Periodic Reporting for period 1 - GREMA (Genetic REcombination and MAting in Mamiellophyceae)
Reporting period: 2022-10-17 to 2024-10-16
Phytoplankton, often referred to as the "grass" of aquatic ecosystems, are the primary producers that capture CO2 and energy from the sun, sustaining all other living beings throughout the aquatic trophic chain. Despite accounting for only an estimated 1% of the global photosynthetic biomass due to their rapid turnover, phytoplankton are responsible for approximately 45% of Earth's net primary production. Their role in capturing CO2 is critical, as they fuel and regulate the global carbon biogeochemical cycle. Phytoplankton encompass a wide variety of microscopic species, ranging from cyanobacteria to large eukaryotic algae such as diatoms.
Among eukaryotic phytoplankton, a group of tiny green algae, the Mamiellophyceae (class Chlorophyta), is particularly noteworthy. These algae are ubiquitous in the sunlit ocean and can comprise up to 80% of phytoplankton abundance in coastal environments. Mamiellophyceae are a diverse group, with the most abundant marine species often being very small in size (0.8 to 3 µm) and primarily represented by the lineages Mantoniella, Micromonas, Ostreococcus, and Bathycoccus. However, early-branching lineages such as Monomastix, Dolichomastix, and Crustomastix, along with the sister lineage Pyramomonas, are less studied. These lineages are typically larger, occupy different ecological niches, and remain underexplored, particularly with respect to their genomic sequences.
Despite their ecological importance, Mamiellophyceae remain poorly studied. This project aims to expand our understanding of Mamiellophyceae diversity by obtaining genomic sequences from early-branching lineages and exploring the genetic basis of recombination and sexual reproduction. This will be achieved through genome sequencing of newly identified species and genetic experiments focusing on well-known genera such as Ostreococcus.
State-of-the-Art
Recombination, the exchange of DNA between homologous chromosomes, is a hallmark of sexual reproduction and has been observed in the vast majority of eukaryotic species. However, the molecular mechanisms underlying partner recognition, such as the determination of mating types, are largely clade-specific and remain unknown for many eukaryotic groups, including unicellular protists like algae.
In the phylum Chlorophyta, much of the knowledge about sexual reproduction comes from the freshwater alga Chlamydomonas reinhardtii (Chlorophyceae). In C. reinhardtii, fertilization is regulated by a mating locus that exists in two genetically divergent forms, "+" and "-". Haploid cells of opposite mating types fuse, undergo meiosis, and engage in recombination, the reciprocal and non-reciprocal exchange of genetic material between chromatids.
In Mamiellophyceae, indirect evidence of recombination has been derived from population genomics studies of Ostreococcus tauri. Patterns of single nucleotide polymorphisms (SNPs) in these studies reveal recombination events and allow the estimation of recombination rates and the ratio of meiotic to mitotic events. A mating-type locus with two alleles (MT+ and MT-) has been recently identified in O. tauri, enabling the description of mating types in other species such as O. lucimarinus, O. mediterraneus, and Micromonas commoda. However, it remains unclear whether this mating-type locus structure is conserved in earlier diverging Mamiellophyceae lineages or whether these groups have entirely distinct mechanisms of sexual reproduction.
Additionally, the relative frequency of mating types in natural populations and the environmental conditions that trigger mating and recombination are poorly understood. These gaps are particularly urgent to address in the context of the Anthropocene and climate change. If environmental changes disproportionately affect one mating type, it could disrupt sexual reproduction in Mamiellophyceae, compromising their genetic diversity and adaptive potential.
This project seeks to address these knowledge gaps by elucidating the evolution of mating types and the prevalence of recombination across natural populations of ecologically relevant Mamiellophyceae species. It also aims to investigate how environmental factors influence mating. To achieve these objectives, we employed a multidisciplinary approach combining bioinformatics and experimental methods, structured around the following specific goals:
Objective. 1: Describe new early branching Mamiellophycean species. We first, will expand the known diversity of Mamiellophycean species by describe new isolated Mamiellophycean species, or describing properly some species already present in culture collections that awaited for a formal description and come from overlooked early branching lineages that are sister to the most common known species that populate marine ecosystems.
