Genome-wide assessment of genetic diversity and transcript variations in 17 accessions of the model diatom Phaeodactylum tricornutum.

Diatoms, a prominent group of phytoplankton, have a significant impact on both the oceanic food chain and carbon sequestration, thereby playing a crucial role in regulating the climate. These highly diverse organisms show a wide geographic distribution across various latitudes. In addition to their ecological significance, diatoms represent a vital source of bioactive compounds that are widely used in biotechnology applications. In the present study, we investigated the genetic and transcriptomic diversity of 17 accessions of the model diatom Phaeodactylum tricornutum including those sampled a century ago as well as more recently collected accessions. The analysis of the data reveals a higher genetic diversity and the emergence of novel clades, indicating an increasing diversity within the P. tricornutum population structure, compared to the previous study and a persistent long-term balancing selection of genes in old and newly sampled accessions. However, the study did not establish a clear link between the year of sampling and genetic diversity, thereby, rejecting the hypothesis of loss of heterozygoty in cultured strains. Transcript analysis identified novel transcript including noncoding RNA and other categories of small RNA such as PiwiRNAs. Additionally, transcripts analysis using differential expression as well as Weighted Gene Correlation Network Analysis has provided evidence that the suppression or downregulation of genes cannot be solely attributed to loss-of-function mutations. This implies that other contributing factors, such as epigenetic modifications, may play a crucial role in regulating gene expression. Our study provides novel genetic resources, which are now accessible through the platform PhaeoEpiview (https://PhaeoEpiView.univ-nantes.fr), that offer both ease of use and advanced tools to further investigate microalgae biology and ecology, consequently enriching our current understanding of these organisms.

A High Frequency of Chromosomal Duplications in Unicellular Algae Is Compensated by Translational Regulation.

Although duplications have long been recognized as a fundamental process driving major evolutionary innovations, direct estimates of spontaneous chromosome duplication rates, leading to aneuploid karyotypes, are scarce. Here, from mutation accumulation (MA) experiments, we provide the first estimates of spontaneous chromosome duplication rates in six unicellular eukaryotic species, which range from 1 × 10-4 to 1 × 10-3 per genome per generation. Although this is ∼5 to ∼60 times less frequent than spontaneous point mutations per genome, chromosome duplication events can affect 1-7% of the total genome size. In duplicated chromosomes, mRNA levels reflected gene copy numbers, but the level of translation estimated by polysome profiling revealed that dosage compensation must be occurring. In particular, one duplicated chromosome showed a 2.1-fold increase of mRNA but translation rates were decreased to 0.7-fold. Altogether, our results support previous observations of chromosome-dependent dosage compensation effects, providing evidence that compensation occurs during translation. We hypothesize that an unknown posttranscriptional mechanism modulates the translation of hundreds of transcripts from genes located on duplicated regions in eukaryotes.

Long-Term Stability of Bacterial Associations in a Microcosm of Ostreococcus tauri (Chlorophyta, Mamiellophyceae).

Phytoplankton-bacteria interactions rule over carbon fixation in the sunlit ocean, yet only a handful of phytoplanktonic-bacteria interactions have been experimentally characterized. In this study, we investigated the effect of three bacterial strains isolated from a long-term microcosm experiment with one Ostreococcus strain (Chlorophyta, Mamiellophyceae). We provided evidence that two Roseovarius strains (Alphaproteobacteria) had a beneficial effect on the long-term survival of the microalgae whereas one Winogradskyella strain (Flavobacteriia) led to the collapse of the microalga culture. Co-cultivation of the beneficial and the antagonistic strains also led to the loss of the microalga cells. Metagenomic analysis of the microcosm is consistent with vitamin B12 synthesis by the Roseovarius strains and unveiled two additional species affiliated to Balneola (Balneolia) and Muricauda (Flavobacteriia), which represent less than 4% of the reads, whereas Roseovarius and Winogradskyella recruit 57 and 39% of the reads, respectively. These results suggest that the low-frequency bacterial species may antagonize the algicidal effect of Winogradskyella in the microbiome of Ostreococcus tauri and thus stabilize the microalga persistence in the microcosm. Altogether, these results open novel perspectives into long-term stability of phytoplankton cultures.

