Nitrogen fixation assessment in a legume-dominant alpine community: comparison of different reference species using the 15N isotope dilution technique

In arctic and alpine environments the highest biologically fixed nitrogen inputs are measured in the presence of leguminous plants, with a maximum annual nitrogen input recorded of about 10 kg ha(-1) year(-1). Among the high-elevation legumes, Trifolium alpinum spreads over many southern European mountain grasslands as a dominant or co-dominant species, accounting for 30-40 % or more of total above-ground phytomass. We estimated the amount of symbiotic nitrogen fixation in T. alpinum-dominant communities using the N-15 isotope dilution technique and comparing the calculations derived from different non-fixing reference species. The estimated percentage of nitrogen derived from the atmosphere varied significantly depending on the reference species used and they were comparable to the ones reported in literature. No differences were detected among years. In the community studied, the use of one reference species instead of another can yield differences of more than 10 kg ha(-1) year(-1), which may represent a considerable difference in the estimation of symbiotic nitrogen fixation in alpine ecosystems. Taking a very precautionary approach and using the grass Nardus stricta as the reference species, we estimated 21.9 kg ha(-1) year(-1) of nitrogen fixation in T. alpinum-dominant communities. This quantity of nitrogen is the highest ever estimated in a natural alpine grassland and it is related to the high annual above-ground phytomass of T. alpinum.