Abstract: Alpine grasslands are cold and hypoxic, typically lacking symbiotic nitrogen fixation. Nonetheless, they often maintain high nitrogen stocks, which suggests an important role for asymbiotic nitrogen fixation (ANF). However, the ANF rates of soil or plant leaves and their driving factors in alpine grasslands remain unclear. We quantified the ANF rates in soil and plant leaves, the soil stoichiometry and the nifH based community composition and abundance across alpine grasslands (swamp meadow, alpine meadow, and alpine steppe) along an altitudinal gradient between 3100 and 4300 m above sea level (a. s. l). The ANF rates in swamp and alpine meadows were faster than in alpine steppe. Diazotroph communities were dominated by Proteobacteria (85%) and Actinobacteria (12%), and 72 nifH OTUs assigned to Cluster II (anfH/vnfH-like) suggested the potential presence of alternative nitrogenase-related homologs. Specialist taxa in soil ANF bacteria were influenced mainly by stochastic processes and exhibited faster transition rates than generalists. Random forest analyses identified soil pH, nifH diversity and composition, soil stoichiometry, and molybdenum as key predictors of soil ANF. Estimated annual nitrogen inputs were 4.0, 3.1, and 0.5 kg N ha-1 yr-1 in alpine meadow, swamp meadow, and alpine steppe, respectively. Our results indicate that ANF represents a significant nitrogen input pathway in alpine grasslands and should be incorporated into nitrogen cycling and regional nitrogen budgets.

Key words: alpine grasslands, asymbiotic nitrogen fixation, niche breadth, nifH gene, stoichiometry