Objective. 2: Capture the ecology and evolution of mating types accross Mamiellophyceae 1) by using a comparative genomics analysis of 10 new different species across Mamiellophyceae and its sister lineage Pyramimonas. In order to to identify the loci of the mating types how it is conserved throught the evolution 2) to estimate the frequency of the two mating types present in Mamiellophyceae natural populations using metagenomic data.
Objective. 3: Detect the abiotic and biotic factors that influence mating in O. tauri by experimental crossing of haploid strains by taking advantage of cell transformation to select for recombinant cells (WP3).
Among eukaryotic phytoplankton, a group of tiny green algae, the Mamiellophyceae (class Chlorophyta), is particularly noteworthy. These algae are ubiquitous in the sunlit ocean and can comprise up to 80% of phytoplankton abundance in coastal environments. Mamiellophyceae are a diverse group, with the most abundant marine species often being very small in size (0.8 to 3 µm) and primarily represented by the lineages Mantoniella, Micromonas, Ostreococcus, and Bathycoccus. However, early-branching lineages such as Monomastix, Dolichomastix, and Crustomastix, along with the sister lineage Pyramomonas, are less studied. These lineages are typically larger, occupy different ecological niches, and remain underexplored, particularly with respect to their genomic sequences.
Despite their ecological importance, Mamiellophyceae remain poorly studied. This project aims to expand our understanding of Mamiellophyceae diversity by obtaining genomic sequences from early-branching lineages and exploring the genetic basis of recombination and sexual reproduction. This will be achieved through genome sequencing of newly identified species and genetic experiments focusing on well-known genera such as Ostreococcus.
State-of-the-Art
Recombination, the exchange of DNA between homologous chromosomes, is a hallmark of sexual reproduction and has been observed in the vast majority of eukaryotic species. However, the molecular mechanisms underlying partner recognition, such as the determination of mating types, are largely clade-specific and remain unknown for many eukaryotic groups, including unicellular protists like algae.
In the phylum Chlorophyta, much of the knowledge about sexual reproduction comes from the freshwater alga Chlamydomonas reinhardtii (Chlorophyceae). In C. reinhardtii, fertilization is regulated by a mating locus that exists in two genetically divergent forms, "+" and "-". Haploid cells of opposite mating types fuse, undergo meiosis, and engage in recombination, the reciprocal and non-reciprocal exchange of genetic material between chromatids.
In Mamiellophyceae, indirect evidence of recombination has been derived from population genomics studies of Ostreococcus tauri. Patterns of single nucleotide polymorphisms (SNPs) in these studies reveal recombination events and allow the estimation of recombination rates and the ratio of meiotic to mitotic events. A mating-type locus with two alleles (MT+ and MT-) has been recently identified in O. tauri, enabling the description of mating types in other species such as O. lucimarinus, O. mediterraneus, and Micromonas commoda. However, it remains unclear whether this mating-type locus structure is conserved in earlier diverging Mamiellophyceae lineages or whether these groups have entirely distinct mechanisms of sexual reproduction.
Additionally, the relative frequency of mating types in natural populations and the environmental conditions that trigger mating and recombination are poorly understood. These gaps are particularly urgent to address in the context of the Anthropocene and climate change. If environmental changes disproportionately affect one mating type, it could disrupt sexual reproduction in Mamiellophyceae, compromising their genetic diversity and adaptive potential.
This project seeks to address these knowledge gaps by elucidating the evolution of mating types and the prevalence of recombination across natural populations of ecologically relevant Mamiellophyceae species. It also aims to investigate how environmental factors influence mating. To achieve these objectives, we employed a multidisciplinary approach combining bioinformatics and experimental methods, structured around the following specific goals:
Objective. 1: Describe new early branching Mamiellophycean species. We first, will expand the known diversity of Mamiellophycean species by describe new isolated Mamiellophycean species, or describing properly some species already present in culture collections that awaited for a formal description and come from overlooked early branching lineages that are sister to the most common known species that populate marine ecosystems.
Objective. 2: Capture the ecology and evolution of mating types accross Mamiellophyceae 1) by using a comparative genomics analysis of 10 new different species across Mamiellophyceae and its sister lineage Pyramimonas. In order to to identify the loci of the mating types how it is conserved throught the evolution 2) to estimate the frequency of the two mating types present in Mamiellophyceae natural populations using metagenomic data.
