2.1 | Experimental design
The dominant species and coexisting species plants in the three grazing treatment samples were collected, and the top healthy, complete, mature leaves (try to pick the same part of each plant) were picked and bagged, and each plant was collected 15-20 leaves (at least 10 individual plants growing independently), mixed and packed into cowhide envelopes. The standard harvesting method was used to collect plant species at the functional group level (25 cm × 25 cm).
The total biomass of the community was investigated using a sample method (25 cm × 25 cm). The above-ground biomass was collected by standard harvesting method, and the soil and below-ground biomass were collected by root drilling method, sampling from 0 to 20 cm, using a 2 mm soil sieve, washing with water to obtain root samples, drying at 65°C to constant weight, weighing. Soil samples are used to determine soil physical and chemical properties. The soil water content is measured by the ring knife method, and the soil evapotranspiration is measured by a small lysimeter, three replicates are set for each plot in the experiment.
Washing the plant leaves with ultrapure water, these were dried at 65 ℃ to constant weight. Then, leaves were smashed through a 100-mesh sieve. Approximately 2 mg of the plant powder sample were burnt in the TOC/TNb analyzer to collect CO2, and injected into a stable carbon isotope mass spectrometer (Thermo, MAT-253) for stable carbon isotope value analysis. This analysis accuracy is ± 0.1‰.
The expression formula of carbon isotope composition is:
δ13C= (Rsam / Rsta-1) ×1000
In this formula, Rsam and Rsta are the13C/12C values of test plants and standard samples, respectively.
Data Analysis
Mean, standard error, and variance analysis are processed by R and Excel 2010. Path analysis is processed by SPSS (Versiton 21.0). Originpro 9.1 software were used to complete relative maps.
3 | RESULT
3.1 |WUE variation characteristics of dominant species in different grazing intensity
The δ13C value of each species differed greatly both in enclosure plots and grazed treatments. The average δ13C values of dominant species in three grazing treatments were -27.61, -26.16 and -27.20‰, respectively. The δ13C value of dominant species increased firstly and then decreased with grazing intensity increasing. The overall range of 9 species is from -24.92 ± 0.07 to -28.08 ± 0.01‰. The former value is 3.16‰ higher than the latter approximately 12.68% reduction range. The highest and lowest values are Gentiana straminea and Leontopodium nanum respectively. The δ13C value of Stipa aliena, Gentiana straminea, Oxytropis ochrocephala are the highest under enclosure, moderate grazing, and heavy grazing gradients (Table 2) Moderate grazing and heavy grazing can increase the δ13C value of dominant species by 5.25% and 1.48% respectively. In other words, both moderate and heavy grazing activities can improve the WUE in alpine meadows.
3.2 |WUE variation characteristics of 11 coexisting species indifferent grazing intensity
The δ13C value of Gentiana straminea andAnaphalis lactea Maxim were highest and lowest respectively. The average δ13C value of functional group plants under moderate grazing were the largest, and the average δ13C value of the functional group plants in three experimental treatments were -27.09, -27.00 and -27.04‰, respectively. The δ13C value of Poa annua, Kobresia humilis, K. pygmaea, and Potentilla nivea increased significantly under moderate grazing (P<0.05, Fig. 2). Leontopodium nanum, Oxytripis ochrocephala, and Anaphalis lactea Maxim had the highest δ13C value under heavy grazing. Stipa aliena, Thermopsis lanceolata, Saussurea superba and Gentiana stramineahad the highest δ13C value under enclosure. In other words, moderate grazing can increase coexisting species plant WUE by 3.32‰ compared with enclosure.
