Sampling and analyses
Considering the effect of fast N transformation (Holst et al. 2007; Song et al. 2007), plant community sampling and species sampling were carried out in each experimental plot 24 hours after injection. A 0.5 × 0.5 m quadrat was selected, and the aboveground part of the plant was sampled by the harvesting method. Root and soil were sampled and merged using a root drill (5 cm in diameter), ”Z”-shaped, 0–30 cm deep, with five drills. This depth contains 95% of the roots in the alpine meadow. The infiltration depth of the injection solution was not more than 10 cm based on a preliminary test. The root samples were separated from the soil and washed with water, and the soil samples were sifted through a sieve (<2 mm) and then air-dried.
Species sampling was carried out in the remaining plot areas after community sampling, and plants that were common to each quadrat were sampled. Seven species were common to each quadrat in the undegraded alpine meadow, including E. nutans , P. annua , K. humilis , S. distigmaticus, P. anserina, P. kansuensis,and L. brachystachya . There were four common species in each degraded alpine meadow quadrat, including E. nutans , P. annua , P. kansuensis, and Ajania tenuifolia . The other plant species were not collected by species owing to their uneven distribution, though they were dominant species in the degraded alpine meadow. Among them, E. nutans , P. annua , and P. kansuensis were common generalist species in the two alpine meadows, and the others were unique species in the two meadow types. We collected 6–12 plants from each species and separated the shoots from the roots.
In the laboratory, all plant samples were washed with distilled water to remove 15N residue on the surface and dried for 48 hours in a 70°C oven. The powder was then ground, and the15N and N concentrations were analyzed by isotope mass spectrometry. The soil 15N and total N concentrations were analyzed by isotope mass spectrometry, the concentrations of NO3- and NH4+ were determined using an autoanalyzer (AA3, Branl- Luebbe, Germany) in 0.5 M K2SO4 extracts, and soluble organic N was determined according to the method described by Chen et al. (2017).