Life cycle strategies in free-living unicellular eukaryotes: Diversity, evolution, and current molecular tools to unravel the private life of microorganisms.

An astonishing range of morphologies and life strategies has arisen across the vast diversity of protists, allowing them to thrive in most environments. In model protists, like Tetrahymena, Dictyostelium, or Trypanosoma, life cycles involving multiple life stages with different morphologies have been well characterized. In contrast, knowledge of the life cycles of free-living protists, which primarily consist of uncultivated environmental lineages, remains largely fragmentary. Various life stages and lineage-specific cellular innovations have been observed in the field for uncultivated protists, but such innovations generally lack functional characterization and have unknown physiological and ecological roles. In the actual state of knowledge, evidence of sexual processes is confirmed for 20% of free-living protist lineages. Nevertheless, at the onset of eukaryotic diversification, common molecular trends emerged to promote genetic recombination, establishing sex as an inherent feature of protists. Here, we review protist life cycles from the viewpoint of life cycle transitions and genetics across major eukaryotic lineages. We focus on the scarcely observed sexual cycle of free-living protists, summarizing evidence for its existence and describing key genes governing its progression, as well as, current methods for studying the genetics of sexual cycles in both cultivable and uncultivated protist groups.

Genomic differentiation of three pico-phytoplankton species in the Mediterranean Sea.

For more than a decade, high-throughput sequencing has transformed the study of marine planktonic communities and has highlighted the extent of protist diversity in these ecosystems. Nevertheless, little is known relative to their genomic diversity at the species-scale as well as their major speciation mechanisms. An increasing number of data obtained from global scale sampling campaigns is becoming publicly available, and we postulate that metagenomic data could contribute to deciphering the processes shaping protist genomic differentiation in the marine realm. As a proof of concept, we developed a findable, accessible, interoperable and reusable (FAIR) pipeline and focused on the Mediterranean Sea to study three a priori abundant protist species: Bathycoccus prasinos, Pelagomonas calceolata and Phaeocystis cordata. We compared the genomic differentiation of each species in light of geographic, environmental and oceanographic distances. We highlighted that isolation-by-environment shapes the genomic differentiation of B. prasinos, whereas P. cordata is impacted by geographic distance (i.e. isolation-by-distance). At present time, the use of metagenomics to accurately estimate the genomic differentiation of protists remains challenging since coverages are lower compared to traditional population surveys. However, our approach sheds light on ecological and evolutionary processes occurring within natural marine populations and paves the way for future protist population metagenomic studies.

Plankton networks driving carbon export in the oligotrophic ocean.

The biological carbon pump is the process by which CO2 is transformed to organic carbon via photosynthesis, exported through sinking particles, and finally sequestered in the deep ocean. While the intensity of the pump correlates with plankton community composition, the underlying ecosystem structure driving the process remains largely uncharacterized. Here we use environmental and metagenomic data gathered during the Tara Oceans expedition to improve our understanding of carbon export in the oligotrophic ocean. We show that specific plankton communities, from the surface and deep chlorophyll maximum, correlate with carbon export at 150 m and highlight unexpected taxa such as Radiolaria and alveolate parasites, as well as Synechococcus and their phages, as lineages most strongly associated with carbon export in the subtropical, nutrient-depleted, oligotrophic ocean. Additionally, we show that the relative abundance of a few bacterial and viral genes can predict a significant fraction of the variability in carbon export in these regions.

Insights into global diatom distribution and diversity in the world's ocean.

Diatoms (Bacillariophyta) constitute one of the most diverse and ecologically important groups of phytoplankton. They are considered to be particularly important in nutrient-rich coastal ecosystems and at high latitudes, but considerably less so in the oligotrophic open ocean. The Tara Oceans circumnavigation collected samples from a wide range of oceanic regions using a standardized sampling procedure. Here, a total of ∼12 million diatom V9-18S ribosomal DNA (rDNA) ribotypes, derived from 293 size-fractionated plankton communities collected at 46 sampling sites across the global ocean euphotic zone, have been analyzed to explore diatom global diversity and community composition. We provide a new estimate of diversity of marine planktonic diatoms at 4,748 operational taxonomic units (OTUs). Based on the total assigned ribotypes, Chaetoceros was the most abundant and diverse genus, followed by Fragilariopsis, Thalassiosira, and Corethron We found only a few cosmopolitan ribotypes displaying an even distribution across stations and high abundance, many of which could not be assigned with confidence to any known genus. Three distinct communities from South Pacific, Mediterranean, and Southern Ocean waters were identified that share a substantial percentage of ribotypes within them. Sudden drops in diversity were observed at Cape Agulhas, which separates the Indian and Atlantic Oceans, and across the Drake Passage between the Atlantic and Southern Oceans, indicating the importance of these ocean circulation choke points in constraining diatom distribution and diversity. We also observed high diatom diversity in the open ocean, suggesting that diatoms may be more relevant in these oceanic systems than generally considered.

