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\begin{document}

\title{Moderate grazing significantly increases water use efficiency in alpine
meadow on the Tibetan Plateau}



\author[1]{Yunying WANG}%
\author[1]{Weiwei PEI}%
\author[2]{Guangmin Cao}%
\author[1]{Xiaowei  Guo}%
\author[2]{H Zhou}%
\author[3]{Yangong Du}%
\affil[1]{Northwest Institute of Plateau Biology Chinese Academy of Sciences}%
\affil[2]{Northwest Plateau Institute of Biology, Chinese Academy of Sciences}%
\affil[3]{Northwest Institute of Plateau Biology}%


\vspace{-1em}



  
  \date{May 20, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Water use efficiency is an important indicator of drought tolerance of
plants. The response was still unclear of water use efficiency to
different grazing intensity and its adaptive mechanisms in alpine
meadow. Here, we analyzed water use efficiency of plant dominant
species, coexisting species and functional group level plants to grazing
intensity by \selectlanguage{greek}δ\selectlanguage{english}13C index in alpine meadow. Furthermore, this study
revealed the driven factors of water use efficiency combined available
nitrogen, soil bulk density, soil organic carbon, soil water content and
evapotranspiration. Grazing increased plants \selectlanguage{greek}δ\selectlanguage{english}13C by 3.37\%. Moderate
grazing significantly increased the \selectlanguage{greek}δ\selectlanguage{english}13C of Gramineae by 4.84\%
(P\textless{}0.05), and increased \selectlanguage{greek}δ\selectlanguage{english}13C of Cyperaceae, Leguminosae, and
Forb by 3.45\%, 0.81\%, and 1.40\% respectively. Some dominant species
and coexisting species have the highest \selectlanguage{greek}δ\selectlanguage{english}13C value under moderate
grazing. This study indicated that moderate grazing significantly
improved the water use efficiency of alpine meadows. Path analysis
resulted showed that the water use efficiency was negatively correlated
with evapotranspiration (P\textless{}0.05), soil water content, soil
organic carbon, and soil bulk density. It was positively correlated with
available nitrogen. Our study provides new insights that moderate
grazing help to increase water use efficiency in alpine meadows. In
addition, evapotranspiration may be the main driving factor affecting
the water use efficiency of alpine meadow.%
\end{abstract}%



\sloppy


Moderate grazing significantly increases water use efficiency in alpine
meadow on the Tibetan Plateau

Yunying Wang\textsuperscript{1,2}, Weiwei Pei\textsuperscript{1,2},
Guangmin Cao\textsuperscript{1}, Xiaowei Guo\textsuperscript{1}, Huakun
Zhou\textsuperscript{1}, Yangong Du\textsuperscript{1,3*}

\textsuperscript{1} Qinghai Provincial Key Laboratory of Restoration
Ecology for Cold Region, Northwest Institute of Plateau Biology, Chinese
Academy of Sciences, Xining, China, 810008

\textsuperscript{2} University of Chinese Academy of Sciences, Beijing,
China, 10049

\textsuperscript{3} College of Geology, Qinghai Normal University,
Xining, China, 810001

Correspondence E-mail: ygdu@nwipb.cas.cn

\textbf{Abstract}

Water use efficiency is an important indicator of drought tolerance of
plants. The response was still unclear of water use efficiency to
different grazing intensity and its adaptive mechanisms in alpine
meadow. Here, we analyzed water use efficiency of plant dominant
species, coexisting species and functional group level plants to grazing
intensity by \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C index in alpine meadow. Furthermore,
this study revealed the driven factors of water use efficiency combined
available nitrogen, soil bulk density, soil organic carbon, soil water
content and evapotranspiration. Grazing increased plants
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C by 3.37\%. Moderate grazing significantly
increased the \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C of Gramineae by 4.84\%
(P\textless{}0.05), and increased \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C of Cyperaceae,
Leguminosae, and Forb by 3.45\%, 0.81\%, and 1.40\% respectively. Some
dominant species and coexisting species have the highest
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value under moderate grazing. This study
indicated that moderate grazing significantly improved the water use
efficiency of alpine meadows. Path analysis resulted showed that the
water use efficiency was negatively correlated with evapotranspiration
(P\textless{}0.05), soil water content, soil organic carbon, and soil
bulk density. It was positively correlated with available nitrogen. Our
study provides new insights that moderate grazing help to increase water
use efficiency in alpine meadows. In addition, evapotranspiration may be
the main driving factor affecting the water use efficiency of alpine
meadow.

