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.