3.3 |Variation characteristics of functional group WUE in different grazing intensity
The δ13C values ​​of the two functional groups of Gramineae and Cyperaceae were the highest in moderate grazing. Both Gramineae and Cyperaceae are regrading as fine forages. Moderate grazing help to increase forages WUE. The δ13C values ​​of the two functional groups of Leguminosae and Forb were the highest in heavy grazing. These drought tolerances increased with grazing intensity increasing. Except for Gramineae plants, the WUE ​​of the other three functional groups Cyperaceae, Leguminosae, and Forb under the three treatments didn’t reach a significant difference. WUE of Gramineae plants is more sensitive and adaptive. Moderate grazing significantly increased Gramineae plants δ13C and its WUE approximately 4.84% comparing with control (P<0.05, Fig. 3). The δ13C value of Gramineae and Cyperaceae plants increased first and then decreased with the increase of grazing intensity. Leguminosae and Forb showed an upward trend.
Overall, grazing activities increased the δ13C value of plants. The average δ13C values ​​of each functional group in the three treatments of enclosure, moderate grazing and heavy grazing were -27.47‰, -26.90‰, and -26.97‰, respectively. Moderate grazing and heavy grazing increased WUE by 2.07% and 1.82% respectively compared with enclosure.
3.4 |Effect of soil physical and chemical properties on WUE variation
Soil evapotranspiration, soil organic carbon, water content and soil bulk density were negative correlate with the δ13C value, and evapotranspiration and soil organic carbon have reached a significant level (P<0.05). However, available nitrogen was significant positive correlate with WUE (P<0.01, Table 3). Grazing intensity is positively correlated with WUE. Grasslands WUE was higher under tolerant environment.
3.5 |Path analysis of direct and indirect effects on WUE in alpine meadow
Path analysis indicated that evapotranspiration has a direct and significant negative correlation effect on δ13C value (P<0.05). Furthermore, available nitrogen, soil organ carbon, soil bulk density and soil water content have indirect effects on the δ13C value (Fig. 4). Meanwhile, the indirect effects of soil available nitrogen, evapotranspiration, and soil bulk density were relative higher through soil organ carbon from -0.85 to 0.51, respectively. Soil organ carbon and available nitrogen were showed positive effect by evapotranspiration approximately 0.27 and 0.22, respectively. Evapotranspiration and available nitrogen were showed effect by soil water content approximately 0.22 and -0.15, respectively. Furthermore, available nitrogen was showed positive effect on WUE by soil water content was 0.38.
4 | DISCUSSION
Previous studies have shown that grazing activities reduced WUE of plant species (Zhu et al., 2015; Huang et al., 2017; Wang, 2017; An et al., 2015). However, our experimental results show that all the dominant species, coexisting species, functional group and community level species, the WUE of plants is the highest under moderate grazing treatment, and grazing activities increase the WUE of plants. Similar results were revealed that moderate grazing WUE increased by 37.72% compared to traditional grazing (Cavalcante et al., 2016). In Inner Mongolia typical grassland community WUE was highest under moderate grazing (Lv et al., 2016), and WUE under grazing treatment increased by 62.86 % Compared to no grazing. In Inner Mongolia Stipabreviflora grassland WUE increased by 0.56% under moderate grazing (Wang, 2018). There may be two reasons: On the one hand, the feeding of plants by livestock causes damage to the leaves of plants under moderate grazing, the canopy structure of plants will also change during the grazing process. These changes may directly limit the transportation and fixation of carbon in plant leaves. The reduction of plant leaf area will weaken the photosynthetic capacity of plants. Plants will increase their net photosynthetic rate to enhance photosynthesis capacity, increase grazing tolerance of grassland plants, and increase the compensatory growth ability of plants. Therefore, the WUE of plants increases (Wang, 2018; Lv, 2016). On the other hand, grazing can increase soil fertility through livestock excrement and urine, improve the microenvironment of plant growth, and enable plants to gain stronger living ability. After plants undergo grazing, their resource utilization capacity increases, so the WUE of plants increases (Pia et al., 1995). Meanwhile, studies have also shown that the saliva remaining on the section of the gnawed plant during the gnawing process of animals can promote the growth of plants, thereby improving WUE of plants (Johnston et al., 2008).