Evolutionary Genomics of Sex-Related Chromosomes at the Base of the Green Lineage.

Although sex is now accepted as a ubiquitous and ancestral feature of eukaryotes, direct observation of sex is still lacking in most unicellular eukaryotic lineages. Evidence of sex is frequently indirect and inferred from the identification of genes involved in meiosis from whole genome data and/or the detection of recombination signatures from genetic diversity in natural populations. In haploid unicellular eukaryotes, sex-related chromosomes are named mating-type (MTs) chromosomes and generally carry large genomic regions where recombination is suppressed. These regions have been characterized in Fungi and Chlorophyta and determine gamete compatibility and fusion. Two candidate MT+ and MT- alleles, spanning 450-650 kb, have recently been described in Ostreococcus tauri, a marine phytoplanktonic alga from the Mamiellophyceae class, an early diverging branch in the green lineage. Here, we investigate the architecture and evolution of these candidate MT+ and MT- alleles. We analyzed the phylogenetic profile and GC content of MT gene families in eight different genomes whose divergence has been previously estimated at up to 640 Myr, and found evidence that the divergence of the two MT alleles predates speciation in the Ostreococcus genus. Phylogenetic profiles of MT trans-specific polymorphisms in gametologs disclosed candidate MTs in two additional species, and possibly a third. These Mamiellales MT candidates are likely to be the oldest mating-type loci described to date, which makes them fascinating models to investigate the evolutionary mechanisms of haploid sex determination in eukaryotes.

Features of the Opportunistic Behaviour of the Marine Bacterium Marinobacter algicola in the Microalga Ostreococcus tauri Phycosphere.

Although interactions between microalgae and bacteria are observed in both natural environment and the laboratory, the modalities of coexistence of bacteria inside microalgae phycospheres in laboratory cultures are mostly unknown. Here, we focused on well-controlled cultures of the model green picoalga Ostreococcus tauri and the most abundant member of its phycosphere, Marinobacter algicola. The prevalence of M. algicola in O. tauri cultures raises questions about how this bacterium maintains itself under laboratory conditions in the microalga culture. The results showed that M. algicola did not promote O. tauri growth in the absence of vitamin B12 while M. algicola depended on O. tauri to grow in synthetic medium, most likely to obtain organic carbon sources provided by the microalgae. M. algicola grew on a range of lipids, including triacylglycerols that are known to be produced by O. tauri in culture during abiotic stress. Genomic screening revealed the absence of genes of two particular modes of quorum-sensing in Marinobacter genomes which refutes the idea that these bacterial communication systems operate in this genus. To date, the 'opportunistic' behaviour of M. algicola in the laboratory is limited to several phytoplanktonic species including Chlorophyta such as O. tauri. This would indicate a preferential occurrence of M. algicola in association with these specific microalgae under optimum laboratory conditions.

Combining Nanopore and Illumina Sequencing Permits Detailed Analysis of Insertion Mutations and Structural Variations Produced by PEG-Mediated Transformation in Ostreococcus tauri.

Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration occurred randomly at a single locus in the genome and mainly as a single copy. Thus, we confirmed the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci. Furthermore, we often observed variations in the vector sequence itself. From these observations, we speculate that a nonhomologous end-joining-like mechanism is employed during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in this ecologically important marine alga.

Author Correction: The Seminavis robusta genome provides insights into the evolutionary adaptations of benthic diatoms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Seminavis robusta genome provides insights into the evolutionary adaptations of benthic diatoms.

Benthic diatoms are the main primary producers in shallow freshwater and coastal environments, fulfilling important ecological functions such as nutrient cycling and sediment stabilization. However, little is known about their evolutionary adaptations to these highly structured but heterogeneous environments. Here, we report a reference genome for the marine biofilm-forming diatom Seminavis robusta, showing that gene family expansions are responsible for a quarter of all 36,254 protein-coding genes. Tandem duplications play a key role in extending the repertoire of specific gene functions, including light and oxygen sensing, which are probably central for its adaptation to benthic habitats. Genes differentially expressed during interactions with bacteria are strongly conserved in other benthic diatoms while many species-specific genes are strongly upregulated during sexual reproduction. Combined with re-sequencing data from 48 strains, our results offer insights into the genetic diversity and gene functions in benthic diatoms.