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.

Testing ecological theories with sequence similarity networks: marine ciliates exhibit similar geographic dispersal patterns as multicellular organisms.

BACKGROUND: High-throughput sequencing technologies are lifting major limitations to molecular-based ecological studies of eukaryotic microbial diversity, but analyses of the resulting millions of short sequences remain a major bottleneck for these approaches. Here, we introduce the analytical and statistical framework of sequence similarity networks, increasingly used in evolutionary studies and graph theory, into the field of ecology to analyze novel pyrosequenced V4 small subunit rDNA (SSU-rDNA) sequence data sets in the context of previous studies, including SSU-rDNA Sanger sequence data from cultured ciliates and from previous environmental diversity inventories. RESULTS: Our broadly applicable protocol quantified the progress in the description of genetic diversity of ciliates by environmental SSU-rDNA surveys, detected a fundamental historical bias in the tendency to recover already known groups in these surveys, and revealed substantial amounts of hidden microbial diversity. Moreover, network measures demonstrated that ciliates are not globally dispersed, but are structured by habitat and geographical location at intermediate geographical scale, as observed for bacteria, plants, and animals. CONCLUSIONS: Currently available 'universal' primers used for local in-depth sequencing surveys provide little hope to exhaust the significantly higher ciliate (and most likely microbial) diversity than previously thought. Network analyses such as presented in this study offer a promising way to guide the design of novel primers and to further explore this vast and structured microbial diversity.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing.

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September-October (autumn) and lowest in April-May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3-5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy.

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR(2), http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

An original mode of symbiosis in open ocean plankton.

Symbiotic relationships are widespread in nature and are fundamental for ecosystem functioning and the evolution of biodiversity. In marine environments, photosymbiosis with microalgae is best known for sustaining benthic coral reef ecosystems. Despite the importance of oceanic microbiota in global ecology and biogeochemical cycles, symbioses are poorly characterized in open ocean plankton. Here, we describe a widespread symbiotic association between Acantharia biomineralizing microorganisms that are abundant grazers in plankton communities, and members of the haptophyte genus Phaeocystis that are cosmopolitan bloom-forming microalgae. Cophylogenetic analyses demonstrate that symbiont biogeography, rather than host taxonomy, is the main determinant of the association. Molecular dating places the origin of this photosymbiosis in the Jurassic (ca. 175 Mya), a period of accentuated marine oligotrophy. Measurements of intracellular dimethylated sulfur indicate that the host likely profits from antioxidant protection provided by the symbionts as an adaptation to life in transparent oligotrophic surface waters. In contrast to terrestrial and marine symbioses characterized to date, the symbiont reported in this association is extremely abundant and ecologically active in its free-living phase. In the vast and barren open ocean, partnership with photosymbionts that have extensive free-living populations is likely an advantageous strategy for hosts that rely on such interactions. Discovery of the Acantharia-Phaeocystis association contrasts with the widely held view that symbionts are specialized organisms that are rare and ecologically passive outside the host.

SENSE-PPI reconstructs interactomes within, across, and between species at the genome scale.

Ab initio computational reconstructions of protein-protein interaction (PPI) networks will provide invaluable insights into cellular systems, enabling the discovery of novel molecular interactions and elucidating biological mechanisms within and between organisms. Leveraging the latest generation protein language models and recurrent neural networks, we present SENSE-PPI, a sequence-based deep learning model that efficiently reconstructs ab initio PPIs, distinguishing partners among tens of thousands of proteins and identifying specific interactions within functionally similar proteins. SENSE-PPI demonstrates high accuracy, limited training requirements, and versatility in cross-species predictions, even with non-model organisms and human-virus interactions. Its performance decreases for phylogenetically more distant model and non-model organisms, but signal alteration is very slow. In this regard, it demonstrates the important role of parameters in protein language models. SENSE-PPI is very fast and can test 10,000 proteins against themselves in a matter of hours, enabling the reconstruction of genome-wide proteomes.

Identification of DNA Viruses in Ancient DNA from Herbarium Samples.

Herbaria encompass millions of plant specimens, mostly collected in the nineteenth and twentieth centuries that can represent a key resource for investigating the history and evolution of phytopathogens. In the last years, the application of high-throughput sequencing technologies for the analysis of ancient nucleic acids has revolutionized the study of ancient pathogens including viruses, allowing the reconstruction of historical genomic viral sequences, improving phylogenetic based molecular dating, and providing essential insight into plant virus ecology. In this chapter, we describe a protocol to reconstruct ancient plant and soil viral sequences starting from highly fragmented ancient DNA extracted from herbarium plants and their associated rhizospheric soil. Following Illumina high-throughput sequencing, sequence data are de novo assembled, and DNA viral sequences are selected, according to their similarity with known viruses.

