Microbial aquatic roommates: Woesearchaeota and photosynthetic organisms in a lake – MARWEL

Within the framework of the ANR JCJC MARWEL project, we implemented an integrated approach combining field-based microbial ecology, state-of-the-art molecular analyses (metagenomics and metatranscriptomics), and cultivation strategies. This multi-scale framework was designed to link in situ microbial diversity to the functional potential and lifestyles of Woesearchaeota Archaea. The project benefited from strong synergies with an IUF project led by M. Hugoni and an ANR PRC coordinated by M. Ader, which allowed a substantial expansion of the sampling effort beyond the scope initially planned. Field campaigns were conducted twice per year in 2022, 2023, and 2024, and two additional sampling campaigns were maintained in 2025 despite the impact of Cyclone Chido in December 2024.

A first major methodological objective was to reconstruct high-quality Woesearchaeota genomes to enable robust phylogenomic and functional analyses. To validate the analytical strategy, we initially worked on metagenomes generated from samples collected in 2017, which allowed the development and optimization of a dedicated bioinformatic pipeline. This approach led to the reconstruction of high-quality genomes and their comprehensive phylogenomic, functional, and transcriptomic characterization. The methodology was validated through a publication (Cloarec et al., 2024, Microbiome) and subsequently deployed at a larger scale to investigate the temporal dynamics of archaeal communities—particularly Woesearchaeota—between 2014 and 2022. These large-scale analyses are currently being finalized, with manuscript submission planned for early 2026.

A second component of the project aimed to establish enrichment cultures of Woesearchaeota in order to explore their potential interaction partners. This effort was strengthened by the recruitment of a postdoctoral researcher funded by Université Claude Bernard Lyon 1, who implemented enrichment cultures under multiple growth conditions. In parallel, specific primers targeting Woesearchaeota were designed, validated by cloning and Sanger sequencing, and used in phylogenetic analyses to confirm their specificity. Enrichment cultures were monitored by quantitative PCR using these primers. At this stage, several cultures show a significant enrichment in Woesearchaeota, although 16S/18S metabarcoding analyses indicate the persistence of an associated microbial diversity. These results represent important first steps toward the cultivation of these still largely unexplored microorganisms and are included in a manuscript currently in preparation, with submission planned for early 2026.

The results obtained within the ANR JCJC MARWEL project demonstrate the relevance of the chosen archaeal model. We showed that Woesearchaeota, although belonging to the rare biosphere, are not only present but also metabolically active in the studied ecosystem, suggesting an effective contribution to ecosystem functioning. These findings are notably embedded in the PhD thesis of Lilian Cloarec (defense scheduled for December 16, 2025), which focuses on the diversity, activity, and ecological strategies of this phylum, first described in 2015. To date, no cultivated representative exists, and its lifestyle remains largely inferred from genomic data.

Analysis of a sampling campaign conducted in November 2017 identified two major physicochemical transition zones within the water column: a first zone at approximately 2 m depth, where the oxycline, thermocline, and chemocline coincide, and a second zone around 14 m, characterized by a chemocline associated with increased concentrations of reduced sulfur compounds (H₂S/HS⁻). Large-scale metagenomic sequencing enabled the reconstruction of 14 Woesearchaeota genomes, including nine high-quality genomes. Phylogenomic analyses based on 15 ribosomal proteins revealed a diversity structured into six subgroups, some of which appear specific to hypersaline environments or even endemic to Lake Dziani Dzaha.

Despite this phylogenetic diversity, all genomes exhibited similar abundance profiles, with a first peak at 2 m, consistent with current knowledge, and a second unexpected peak between 14 and 16 m. Metatranscriptomic data confirmed high Woesearchaeota activity in both zones and revealed an active fermentative metabolism producing acetate, lactate, and hydrogen. These results support the hypothesis of a syntrophic lifestyle and identify transition zones as hotspots of Woesearchaeota activity. Fine-scale transcriptomic analyses further revealed distinct ecotypes within phylogenetic subgroups, indicating differentiated ecological strategies in response to environmental conditions and/or associated partners.

Finally, temporal analyses of communities spanning 2014 to 2022 showed that Woesearchaeota populations are highly dynamic and responsive to major disturbances, particularly the seismo-volcanic crisis that affected the Mayotte archipelago from 2018 onward. Our results indicate a shift in the balance between stochastic and deterministic assembly processes while highlighting the maintenance of functional redundancy for key carbon recycling processes, despite substantial taxonomic reorganization.

This ANR JCJC project opens up major scientific perspectives. First, it highlights the ecological value of long-term spatio-temporal monitoring of natural ecosystems, particularly in the context of rapid climate change and increasing environmental disturbances. In this framework, the See-Life CNRS label (led by Cécile Bernard, Professor at MNHN) will enable the continuation of monitoring activities at Lake Dziani Dzaha. This effort is especially timely, as the passage of Cyclone Chido in December 2024 profoundly altered the lake’s watershed, causing extensive removal of surrounding vegetation and a massive input of organic matter into the lake, which is likely to have long-lasting consequences for its biogeochemical and microbial functioning.

From a conceptual standpoint, this project has revealed the limitations of current approaches to characterizing the lifestyle of Woesearchaeota. Although no definitive conclusion could be reached regarding a strictly free-living or host-attached lifestyle, the combination of genomic and transcriptomic signatures related to microbial interactions (notably surface-associated features such as the S-layer) and motility (e.g. archaellins), together with an in-depth review of the literature, suggests a lifestyle far more flexible than previously assumed. We hypothesize that Woesearchaeota may alternate between free-living and host-associated states, depending on host availability and specificity, as well as on environmental conditions.

More broadly, the role of Woesearchaeota in moderately saline ecosystems—which are expected to be increasingly affected by climate change, including potential salinity increases driven by rising temperatures—remains largely unexplored. In this context, I submitted a proposal to the 2025 ANR call aiming to identify the mechanisms underlying the ecological success of Woesearchaeota, despite their reduced genomes and apparent metabolic limitations. Finally, the ERC Consolidator project planned for submission in January 2026 will specifically address the role of microbial interactions in shaping Woesearchaeota ecological success, with a particular focus on genes lacking homologs in current databases. The functional potential of these genes will be explored using innovative approaches that combine 3D protein structure–based geometric analyses with machine learning methods.

The MARWEL project proposes to unravel specific genomic adaptations and interactions related to Woesearchaeota ecology. Indeed, we benefit from a unique case study in an extreme microbial lacustrine ecosystem in which previous work reported a strong niche partitioning of Woesearchaeota between a free-living lifestyle and an associated lifestyle to photosynthetic microorganisms (i.e. the cyanobacteria Arthrospira fusiformis and/or the picoeukaryote Picocystis salinarum). Using an original microbial cytometry cell-sorting coupled to meta-omics approaches (metagenomics and metatranscriptomics), we aim at (1) deciphering which genomic specificities of Woesearchaeota are related to free-living or associated lifestyle and (2) to evidence the type of interactions that took place at the microscale (i.e. phycosphere) in the case of Woesearchaeota associated with photosynthetic members, A. fusiformis and/or P. salinarum.