Objective. 3: Detect the abiotic and biotic factors that influence mating in O. tauri by experimental crossing of haploid strains by taking advantage of cell transformation to select for recombinant cells (WP3).
1) Expanding the diversity of Mamiellophycean described species and their organellar genomes
First, I put our efforts in properly describing the 8 new Mamiellophycean and two species or Pyramimonas, which this action aimed to produce high quality genomes.
We have generated electron microscopy data from the new isolated species, and from the species already present in culture collections we have used the ribosomal genes and the plastid and mitochondrial organellar genomes present in the genomes sequenced to properly place them in the tree of life and demonstrate that are new and different species from previous studies. Therefore, we not only have properly described new Mamiellophycean species we have provided new organellar genomes, which for early branching lineages there was only one Monomastix species, the same for Pyramimonas.
2) Capture the ecology and evolution of mating types accross Mamiellophyceae
We have sequenced and annotated the genomes of 10 new species of Mamiellophyceans (8) and Pyramimonas (2). In the general genomic statistics, it is clear the path to genome reduction towards the genus Ostreococcus and Micromonas as previously described. However now we can track with much resolution in which genes and pathways the genomes have been reduced. First, we tracked the evolution of the mating type identified in Ostreococcus throughout all the green algae genomes sequenced. To do so we have performed homology-based search to detect if the genes present in the mating types were conserved. We have observed that the mating type described in Ostreococcus it is conserved ot terms of gene content with a considerably high similarity within Mamiellales (Micromonas, Mantoniella, Ostreococcus and Bathycoccus genera), in earlier branching lineages, they seem to contain a genes related to mating, but the mating loci that they might display it is not homologous to Mamiellales species. Therefore, such mating type structure it is a Mamiealles innovation.
We are still, improving the analysis of ancestral reconstruction and evolution of the mating locus together with the reconstruction of the whole genome evolution to better understand the expansions and losses suffered from Mamiellophycean species and the impact that can have this with its ecological distribution.
3): Environmental factors that stimulate recombination in O. mediterraneus and cellular and molecular mechanisms behind fertilisation:
The objective of this research was to identify the abiotic conditions that trigger mating in Ostreococcus, with a specific focus on Ostreococcus mediterraneus. This species was chosen because its cultures were more recently isolated from the environment compared to O. tauri, making it more likely that its sexual functionality had been retained, unlike in strains subjected to prolonged asexual growth in laboratory conditions. The work was carried out using the O. mediterraneusstrains RCC2590, representing the M+ mating type, and RCC2582, representing the M- mating type.
To achieve this goal, two complementary approaches were pursued. The first involved the development of selection vectors to enable detection of hybrid daughter cells containing genetic material from both parental strains. The second approach involved directly mixing the M+ and M- strains under various abiotic conditions to observe the presence of a 4n meiotic stage, which would be indicative of sexual reproduction. This stage was to be identified through flow cytometry using SYBR staining.
For the first approach, two antibiotic resistance vectors were designed and produced. One vector contained a G418 resistance gene, which was already present in the existing POCK11 vector used in the laboratory. This vector was utilized to transform the M+ strain. The second vector, containing a cloNat resistance gene, was designed and ordered specifically for this work. While awaiting the arrival of the cloNat vector, efforts were directed toward testing the second approach, which involved culturing the M+ and M- strains together under different abiotic conditions to induce mating.
The abiotic conditions tested included starvation, achieved by withholding media changes for ten days; nitrogen depletion; and maintenance of the cultures in complete darkness. Additionally, nitrogen depletion was combined with darkness, as these conditions are known to trigger gamete formation in Chlamydomonas. Despite these efforts, no clear evidence of a 4n stage was observed under any of these conditions. It was hypothesized that either the tested conditions do not trigger mating in O. mediterraneus, or that the 4n stage occurs very rapidly and asynchronously, making it difficult to detect within the three-hour sampling intervals used. Furthermore, if the proportion of 4n cells relative to other stages is low, it could further complicate detection.
The results of this work underscore the need to conduct further experiments using the selection vectors to conclusively determine whether mating occurs under specific conditions. These vectors will provide a robust method for selecting and identifying hybrid cells, overcoming the challenges associated with direct observation of meiotic stages. Future experiments are planned in collaboration with the undergraduate student Gaokang Liu from the Genophy Lab and the supervisor, Sheree Yau. Unfortunately, progress on this work package had to be temporarily paused due to parental leave, but efforts will resume in the coming months to accomplish project milestones D.3.1 and M3.1.