KEYWORDS: alpine meadow, grazing activity, water use efficiency,
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C

\textbf{1 \textbar{} INTRODUCTION}

Plant leaf carbon isotope composition (\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C) is the
integration of photosynthetic activities during plant leaf tissue
synthesis, which can reflect the relative relationship between plant
water loss and carbon harvest (Shen et al., 2019). It is often used to
indirectly indicate the long-term water-use efficiency (WUE) of plants
(Liu et al., 2016; Stewart et al., 1995; Farquhar et al., 1982). A large
number of studies have shown that the \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value of
plant leaves has a robust positive correlation with water use
efficiency. Larger of \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C indicated higher plants WUE
(Su et al., 2005; Qu et al., 2001; Farquhar et al., Shen et al., 2017;
Pan et al., 2011). Grazing is the main utilization mode of grassland
ecosystems. The species richness and coverage of grassland will decrease
significantly during grassland degradation processes (Su et al., 2005).

More than 60\% of alpine meadows were degraded seriously than moderate
degradation, and grazing is the leading factor in the degradation of
alpine meadows (Cao et al., 2018). Previous studies focused on impacts
of grazing intensities on grassland multi-functions (Du et al., 2019;
Shen et al., 2019; Zhang et al., 2019). The grazing activities played a
role in balancing and stabilizing the plant community (Wang, 2018).
Grazing activities decreased the biomass and diversity of grassland
ecosystems (Lin et al., 2017; Xu et al., 2015; Gang et al., 2019), soil
water content (Zhang et al., 2019; Lin et al., 2011), and
evapotranspiration process (Zhang et al., 2019; He et al., 2014; Shu et
al., 2019). The stable carbon isotope technology is fast and accurate
due to its labeling, integration and indication functions (Jiang et al.,
1996). Some studies based on the \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C stable isotope
technology have found that the long-term WUE of plants is studied
through the monthly and annual changes of the WUE in desert plants (Su
et al., 2005). Grazing has a significant impact on plants stable isotope
indicators (Chen, 2013). Grazing pastures in Guinea, Brazil, Tanzania,
plant WUE shows an increasing trend under moderate grazing (Cavalcante
et al., 2016; Wang, 2016). The \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C of \emph{Stipa
breviflora} plants showed a downward trend with the grazing activities
increasing (Wang, 2016). The WUE of typical plants showed a downward
trend under grazing conditions, but the soil shows an upward trend in
the desert steppe of Inner Mongolia (Zhu et al., 2015).

The \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value is affected by environmental factors
such as evapotranspiration, soil water content, soil organic carbon, and
soil bulk density, which in turn affects plant WUE. Zhang (2003) studied
the changes in the \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C values of terrestrial
C\textsubscript{3} plants in 72 regions of China's Xinjiang, Gansu,
Inner Mongolia, Qaidam Basin and the Qinghai-Tibet Plateau and found
that precipitation and \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C values are negatively
correlated. With an increase of 100 mm, the \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value
of the plant is negative 0.33%0 (Stewart et al., 1995). Soil water
content has a weak negative correlation with WUE (Wu et al., 2019). The
WUE of Haibei alpine meadow in Qinghai is positively correlated with the
change of evapotranspiration (Shi et al., 2020). Zhu (2020) et al
studied the correlation between the \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value and
soil organic carbon under different grazing treatments in the Xilamuren
desert steppe in central and western Inner Mongolia, and found that they
are negatively correlated. The content of soil organic carbon in Panyang
Lake is different from \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C. The relationship between
the two was also found to be negative (Wang et al., 2016).

