Adaptive pangenomic remodeling in the Azolla cyanobiont amid a transient microbiome

Abstract

Plants fix nitrogen in concert with diverse microbial symbionts, often recruiting them from the surrounding environment each generation. Vertical transmission of a microbial symbiont from parent to offspring can produce extreme evolutionary consequences, including metabolic codependence, genome reduction, and synchronized life cycles. One of the few examples of vertical transmission of N-fixing symbionts occurs in Azolla ferns, which maintain an obligate mutualism with the cyanobacterium Trichormus azollae--but the genomic consequences of this interaction, and whether the symbiosis involves other vertically transmitted microbial partners, are currently unknown. We generated high-coverage metagenomes across the genus Azolla and reconstructed metagenome assembled genomes to investigate whether a core microbiome exists within Azolla leaf cavities, and how the genomes of T. azollae diverged from their free-living relatives. Our results suggest that T. azollae is the only consistent symbiont across all Azolla accessions, and that other bacterial groups are transient or facultative associates. Pangenomic analyses of T. azollae indicate extreme pseudogenization and gene loss compared to free-living relatives--especially in defensive, stress-tolerance, and secondary metabolite pathways--yet the key functions of nitrogen fixation and photosynthesis remain intact. Additionally, differential codon bias and intensified (rather than relaxed) selection on photosynthesis, intracellular transport, and carbohydrate metabolism genes suggest ongoing evolution in response to the unique conditions within Azolla leaf cavities. These findings highlight how genome erosion and shifting selection pressures jointly drive the evolution of this unique mutualism, while broadening the taxonomic scope of genomic studies on vertically transmitted symbioses.