First, I put our efforts in properly describing the 8 new Mamiellophycean and two species or Pyramimonas, which this action aimed to produce high quality genomes.
We have generated electron microscopy data from the new isolated species, and from the species already present in culture collections we have used the ribosomal genes and the plastid and mitochondrial organellar genomes present in the genomes sequenced to properly place them in the tree of life and demonstrate that are new and different species from previous studies. Therefore, we not only have properly described new Mamiellophycean species we have provided new organellar genomes, which for early branching lineages there was only one Monomastix species, the same for Pyramimonas.
2) Capture the ecology and evolution of mating types accross Mamiellophyceae
We have sequenced and annotated the genomes of 10 new species of Mamiellophyceans (8) and Pyramimonas (2). In the general genomic statistics, it is clear the path to genome reduction towards the genus Ostreococcus and Micromonas as previously described. However now we can track with much resolution in which genes and pathways the genomes have been reduced. First, we tracked the evolution of the mating type identified in Ostreococcus throughout all the green algae genomes sequenced. To do so we have performed homology-based search to detect if the genes present in the mating types were conserved. We have observed that the mating type described in Ostreococcus it is conserved ot terms of gene content with a considerably high similarity within Mamiellales (Micromonas, Mantoniella, Ostreococcus and Bathycoccus genera), in earlier branching lineages, they seem to contain a genes related to mating, but the mating loci that they might display it is not homologous to Mamiellales species. Therefore, such mating type structure it is a Mamiealles innovation.
We are still, improving the analysis of ancestral reconstruction and evolution of the mating locus together with the reconstruction of the whole genome evolution to better understand the expansions and losses suffered from Mamiellophycean species and the impact that can have this with its ecological distribution.
3): Environmental factors that stimulate recombination in O. mediterraneus and cellular and molecular mechanisms behind fertilisation:
The objective of this research was to identify the abiotic conditions that trigger mating in Ostreococcus, with a specific focus on Ostreococcus mediterraneus. This species was chosen because its cultures were more recently isolated from the environment compared to O. tauri, making it more likely that its sexual functionality had been retained, unlike in strains subjected to prolonged asexual growth in laboratory conditions. The work was carried out using the O. mediterraneusstrains RCC2590, representing the M+ mating type, and RCC2582, representing the M- mating type.
To achieve this goal, two complementary approaches were pursued. The first involved the development of selection vectors to enable detection of hybrid daughter cells containing genetic material from both parental strains. The second approach involved directly mixing the M+ and M- strains under various abiotic conditions to observe the presence of a 4n meiotic stage, which would be indicative of sexual reproduction. This stage was to be identified through flow cytometry using SYBR staining.
For the first approach, two antibiotic resistance vectors were designed and produced. One vector contained a G418 resistance gene, which was already present in the existing POCK11 vector used in the laboratory. This vector was utilized to transform the M+ strain. The second vector, containing a cloNat resistance gene, was designed and ordered specifically for this work. While awaiting the arrival of the cloNat vector, efforts were directed toward testing the second approach, which involved culturing the M+ and M- strains together under different abiotic conditions to induce mating.
The abiotic conditions tested included starvation, achieved by withholding media changes for ten days; nitrogen depletion; and maintenance of the cultures in complete darkness. Additionally, nitrogen depletion was combined with darkness, as these conditions are known to trigger gamete formation in Chlamydomonas. Despite these efforts, no clear evidence of a 4n stage was observed under any of these conditions. It was hypothesized that either the tested conditions do not trigger mating in O. mediterraneus, or that the 4n stage occurs very rapidly and asynchronously, making it difficult to detect within the three-hour sampling intervals used. Furthermore, if the proportion of 4n cells relative to other stages is low, it could further complicate detection.
The results of this work underscore the need to conduct further experiments using the selection vectors to conclusively determine whether mating occurs under specific conditions. These vectors will provide a robust method for selecting and identifying hybrid cells, overcoming the challenges associated with direct observation of meiotic stages. Future experiments are planned in collaboration with the undergraduate student Gaokang Liu from the Genophy Lab and the supervisor, Sheree Yau. Unfortunately, progress on this work package had to be temporarily paused due to parental leave, but efforts will resume in the coming months to accomplish project milestones D.3.1 and M3.1.
