Aims. Terrestrial mosses contribute significantly to nitrogen budgets in boreal forests through symbiotic associations with N₂-fixing cyanobacteria, but few studies have considered these interactions in temperate systems. We investigated how N₂-fixation by moss-cyanobacterial associations contribute spatio-temporally to site-level N dynamics in a western North American prairie ecosystem.

Methods. We first tested for the presence of N₂-fixing cyanobacteria on six moss species using epi-fluorescence light microscopy. We then used an acetylene reduction assay to estimate monthly N₂-fixation rates in moss-cyanobacteria associations across three prairies located in the Puget Sound, Washington State, USA. We evaluated temperature and precipitation effects on N₂-fixation activity. 

Results. We confirmed the presence of N₂-fixing cyanobacteria on three moss species (Pleurozium schreberi (Brid.) Mitt., Racomitrium elongatum Frisvoll, Rhytidiadelphus triquetrus (Hedw) Warnst.) and found species, site and seasonal variation in N₂-fixation rates. Racomitrium elongatum exhibited the highest N₂-fixation rates, with peaks in April and August. We found that temperature and precipitation were strongly correlated with N₂-fixation rates and likelihood of activity. 

Conclusions. Our results highlight a previously undescribed source of biological N₂-fixation in temperate grasslands. Changes in the distribution and activity of these species due to climate change and management practices could impact stand-level nitrogen dynamics.