Virus-host coexistence in phytoplankton through the genomic lens.

Virus-microbe interactions in the ocean are commonly described by "boom and bust" dynamics, whereby a numerically dominant microorganism is lysed and replaced by a virus-resistant one. Here, we isolated a microalga strain and its infective dsDNA virus whose dynamics are characterized instead by parallel growth of both the microalga and the virus. Experimental evolution of clonal lines revealed that this viral production originates from the lysis of a minority of virus-susceptible cells, which are regenerated from resistant cells. Whole-genome sequencing demonstrated that this resistant-susceptible switch involved a large deletion on one chromosome. Mathematical modeling explained how the switch maintains stable microalga-virus population dynamics consistent with their observed growth pattern. Comparative genomics confirmed an ancient origin of this "accordion" chromosome despite a lack of sequence conservation. Together, our results show how dynamic genomic rearrangements may account for a previously overlooked coexistence mechanism in microalgae-virus interactions.

Metabolomic Insights into Marine Phytoplankton Diversity.

The democratization of sequencing technologies fostered a leap in our knowledge of the diversity of marine phytoplanktonic microalgae, revealing many previously unknown species and lineages. The evolutionary history of the diversification of microalgae can be inferred from the analysis of their genome sequences. However, the link between the DNA sequence and the associated phenotype is notoriously difficult to assess, all the more so for marine phytoplanktonic microalgae for which the lab culture and, thus, biological experimentation is very tedious. Here, we explore the potential of a high-throughput untargeted metabolomic approach to explore the phenotypic-genotypic gap in 12 marine microalgae encompassing 1.2 billion years of evolution. We identified species- and lineage-specific metabolites. We also provide evidence of a very good correlation between the molecular divergence, inferred from the DNA sequences, and the metabolomic divergence, inferred from the complete metabolomic profiles. These results provide novel insights into the potential of chemotaxonomy in marine phytoplankton and support the hypothesis of a metabolomic clock, suggesting that DNA and metabolomic profiles co-evolve.

Genome Resolved Biogeography of Mamiellales.

Among marine phytoplankton, Mamiellales encompass several species from the genera Micromonas, Ostreococcus and Bathycoccus, which are important contributors to primary production. Previous studies based on single gene markers described their wide geographical distribution but led to discussion because of the uneven taxonomic resolution of the method. Here, we leverage genome sequences for six Mamiellales species, two from each genus Micromonas, Ostreococcus and Bathycoccus, to investigate their distribution across 133 stations sampled during the Tara Oceans expedition. Our study confirms the cosmopolitan distribution of Mamiellales and further suggests non-random distribution of species, with two triplets of co-occurring genomes associated with different temperatures: Ostreococcuslucimarinus, Bathycoccusprasinos and Micromonaspusilla were found in colder waters, whereas Ostreococcus spp. RCC809, Bathycoccus spp. TOSAG39-1 and Micromonascommoda were more abundant in warmer conditions. We also report the distribution of the two candidate mating-types of Ostreococcus for which the frequency of sexual reproduction was previously assumed to be very low. Indeed, both mating types were systematically detected together in agreement with either frequent sexual reproduction or the high prevalence of a diploid stage. Altogether, these analyses provide novel insights into Mamiellales' biogeography and raise novel testable hypotheses about their life cycle and ecology.

A genomics approach reveals the global genetic polymorphism, structure, and functional diversity of ten accessions of the marine model diatom Phaeodactylum tricornutum.