The genomic potential of photosynthesis in piconanoplankton is functionally redundant but taxonomically structured at a global scale.

Carbon fixation is a key metabolic function shaping marine life, but the underlying taxonomic and functional diversity involved is only partially understood. Using metagenomic resources targeted at marine piconanoplankton, we provide a reproducible machine learning framework to derive the potential biogeography of genomic functions through the multi-output regression of gene read counts on environmental climatologies. Leveraging the Marine Atlas of Tara Oceans Unigenes, we investigate the genomic potential of primary production in the global ocean. The latter is performed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and is often associated with carbon concentration mechanisms in piconanoplankton, major marine unicellular photosynthetic organisms. We show that the genomic potential supporting C4 enzymes and RUBISCO exhibits strong functional redundancy and important affinity toward tropical oligotrophic waters. This redundancy is taxonomically structured by the dominance of Mamiellophyceae and Prymnesiophyceae in mid and high latitudes. These findings enhance our understanding of the relationship between functional and taxonomic diversity of microorganisms and environmental drivers of key biogeochemical cycles.

Diversity patterns of uncultured Haptophytes unravelled by pyrosequencing in Naples Bay.

Haptophytes are a key phylum of marine protists, including ~300 described morphospecies and 80 morphogenera. We used 454 pyrosequencing on large subunit ribosomal DNA (LSU rDNA) fragments to assess the diversity from size-fractioned plankton samples collected in the Bay of Naples. One group-specific primer set targeting the LSU rDNA D1/D2 region was designed to amplify Haptophyte sequences from nucleic acid extracts (total DNA or RNA) of two size fractions (0.8-3 or 3-20 µm) and two sampling depths [subsurface, at 1 m, or deep chlorophyll maximum (DCM) at 23 m]. 454 reads were identified using a database covering the entire Haptophyta diversity currently sequenced. Our data set revealed several hundreds of Haptophyte clusters. However, most of these clusters could not be linked to taxonomically known sequences: considering OTUs(97%) (clusters build at a sequence identity level of 97%) on our global data set, less than 1% of the reads clustered with sequences from cultures, and less than 12% clustered with reference sequences obtained previously from cloning and Sanger sequencing of environmental samples. Thus, we highlighted a large uncharacterized environmental genetic diversity, which clearly shows that currently cultivated species poorly reflect the actual diversity present in the natural environment. Haptophyte community appeared to be significantly structured according to the depth. The highest diversity and evenness were obtained in samples from the DCM, and samples from the large size fraction (3-20 µm) taken at the DCM shared a lower proportion of common OTUs(97%) with the other samples. Reads from the species Chrysoculter romboideus were notably found at the DCM, while they could be detected at the subsurface. The highest proportion of totally unknown OTUs(97%) was collected at the DCM in the smallest size fraction (0.8-3 µm). Overall, this study emphasized several technical and theoretical barriers inherent to the exploration of the large and largely unknown diversity of unicellular eukaryotes.

Beyond the limits of the unassigned protist microbiome: inferring large-scale spatio-temporal patterns of Syndiniales marine parasites.

Marine protists are major components of the oceanic microbiome that remain largely unrepresented in culture collections and genomic reference databases. The exploration of this uncharted protist diversity in oceanic communities relies essentially on studying genetic markers from the environment as taxonomic barcodes. Here we report that across 6 large scale spatio-temporal planktonic surveys, half of the genetic barcodes remain taxonomically unassigned at the genus level, preventing a fine ecological understanding for numerous protist lineages. Among them, parasitic Syndiniales (Dinoflagellata) appear as the least described protist group. We have developed a computational workflow, integrating diverse 18S rDNA gene metabarcoding datasets, in order to infer large-scale ecological patterns at 100% similarity of the genetic marker, overcoming the limitation of taxonomic assignment. From a spatial perspective, we identified 2171 unassigned clusters, i.e., Syndiniales sequences with 100% similarity, exclusively shared between the Tropical/Subtropical Ocean and the Mediterranean Sea among all Syndiniales orders and 25 ubiquitous clusters shared within all the studied marine regions. From a temporal perspective, over 3 time-series, we highlighted 39 unassigned clusters that follow rhythmic patterns of recurrence and are the best indicators of parasite community's variation. These clusters withhold potential as ecosystem change indicators, mirroring their associated host community responses. Our results underline the importance of Syndiniales in structuring planktonic communities through space and time, raising questions regarding host-parasite association specificity and the trophic mode of persistent Syndiniales, while providing an innovative framework for prioritizing unassigned protist taxa for further description.

Evolutionary history of the Corallinales (Corallinophycidae, Rhodophyta) inferred from nuclear, plastidial and mitochondrial genomes.

