The path to dependence: stepwise genome evolution from a facultative symbiont to an endosymbiont in the N2-fixing diatom-Richelia symbioses

A few genera of diatoms are widespread in the oceans and form stable partnerships with N2-fixing filamentous cyanobacteria Richelia spp. A unique feature of the diatom-Richelia symbioses is the symbiont cellular location spans a continuum of integration (epibiont, periplasmic, endobiont) that is reflected in the symbiont genome size and content. In this study we analyzed genomes derived from cultures and environmental metagenome-assembled genomes focusing on characters indicative of genome evolution. Our results show an enrichment of short length transposable elements (TEs) and pseudogenes in the periplasmic endosymbiont genomes, suggesting an active and transitionary period in genome evolution. In contrast, genomes of endobionts exhibited fewer TEs and pseudogenes, reflecting advanced stages of genome reduction and increased host dependency in the endobionts. Pangenome analyses identified that endobionts streamline their genomes and conserve a majority of their genes in the core, whereas periplasmic endobionts and epibionts maintain larger flexible genomes, suggesting increased genomic plasticity. Functional gene comparisons with other N2-fixing cyanobacteria revealed that Richelia endobionts have similar patterns of metabolic loss but are distinguished by absence of specific pathways (e.g. cytochrome bd ubiquinol oxidase, lipid-A) that increase dependency and direct interaction with their respective hosts. In conclusion, our findings underscore the dynamic nature of genome reduction in N2-fixing cyanobacterial symbionts and demonstrate the diatom-Richelia symbioses as a valuable and rare model to study genome evolution in the transitional stages from a free-living facultative symbiont to a host-dependent endobiont.