The cyanobacteria belonging to the genus Nostoc comprise free-living strains but also facultative plant-symbionts. Symbiotic strains can enter into symbiosis with a taxonomically diverse range of host plants. Little is known about genomic changes associated with evolutionary transition of Nostoc from free-living to plant symbiont. Here we compared the genomes derived from eleven symbiotic Nostoc strains isolated from different host plants and infer phylogenetic relationships between strains. Phylogenetic reconstructions of 89 Nostocales showed that symbiotic Nostoc strains with a broad host range, entering epiphytic and intracellular or extracellular endophytic interactions, form a monophyletic clade indicating a common evolutionary history. A polyphyletic origin was found for Nostoc strains which enter only extracellular symbioses, suggesting that this trait was most likely gained several times in the evolution of the Nostocales. Facultative symbiotic Nostoc strains showed enriched functions in the transport and metabolism of organic sulfur, chemotaxis and motility, as well as the uptake of phosphate, amino acid and ammonium. The genomes of the intracellular clade differ from that of other Nostoc strains by a gain/enrichment of genes encoding proteins to generate L-methionine from sulfite and pathways for the degradation of the plant metabolites vanillin and vanillate, and of the macromolecule xylan present in plant cell-walls. These compounds could function as C sources for members of the intracellular clade. Molecular clock analysis suggested that the intracellular clade emerged ~600 million years ago, which would predate the origin of land plants. This suggest that intracellular cyanobacterial symbioses may have even predated the emergence of extant terrestrial plants.

Dinitrogen (N₂)-fixation by cyanobacteria in symbiosis with feathermosses is the primary pathway of biological N input into boreal forests. Despite its significance, little is known about the cyanobacterial gene repertoire and regulatory rewiring needed for the establishment and maintenance of the symbiosis. To determine gene acquisitions and regulatory changes allowing cyanobacteria to form and maintain this symbiosis, we compared genomically closely related symbiotic-competent and incompetent Nostoc strains, using a proteogenomics approach and an experimental setup allowing for controlled chemical and physical contact between partners. Thirty-two gene families were found only in the genomes of symbiotic strains, including some never before associated with cyanobacterial symbiosis. We identified conserved orthologs that were differentially expressed in symbiotic strains, including protein families involved in chemotaxis and motility, NO regulation, sulfate/phosphate transport, and glycosyl-modifying and oxidative stress-mediating exoenzymes. The physical moss-cyanobacteria epiphytic symbiosis is distinct from other cyanobacteria-plant symbioses, with Nostoc retaining motility, and lacking modulation of N₂-fixation, photosynthesis, GS-GOGAT cycle, and heterocyst formation. The results expand our knowledgebase of plant-cyanobacterial symbioses, provide a model of information and material exchange in this ecologically significant symbiosis, and suggest new currencies, namely nitric oxide and aliphatic sulfonates, may be involved in establishing and maintaining the cyanobacteria-feathermoss symbiosis. 

Dinitrogen (N-2)-fixation by cyanobacteria living in symbiosis with pleurocarpous feather mosses (for example, Pleurozium schreberi and Hylocomium splendens) represents the main pathway of biological N input into N-depleted boreal forests. Little is known about the role of the cyanobacterial community in contributing to the observed temporal variability of N-2-fixation. Using specific nifH primers targeting four major cyanobacterial clusters and quantitative PCR, we investigated how community composition, abundance and nifH expression varied by moss species and over the growing seasons. We evaluated N-2-fixation rates across nine forest sites in June and September and explored the abundance and nifH expression of individual cyanobacterial clusters when N-2-fixation is highest. Our results showed temporal and host-dependent variations of cyanobacterial community composition, nifH gene abundance and expression. N2-fixation was higher in September than June for both moss species, explained by higher nifH gene expression of individual clusters rather than higher nifH gene abundance or differences in cyanobacterial community composition. In most cases, 'Stigonema cluster' made up less than 29% of the total cyanobacterial community, but accounted for the majority of nifH gene expression (82-94% of total nifH expression), irrespective of sampling date or moss species. Stepwise multiple regressions showed temporal variations in N-2-fixation being greatly explained by variations in nifH expression of the 'Stigonema cluster'. These results suggest that Stigonema is potentially the most influential N-2-fixer in symbiosis with boreal forest feather mosses.

In boreal forests, N₂-fixation by filamentous heterocystous cyanobacteria in symbiosis with pleurocarpous feathermosses represent the main biological input of new nitrogen (N), and greatly support the surrounding forest productivity. In these ecosystems, climate change is expected to result in 2-8°C increase in temperature and two times greater carbon dioxide (CO₂) over the next century, yet little is known about the effects of these factors on the community composition of cyanobacteria in symbiosis with feathermosses. In particular, it is unknown how N₂-fixation activity will change and the resulting impact on the moss growth rate (MGR). Here, we reconstructed the symbiosis between the common feathermoss Pleurozium schreberi and different cyanobacteria isolated from feathermosses. Feathermosses were inoculated with different strains of cyanobacteria and exposed to different temperature (11°C and 19°C) and CO₂ (500 ppm and 1000 ppm) conditions. Changes in MGR, N₂-fixation rate, and cyanobacterial community structure and composition were measured at distinct time points: 5, 10, 20, and 30 weeks of exposure. Our results indicate that both elevated temperature and CO₂ levels can individually and combined have positive effects on the N₂-fixation activities, and consequently on MGR. The underlying biotic mechanisms for increased N₂-fixation and MGR were changes in the cyanobacterial community diversity and composition.  In general, we observed a reduction in cyanobacterial diversity and an increase in cyanobacterial strain abundance that have adapted and dominated under the elevated temperature and CO₂ levels.