Biological nitrogen fixation (BNF) is an important source of nitrogen in ecosystem, therefore, several experimental techniques and calculating methods have been developed to quantify the contribution of BNF to plants and vegetations. In this study, percentage of plant N derived from N2-fixation (%Ndfa) was evaluated for a leguminous species, Astragalus arnoldii, in an alpine steppe in Qinghai-Tibet Plateau in order to compare two dominant in situ experimental techniques with different calculating approaches, reference plants, and sampling times. The δ15N values of four reference plants, i.e., Stipa purpurea, Kobresia pygmaea, Leontopodium nanum, and Carex moorcroftii, were significantly lower in late August than in late July (P<0.05). However, they were significantly higher in later August, 30 days after application of (15NH4)2SO4 to soil, than late July which was 24h after labeling (P<0.05). In either site with or without (15NH4)2SO4 application, δ15N of A. arnoldii did not differ in the two sampling times. Besides,%Ndfa of A. arnoldii did not differ between calculation based on 15N excess relative to atmospheric N2 and that relative to unlabeled plants. Nevertheless,%Ndfa measured by natural abundance technique was significantly higher than that by isotope dilution technique (P<0.05) except for L. nanum as the reference species. The sampling time had a significant effect on estimated%Ndfa values in both the naturalabundance technique (F=89.906, P<0.01), and isotope dilution technique (F=496.712, P<0.01).
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