Grazing plays an important role in balancing ecosystem plants (Zhu, 2017). Moderate grazing could improve grasslands biomasses and richness, but heavy grazing significantly decreased biomasses and richness (Du et al., 2019). The WUE of plants is the highest under the moderate grazing in this study. This indicated that the plants are highly tolerant to drought environment under moderate grazing. When suffering from water stress, they can improve their WUE to ensure water supply (Sun et al., 2005). Severe grazing state WUE shows a downward trend compared to moderate grazing. This may be because grassland degradation is more serious and biomass is reduced (Du et al., 2019), the bare grassland area is larger, and vegetation is scarce under severe grazing treatment. Therefore, plant WUE showed a downward trend. But the WUE of Forb was the highest in the state of heavy grazing. Due to the large amount of grazing by livestock, the growth space of Forb increased, which led to the dominant position of Forb in the sample plot. Therefore, Forb has the highest WUE and strong ability to adapt to the environment at the level of functional groups. Gramineae, which are fond of livestock, have the lowest WUE.
Some environmental factors that affect the process of gas exchange metabolism also affect the WUE of many plants, including soil water content (Ehleringer, 1994; Stewart et al., 1995; Korol et al., 1999; Wu et al., 2019), precipitation (Anderson et al., 2000; Stewart et al., 1995), evapotranspiration (Xu et al., 1998), nitrogen availability (Hogberg et al., 1993; Guehl et al., 1995). Among them, soil water content, precipitation and evapotranspiration have a negative correlation with WUE. This is consistent with the results of this experiment. It is also because when the water content decreases, plants will better adapt to the environment by increasing their own WUE (Sun et al., 2005; Chen, 2003). Studies have shown that evapotranspiration will decrease with the increase of grazing intensity (Shu et al., 2019), and the increase of WUE is related to lower evapotranspiration and the coverage and biomass of dominant species in the community. This research result has also been verified by many scholars (Xu et al., 1998; Cavalcante et al., 2016; Varnamkhasti et al., 1995). There is a negative correlation between the WUE and the soil bulk density. It may be speculated that after the soil bulk density increases, the soil water content shows a downward trend (Shu et al., 2019). When plants are subjected to water stress, they will increase their water use efficiency to survive better. There is a negative correlation with soil organic carbon content, which may be due to the increase of soil organic matter content in the enclosure. Microorganisms preferentially use12C to enrich the residual organic matter with13C in the process of soil organic carbon decomposition. Because enclosure slows down this decomposition process, the WUE in the enclosed plot is lower than that in the free grazing plot (Zhu et al., 2020). Nitrogen plays a vital role in the growth and development of plants, and the increase in soil nitrogen can improve plant WUE (Chen, 2003).
5 |CONCLUSION 
Considering that there are certain differences in the WUE of different plant species under different grazing intensity. Through the study of the changes in the WUE of plants at the level of dominant species, coexisting species and functional groups of alpine meadows under the three treatments of enclosure, moderate grazing and heavy grazing in Haibei, Qinghai, and the adaptation mechanism of WUE of alpine meadow the study. Draws the following conclusions:
1) The order of the WUE is Gramineae > Cyperaceae > Forb > Leguminous analysis from the perspective of functional groups.
2) The WUE of the dominant species, coexisting species and functional group level species are the highest under moderate grazing, followed by heavy grazing, and the lowest under enclosure. Grazing activities increased the WUE of plants.
3) There is a negative correlation between soil water content, evapotranspiration, soil bulk density, soil organic carbon and WUE, and the evapotranspiration will significantly affect WUE. There is a positive correlation between available nitrogen and grazing intensity.
This research shows that evapotranspiration was perhaps the main driven factor in the WUE of alpine meadow in Qinghai. Our results provide new insights that Moderate grazing help to increase forages WUE. These drought tolerances of Leguminosae and Forb increased with grazing intensity increasing.
ACKNOWLEDGEMENTS
This work is supported by Qinghai innovation platform construction project (2020-KF-12), western light of Chinese Academy of Science for talents, and the national natural science foundation of China (31770532).