Diatoms emerged in the Mesozoic period and presently constitute one of the main primary producers in the world's ocean and are of a major economic importance. In the current study, using whole genome sequencing of ten accessions of the model diatom Phaeodactylum tricornutum, sampled at broad geospatial and temporal scales, we draw a comprehensive landscape of the genomic diversity within the species. We describe strong genetic subdivisions of the accessions into four genetic clades (A-D) with constituent populations of each clade possessing a conserved genetic and functional makeup, likely a consequence of the limited dispersal of P. tricornutum in the open ocean. We further suggest dominance of asexual reproduction across all the populations, as implied by high linkage disequilibrium. Finally, we show limited yet compelling signatures of genetic and functional convergence inducing changes in the selection pressure on many genes and metabolic pathways. We propose these findings to have significant implications for understanding the genetic structure of diatom populations in nature and provide a framework to assess the genomic underpinnings of their ecological success and impact on aquatic ecosystems where they play a major role. Our work provides valuable resources for functional genomics and for exploiting the biotechnological potential of this model diatom species.

Single cell ecogenomics reveals mating types of individual cells and ssDNA viral infections in the smallest photosynthetic eukaryotes.

Planktonic photosynthetic organisms of the class Mamiellophyceae include the smallest eukaryotes (less than 2 µm), are globally distributed and form the basis of coastal marine ecosystems. Eight complete fully annotated 13-22 Mb genomes from three genera, Ostreococcus, Bathycoccus and Micromonas, are available from previously isolated clonal cultured strains and provide an ideal resource to explore the scope and challenges of analysing single cell amplified genomes (SAGs) isolated from a natural environment. We assembled data from 12 SAGs sampled during the Tara Oceans expedition to gain biological insights about their in situ ecology, which might be lost by isolation and strain culture. Although the assembled nuclear genomes were incomplete, they were large enough to infer the mating types of four Ostreococcus SAGs. The systematic occurrence of sequences from the mitochondria and chloroplast, representing less than 3% of the total cell's DNA, intimates that SAGs provide suitable substrates for detection of non-target sequences, such as those of virions. Analysis of the non-Mamiellophyceae assemblies, following filtering out cross-contaminations during the sequencing process, revealed two novel 1.6 and 1.8 kb circular DNA viruses, and the presence of specific Bacterial and Oomycete sequences suggests that these organisms might co-occur with the Mamiellales. This article is part of a discussion meeting issue 'Single cell ecology'.

First Estimation of the Spontaneous Mutation Rate in Diatoms.

Mutations are the origin of genetic diversity, and the mutation rate is a fundamental parameter to understand all aspects of molecular evolution. The combination of mutation-accumulation experiments and high-throughput sequencing enabled the estimation of mutation rates in most model organisms, but several major eukaryotic lineages remain unexplored. Here, we report the first estimation of the spontaneous mutation rate in a model unicellular eukaryote from the Stramenopile kingdom, the diatom Phaeodactylum tricornutum (strain RCC2967). We sequenced 36 mutation accumulation lines for an average of 181 generations per line and identified 156 de novo mutations. The base substitution mutation rate per site per generation is µbs = 4.77 × 10-10 and the insertion-deletion mutation rate is µid = 1.58 × 10-11. The mutation rate varies as a function of the nucleotide context and is biased toward an excess of mutations from GC to AT, consistent with previous observations in other species. Interestingly, the mutation rates between the genomes of organelles and the nucleus differ, with a significantly higher mutation rate in the mitochondria. This confirms previous claims based on indirect estimations of the mutation rate in mitochondria of photosynthetic eukaryotes that acquired their plastid through a secondary endosymbiosis. This novel estimate enables us to infer the effective population size of P. tricornutum to be Ne∼8.72 × 106.

Genome Analyses of the Microalga Picochlorum Provide Insights into the Evolution of Thermotolerance in the Green Lineage.

While the molecular events involved in cell responses to heat stress have been extensively studied, our understanding of the genetic basis of basal thermotolerance, and particularly its evolution within the green lineage, remains limited. Here, we present the 13.3-Mb haploid genome and transcriptomes of a halotolerant and thermotolerant unicellular green alga, Picochlorum costavermella (Trebouxiophyceae) to investigate the evolution of the genomic basis of thermotolerance. Differential gene expression at high and standard temperatures revealed that more of the gene families containing up-regulated genes at high temperature were recently evolved, and less originated at the ancestor of green plants. Inversely, there was an excess of ancient gene families containing transcriptionally repressed genes. Interestingly, there is a striking overlap between the thermotolerance and halotolerance transcriptional rewiring, as more than one-third of the gene families up-regulated at 35 °C were also up-regulated under variable salt concentrations in Picochlorum SE3. Moreover, phylogenetic analysis of the 9,304 protein coding genes revealed 26 genes of horizontally transferred origin in P. costavermella, of which five were differentially expressed at higher temperature. Altogether, these results provide new insights about how the genomic basis of adaptation to halo- and thermotolerance evolved in the green lineage.