Although, the responses of WUE to grazing at the species level and
community level were vastly researched. However, few studies focused on
the response of WUE of alpine meadows to grazing intensity and
adaptation mechanisms. This study aimed to reveal the long-term WUE
variation characteristics under different grazing intensities and its
driven factors. Thus, we study different species, function groups and
community level WUE under long-term grazing. Furthermore, main driven
factors were discussed including soil available nitrogen, organic
carbon, evapotranspiration, soil water content, and bulk density. The
following scientific questions were answered: (1) How did the WUE of
the dominant species and coexisting species respond to different grazing
intensities? (2) How did plant functional group species WUE respond to
different grazing intensities? (3) How did the coupling factors affect
WUE in different grazing intensities?

\textbf{2 \textbar{} MATERIALS AND METHOD}

\textbf{2.1 \textbar{} Site description}

The research plots were carried out in Qinghai Haibei alpine grassland
ecosystem national observation and research station (Haibei Station,
37\selectlanguage{ngerman}°39.876\selectlanguage{english}' N, 101deg10.748\selectlanguage{english}' E), with an altitude of 3227.6 m. The sample
plot is a winter pasture. The grazing period is from the beginning of
October to the end of April of the following year, so the plant growth
season is not disturbed by grazing. It belongs to typical plateau
continental climate, the annual average precipitation and temperature
are 560 mm and -1.7 degC, respectively. The precipitation from May to
September during the growing season accounts for about 80\% of the total
annual precipitation.

The plant growing season has rain and heat at the same time, and soil
type is alpine meadow soil. Since the 1980s, the three pastures and
grazing channel grasslands have carried relatively stable grazing
intensity, and formed obvious grazing gradients in terms of surface
characteristics and species composition (Table 1, Fig. 1).

The native vegetation of the alpine meadow in this area is cold
mesophytic, wet mesophytic and dry mesophytic perennial Kobresia as a
constructive vegetation type. The community structure is relatively
complex and the species composition is rich. There are about 30 species
m\textsuperscript{-2}, and the dominant species are mainly
including\emph{Kobresia humilis, K. pygmaea, Elymus nutants, Festuca
rubra, Poa annua, Oxytripis ochrocephala, Gentiana straminea, Potentilla
nivea, Saussurea superba, Thermopsis lanceolate} . Coexisting species
including\emph{Stipa aliena, Poa annua, Kobresia humilis, Kobresia
pygmaea, Oxytropis ochrocephala, Thermopsis lanceolata, Saussurea
superba, Leontopodium nanum, Gentiana straminea, Potentilla nivea,
Anaphalis lactea Maxim.} Among them, \emph{Stipa aliena} and \emph{Poa
annua}belong to Gramineae, \emph{Kobresia humilis} and \emph{Kobresia
pygmaea}belong to Cyperaceae, \emph{Oxytropis ochrocephala} and
\emph{Thermopsis lanceolata} belong to Leguminous, \emph{Saussurea
superba, Leontopodium nanum, Gentiana straminea, Potentilla nivea} and
\emph{Anaphalis lactea Maxim} belong to Forb group.

\selectlanguage{english}\textbf{2.1 \textbar{} 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 x 25 cm).

The total biomass of the community was investigated using a sample
method (25 cm x 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 65degC
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 \selectlanguage{english}
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 CO\textsubscript{2}, and injected into a stable
carbon isotope mass spectrometer (Thermo, MAT-253) for stable carbon
isotope value analysis. This analysis accuracy is +- 0.1\selectlanguage{english}%0.

The expression formula of carbon isotope composition is:

\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C= (R\textsubscript{sam} / R\textsubscript{sta}-1)
\selectlanguage{ngerman}×1000

In this formula, R\textsubscript{sam} and R\textsubscript{sta} are
the\textsuperscript{13}C/\textsuperscript{12}C 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.