Through genome sequencing and annotation efforts, significant progress has been made in understanding the diversity and evolution of mating systems in Mamiellophyceae. We sequenced and assembled the genomes of multiple species, including three entirely new species isolated in our lab: two Pyramimonas and one Mantoniella. The discovery and detailed description of the novel Mantoniella species, along with its phylogenetic position and organellar genome information, represent a substantial contribution to our understanding of microalgal diversity. Additionally, we identified taxonomic misassignments in culture collections, such as Dolichomastigales RCC1513, underscoring the importance of genomic data in correcting and refining species classifications. These findings have led to the preparation of a manuscript that will formally describe these new species, providing critical insights into their evolutionary and ecological roles.
In the context of mating systems, we generated high-quality genomic data from ten species of Mamiellophyceae and their sister group Pyramimonas. Using advanced sequencing technologies, such as Oxford Nanopore, we identified the conservation of mating loci across Mamiellophyceae through homology-based searches. This work demonstrates that the mating loci structure previously discovered in Ostreococcus is conserved within the Mamiellales but exhibits divergence in early-branching groups of Mamiellophyceae. These early-branching groups appear to retain sexual activity, but likely through a distinct mating locus, suggesting evolutionary innovations in mating systems within this lineage. These findings push the boundaries of our understanding by elucidating the genomic underpinnings of mating loci evolution and their ecological implications across diverse lineages.
Furthermore, environmental data analysis has begun to shed light on the distribution of mating types in natural populations of Mamiellophyceae. As part of the TARA-TREC consortium, which integrates efforts from leading institutions like EMBL and Tara Oceans Foundation, we are exploring the diversity of microbial communities from European coasts. Leveraging this data, we aim to understand the ratio of mating types within natural populations of Mamiellales, a group comprising some of the most abundant species in coastal marine ecosystems. This work will soon result in the publication of D2.3 advancing knowledge on how mating systems function and persist in natural marine environments.
Overall, this research goes beyond the current state of the art by providing (1) the discovery and formal description of new species, (2) insights into the evolution and conservation of mating loci, (3) genomic data for understanding ecological and evolutionary strategies of marine microalgae, and (4) novel perspectives on the distribution and dynamics of mating types in natural ecosystems. These achievements not only address fundamental questions about the evolution of sexual reproduction in microalgae but also contribute to a broader understanding of microbial diversity and function in marine ecosystems.
In the context of mating systems, we generated high-quality genomic data from ten species of Mamiellophyceae and their sister group Pyramimonas. Using advanced sequencing technologies, such as Oxford Nanopore, we identified the conservation of mating loci across Mamiellophyceae through homology-based searches. This work demonstrates that the mating loci structure previously discovered in Ostreococcus is conserved within the Mamiellales but exhibits divergence in early-branching groups of Mamiellophyceae. These early-branching groups appear to retain sexual activity, but likely through a distinct mating locus, suggesting evolutionary innovations in mating systems within this lineage. These findings push the boundaries of our understanding by elucidating the genomic underpinnings of mating loci evolution and their ecological implications across diverse lineages.
Furthermore, environmental data analysis has begun to shed light on the distribution of mating types in natural populations of Mamiellophyceae. As part of the TARA-TREC consortium, which integrates efforts from leading institutions like EMBL and Tara Oceans Foundation, we are exploring the diversity of microbial communities from European coasts. Leveraging this data, we aim to understand the ratio of mating types within natural populations of Mamiellales, a group comprising some of the most abundant species in coastal marine ecosystems. This work will soon result in the publication of D2.3 advancing knowledge on how mating systems function and persist in natural marine environments.
Overall, this research goes beyond the current state of the art by providing (1) the discovery and formal description of new species, (2) insights into the evolution and conservation of mating loci, (3) genomic data for understanding ecological and evolutionary strategies of marine microalgae, and (4) novel perspectives on the distribution and dynamics of mating types in natural ecosystems. These achievements not only address fundamental questions about the evolution of sexual reproduction in microalgae but also contribute to a broader understanding of microbial diversity and function in marine ecosystems.