Fish larval recruitment to reefs is a thyroid hormone-mediated metamorphosis sensitive to the pesticide chlorpyrifos.

Larval recruitment, the transition of pelagic larvae into reef-associated juveniles, is a critical step for the resilience of marine fish populations but its molecular control is unknown. Here, we investigate whether thyroid-hormones (TH) and their receptors (TR) coordinate the larval recruitment of the coral-reef-fish Acanthurus triostegus. We demonstrate an increase of TH-levels and TR-expressions in pelagic-larvae, followed by a decrease in recruiting juveniles. We generalize these observations in four other coral reef-fish species. Treatments with TH or TR-antagonist, as well as relocation to the open-ocean, disturb A. triostegus larvae transformation and grazing activity. Likewise, chlorpyrifos, a pesticide often encountered in coral-reefs, impairs A. triostegus TH-levels, transformation, and grazing activity, hence diminishing this herbivore's ability to control the spread of reef-algae. Larval recruitment therefore corresponds to a TH-controlled metamorphosis, sensitive to endocrine disruption. This provides a framework to understand how larval recruitment, critical to reef-ecosystems maintenance, is altered by anthropogenic stressors.

Population genomics of picophytoplankton unveils novel chromosome hypervariability.

Tiny photosynthetic microorganisms that form the picoplankton (between 0.3 and 3 µm in diameter) are at the base of the food web in many marine ecosystems, and their adaptability to environmental change hinges on standing genetic variation. Although the genomic and phenotypic diversity of the bacterial component of the oceans has been intensively studied, little is known about the genomic and phenotypic diversity within each of the diverse eukaryotic species present. We report the level of genomic diversity in a natural population of Ostreococcus tauri (Chlorophyta, Mamiellophyceae), the smallest photosynthetic eukaryote. Contrary to the expectations of clonal evolution or cryptic species, the spectrum of genomic polymorphism observed suggests a large panmictic population (an effective population size of 1.2 × 107) with pervasive evidence of sexual reproduction. De novo assemblies of low-coverage chromosomes reveal two large candidate mating-type loci with suppressed recombination, whose origin may pre-date the speciation events in the class Mamiellophyceae. This high genetic diversity is associated with large phenotypic differences between strains. Strikingly, resistance of isolates to large double-stranded DNA viruses, which abound in their natural environment, is positively correlated with the size of a single hypervariable chromosome, which contains 44 to 156 kb of strain-specific sequences. Our findings highlight the role of viruses in shaping genome diversity in marine picoeukaryotes.

Spontaneous Mutation Rate in the Smallest Photosynthetic Eukaryotes.

Mutation is the ultimate source of genetic variation, and knowledge of mutation rates is fundamental for our understanding of all evolutionary processes. High throughput sequencing of mutation accumulation lines has provided genome wide spontaneous mutation rates in a dozen model species, but estimates from nonmodel organisms from much of the diversity of life are very limited. Here, we report mutation rates in four haploid marine bacterial-sized photosynthetic eukaryotic algae; Bathycoccus prasinos, Ostreococcus tauri, Ostreococcus mediterraneus, and Micromonas pusilla. The spontaneous mutation rate between species varies from µ = 4.4 × 10-10 to 9.8 × 10-10 mutations per nucleotide per generation. Within genomes, there is a two-fold increase of the mutation rate in intergenic regions, consistent with an optimization of mismatch and transcription-coupled DNA repair in coding sequences. Additionally, we show that deviation from the equilibrium GC content increases the mutation rate by ∼2% to ∼12% because of a GC bias in coding sequences. More generally, the difference between the observed and equilibrium GC content of genomes explains some of the inter-specific variation in mutation rates.