Systematics of the red algal order Corallinales has a long and convoluted history. In the present study, molecular approaches were used to assess the phylogenetic relationships based on the analyses of two datasets: a large dataset of SSU sequences including mainly sequences from GenBank; and a combined dataset including four molecular markers (two nuclear: SSU, LSU; one plastidial: psbA; and one mitochondrial: COI). Phylogenetic analyses of both datasets re-affirmed the monophyly of the Corallinales as well as the two families (Corallinaceae and Hapalidiaceae) currently recognized within the order. Three of the four subfamilies of the Corallinaceae (Corallinoideae, Lithophylloideae, Metagoniolithoideae) were also resolved as a monophyletic lineage whereas members of the Mastophoroideae were resolved as four distinct lineages. We therefore propose to restrict the Mastophoroideae to the genera Mastophora, Metamastophora, and possibly Lithoporella in the aim of rendering this subfamily monophyletic. In addition, our phylogenies resolved the genus Hydrolithon in two unrelated lineages, one containing the generitype Hydrolithon reinboldii and the second containing Hydrolithon onkodes, which used to be the generitype of the now defunct genus Porolithon. We therefore propose to resurrect the genus Porolithon for the second lineage encompassing those species with primarily monomerous thalli, and trichocyte arrangements in large pustulate horizontal rows. Moreover, our phylogenetic analyses revealed the presence of cryptic diversity in several taxa, shedding light on the need for further studies to better circumscribe species frontiers within the diverse order Corallinales, especially in the genera Mesophyllum and Neogoniolithon.

Towards omics-based predictions of planktonic functional composition from environmental data.

Marine microbes play a crucial role in climate regulation, biogeochemical cycles, and trophic networks. Unprecedented amounts of data on planktonic communities were recently collected, sparking a need for innovative data-driven methodologies to quantify and predict their ecosystemic functions. We reanalyze 885 marine metagenome-assembled genomes through a network-based approach and detect 233,756 protein functional clusters, from which 15% are functionally unannotated. We investigate all clusters' distributions across the global ocean through machine learning, identifying biogeographical provinces as the best predictors of protein functional clusters' abundance. The abundances of 14,585 clusters are predictable from the environmental context, including 1347 functionally unannotated clusters. We analyze the biogeography of these 14,585 clusters, identifying the Mediterranean Sea as an outlier in terms of protein functional clusters composition. Applicable to any set of sequences, our approach constitutes a step towards quantitative predictions of functional composition from the environmental context.

Mixotrophic protists display contrasted biogeographies in the global ocean.

Mixotrophy, or the ability to acquire carbon from both auto- and heterotrophy, is a widespread ecological trait in marine protists. Using a metabarcoding dataset of marine plankton from the global ocean, 318,054 mixotrophic metabarcodes represented by 89,951,866 sequences and belonging to 133 taxonomic lineages were identified and classified into four mixotrophic functional types: constitutive mixotrophs (CM), generalist non-constitutive mixotrophs (GNCM), endo-symbiotic specialist non-constitutive mixotrophs (eSNCM), and plastidic specialist non-constitutive mixotrophs (pSNCM). Mixotrophy appeared ubiquitous, and the distributions of the four mixotypes were analyzed to identify the abiotic factors shaping their biogeographies. Kleptoplastidic mixotrophs (GNCM and pSNCM) were detected in new zones compared to previous morphological studies. Constitutive and non-constitutive mixotrophs had similar ranges of distributions. Most lineages were evenly found in the samples, yet some of them displayed strongly contrasted distributions, both across and within mixotypes. Particularly divergent biogeographies were found within endo-symbiotic mixotrophs, depending on the ability to form colonies or the mode of symbiosis. We showed how metabarcoding can be used in a complementary way with previous morphological observations to study the biogeography of mixotrophic protists and to identify key drivers of their biogeography.

Author Correction: Clade-specific diversification dynamics of marine diatoms since the Jurassic.

In the version of this Article originally published, the authors did not give credit to David G. Mann for the four microscopic images used in Fig. 1a. This has now been amended in all versions of the Article.

Clade-specific diversification dynamics of marine diatoms since the Jurassic.

Diatoms are one of the most abundant and diverse groups of phytoplankton and play a major role in marine ecosystems and the Earth's biogeochemical cycles. Here we combine DNA metabarcoding data from the Tara Oceans expedition with palaeoenvironmental data and phylogenetic models of diversification to analyse the diversity dynamics of marine diatoms. We reveal a primary effect of variation in carbon dioxide partial pressure (pCO2) on early diatom diversification, followed by a major burst of diversification in the late Eocene epoch, after which diversification is chiefly affected by sea level, an influx of silica availability and competition with other planktonic groups. Our results demonstrate a remarkable heterogeneity of diversification dynamics across diatoms and suggest that a changing climate will favour some clades at the expense of others.