Study site
The experiment was carried out in Maqin County, Qinghai Province (34° 18’N, 100° 27’E) at an altitude of 4120 m. The plateau has a continental climate with strong solar radiation, long cold winters, and short cool summers. The average annual temperature is -0.6 °C, and the average annual precipitation is 513 mm, mainly from May to September. There was no complete frost-free period in this region. The natural vegetation type in this region is Kobresia meadow, the soil is alpine meadow soil, and the soil surface has a mattic epipedon. The dominant species isKobresia humilis , and the main associated species areScirpus distigmaticus , Elymus nutans , Poa annua ,Lagotis brachystachya , Pedicularis kansuensis , andPotentilla anserina . Due to global change and overgrazing, there was a large area of degraded Kobresia meadow in this region. When the meadow begins to degrade, first the original habitat is fragmented, patches appear, and forbs occupy the patches, until finally the original mattic epipedons disappears completely, the soil surface is soft, and becomes the most serious degraded state ”black soil beach”. Compared with the undegraded meadow, the plant coverage of the degraded alpine meadow decreased, and the plant distribution changed from compact and uniform to loose and uneven, thus becoming a typical degraded alpine meadow landscape (Supplementary figure 1). Degraded alpine meadow have a small number of residual Gramineae and Cyperaceae and the dominant species are mainly forbs. In this study, an undegraded Kobresiaalpine meadow and a degraded Kobresia alpine meadow were selected as the grazing meadows in the winter and spring in this region.
15N isotope labeling
15N labeling was carried out on a sunny day on August 6, 2019. August shows the maximum annual plant biomass in this region (Shi et al. 2022). Twenty 1 m × 1 m plots were randomly set up for each grassland type, among which 15 were 15N isotope injection plots and five were control plots. In 15N isotope injection plots, 5 plots of each N type were randomly injected with equal molar concentrations of15NH4NO3, NH415NO3, and15N-glycine (98% abundance) . During15N isotope injection, each cell was fixed with a grid of 4 cm × 4 cm, and each grid crossing point was used as the injection point. Then, a syringe was used to inject 20 ml of the15N isotope solution diluted with distilled water into the soil at a depth of 20 cm such that 100 mg 15N was injected into each treated plot. The control plots were injected with the same volume and amount of unlabeled N solutions following the same method.