused on planktonic environments, while benthic protists have received relatively little attention. We therefore hypothesize that current diversity surveys have only skimmed the surface of protist diversity in marine sediments, which may harbor greater diversity than planktonic environments. We tested this by analyzing sequences of the hypervariable V4 18S rRNA from benthic and planktonic protist communities sampled in European coastal regions. Despite a similar number of OTUs in both realms, richness estimations indicated that we recovered at least 70% of the diversity in planktonic protist communities, but only 33% in benthic communities. There was also little overlap of OTUs between planktonic and benthic communities, as well as between separate benthic communities. We argue that these patterns reflect the heterogeneity and diversity of benthic habitats. A comparison of all OTUs against the Protist Ribosomal Reference database showed that a higher proportion of benthic than planktonic protist diversity is missing from public databases; similar results were obtained by comparing all OTUs against environmental references from NCBI's Short Read Archive. We suggest that the benthic realm may therefore be the world's largest reservoir of marine protist diversity, with most taxa at present undescribed.

Historical records from dated sediment cores reveal the multidecadal dynamic of the toxic dinoflagellate Alexandrium minutum in the Bay of Brest (France).

The multiannual dynamic of the cyst-forming and toxic marine dinoflagellate Alexandrium minutum was studied over a time scale of about 150 years by a paleoecological approach based on ancient DNA (aDNA) quantification and cyst revivification data obtained from two dated sediment cores of the Bay of Brest (Brittany, France). The first genetic traces of the species presence in the study area dated back to 1873 ± 6. Specific aDNA could be quantified by a newly developed real-time PCR assay in the upper core layers, in which the germination of the species (in up to 17-19-year-old sediments) was also obtained. In both cores studied, our quantitative paleogenetic data showed a statistically significant increasing trend in the abundance of A. minutum ITS1 rDNA copies over time, corroborating three decades of local plankton data that have documented an increasing trend in the species cell abundance. By comparison, paleogenetic data of the dinoflagellate Scrippsiella donghaienis did not show a coherent trend between the cores studied, supporting the hypothesis of the existence of a species-specific dynamic of A. minutum in the study area. This work contributes to the development of paleoecological research, further showing its potential for biogeographical, ecological and evolutionary studies on marine microbes.