Long-Term Grazing Exclusion Affects the Biomass Rather than the Percentage of Nitrogen Derived from the Atmosphere in Astragalus arnoldii

The nitrogen derived from the atmosphere (%Ndfa) serves as a critical parameter in assessing the biological nitrogen fixation of legumes. Nevertheless, our comprehension of the impact of grazing exclusion primarily relies on productivity and nitrogen content. A comprehensive understanding of the response of %Ndfa to long-term grazing exclusion in the alpine grassland of the Qinghai-Tibet Plateau remains elusive. In this study, we employed the natural ¹⁵N abundance technique (NA) and the ¹⁵N dilution technique (ID) to compare the %Ndfa of Astragalus arnoldii inside and outside the animal exclusion fences. For NA, plant samples were collected at three time points, whereas for ID, plants were sampled after one day, one month, and one year after labeling. The total biomass of A. arnoldii outside the fences (27.18 ± 2.52 g m^− 2) was significantly higher than that inside the fences (24.28 ± 1.47 g m^− 2). No significant differences in %Ndfa inside (74.36 ± 2.36%) and outside (75.57 ± 2.26%) the fences were found. Both δ¹⁵N and total plant nitrogen concentration varied widely between A. arnoldii and non-legumes. There was no relationship between plants nitrogen concentration and δ¹⁵N. NA and ID were consistent in estimating %Ndfa (77.52 ± 1.96% vs. 72.39 ± 2.59%). Long-term grazing exclusion led to a reduction in biological nitrogen fixation primarily through the population size of legumes rather than %Ndfa. The variations in plants δ¹⁵N and nitrogen concentration suggested the distinctions between legumes and non-fixing plants. It could be reasonable to evaluate the effects of long-term grazing exclusion on %Ndfa by either NA or ID. However, when estimating %Ndfa, factors such as methods, sampling time points and labeling duration should be carefully considered.