\selectlanguage{english}\textbf{3 \textbar{} RESULT}

\selectlanguage{english}\textbf{3.1 \textbar{}} \selectlanguage{english} \textbf{WUE variation characteristics of
dominant species in different grazing intensity}

The \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value of each species differed greatly both
in enclosure plots and grazed treatments. The average
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C values of dominant species in three grazing
treatments were -27.61, -26.16 and -27.20%0, respectively. The
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value of dominant species increased firstly and
then decreased with grazing intensity increasing. The overall range of 9
species is from -24.92 \selectlanguage{ngerman}± 0.07 to -28.08 ± 0.01\selectlanguage{english}%0. The former value is
3.16\selectlanguage{english}%0 higher than the latter approximately 12.68\% reduction range. The
highest and lowest values are Gentiana \emph{straminea} and Leontopodium
nanum respectively. The \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value of \emph{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
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C 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.

\textbf{3.2 \textbar{}}  \textbf{WUE variation characteristics of 11
coexisting species indifferent grazing intensity}

The \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value of \emph{Gentiana straminea}
and\emph{Anaphalis lactea Maxim} were highest and lowest respectively.
The average \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value of functional group plants
under moderate grazing were the largest, and the average
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value of the functional group plants in three
experimental treatments were -27.09, -27.00 and -27.04%0, respectively.
The \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value of \emph{Poa annua, Kobresia humilis,
K. pygmaea,} and \emph{Potentilla nivea} increased significantly under
moderate grazing (P\textless{}0.05, Fig. 2). \emph{Leontopodium nanum,
Oxytripis ochrocephala, and Anaphalis lactea Maxim} had the highest
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value under heavy grazing. \emph{Stipa aliena,
Thermopsis lanceolata, Saussurea superba} and \emph{Gentiana
straminea}had the highest \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value under enclosure.
In other words, moderate grazing can increase coexisting species plant
WUE by 3.32%0 compared with enclosure.

\textbf{3.3 \textbar{}}  \textbf{Variation characteristics of functional
group WUE in different grazing intensity}

The \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C 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 \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C 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 \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C and its WUE
approximately 4.84\% comparing with control (P\textless{}0.05, Fig. 3).
The \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C 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 \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value
of plants. The average \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C values of each
functional group in the three treatments of enclosure, moderate grazing
and heavy grazing were -27.47%0, -26.90%0, and -26.97%0, respectively.
Moderate grazing and heavy grazing increased WUE by 2.07\% and 1.82\%
respectively compared with enclosure.

\textbf{3.4 \textbar{}}  \textbf{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 \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C
value, and evapotranspiration and soil organic carbon have reached a
significant level (P\textless{}0.05). However, available nitrogen was
significant positive correlate with WUE (P\textless{}0.01, Table 3).
Grazing intensity is positively correlated with WUE. Grasslands WUE was
higher under tolerant environment.

\textbf{3.5 \textbar{}}  \textbf{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 \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C value
(P\textless{}0.05). Furthermore, available nitrogen, soil organ carbon,
soil bulk density and soil water content have indirect effects on the
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C 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.

\textbf{4 \textbar{} 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
Stipa\emph{breviflora} 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
use\textsuperscript{12}C to enrich the residual organic matter
with\textsuperscript{13}C 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).

\textbf{5 \textbar{}}  \textbf{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 \textgreater{} Cyperaceae
\textgreater{} Forb \textgreater{} 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.

\textbf{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).

\textbf{DATA AVAILABILITY STATEMENT}

Grassland plant leaves isotopte data in this paper would be upload in
dryad when this manuscript could be accepted.

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\textbf{Hosted file}

\verb`Fig. 1-4.docx`  available at \url{https://authorea.com/users/472039/articles/709273-moderate-grazing-significantly-increases-water-use-efficiency-in-alpine-meadow-on-the-tibetan-plateau}

\textbf{Hosted file}

\verb`Table 1-3.docx`  available at \url{https://authorea.com/users/472039/articles/709273-moderate-grazing-significantly-increases-water-use-efficiency-in-alpine-meadow-on-the-tibetan-plateau}

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