Global Ecology and Conservation 22 (2020) e01003

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Global Ecology and Conservation

journal homepage: http://www.elsevier.com/locate/gecco

Original Research Article

Responses of plant diversity and soil microorganism diversity

to water and nitrogen additions in the Qinghai-Tibetan
Plateau
Jingjing Li a, Chao Yang b, Huakun Zhou c, Xinqing Shao a, *, 1
a
College of Grassland Science and Technology, China Agricultural University, Beijing, 100193, China
b
Grassland Agri-Husbandry Research Center, College of Grassland Science, Qingdao Agricultural University, Qingdao, 266109, China
c
Key Laboratory of Restoration Ecology of Cold Area in Qinghai Province, Northwest Institute of Plateau Biology, Chinese Academy of
Sciences, Xining, 810008, Qinghai, China

a r t i c l e i n f o a b s t r a c t

Article history: The Qinghai-Tibetan Plateau is experiencing significant nitrogen (N) deposition and
Received 9 September 2019 increased precipitation. Although changes in N deposition and precipitation may cause
Received in revised form 3 March 2020 changes in the composition and diversity of plants, the relationships between plant di-
Accepted 3 March 2020
versity and soil microbial diversity still has not been fully researched. Thus, we conducted
a field simulation experiment in an alpine meadow that included three N and two water
Keywords:
(W) addition levels, as well as their interactions. The abbreviations of all treatments are
Precipitation changes
shown below: CK, control; W, added water; N5, added 5 g N m
2 yr
1; N10, added 10 g N
Nitrogen deposition
Alpine meadow ecosystem
m
2 yr
1; N5W, added N5 and W; N10W, added N10 and W. The dominant plant species
Plant diversity belong to the Gramineae family and include Elymus dahuricus, Stipa capillata, Poa pratensis
Soil microbial diversity and Agropyron cristatum. One year later after N and water addition, the results indicated
that soil pH decreased with N addition, and with a combination of N and W addition
together. High rate of N addition significantly lowered plant diversity. For different plant
functional groups, the relative abundance of grasses significantly increased, while the
relative abundance of forbs significantly decreased under N10, N5W and N10W treatments.
Under N10W treatment, the relative abundance of legumes was significantly reduced,
while the relative abundance of cyperaceae was significantly increased. W and N in-
teractions significantly decreased soil bacterial and fungal diversity. N addition showed an
indirect effect on the fungal diversity by directly affecting plant productivity. Water
addition showed an indirect effect on bacterial diversity by directly affecting plant di-
versity. Soil bacterial diversity showed a positive correlation with plant diversity, while soil
fungal diversity had no significant correlation with plant diversity, but had a negative
correlation with plant productivity. It also indicates that the strength of feedbacks between
aboveground and belowground biodiversity will vary depending on which groups of soil
biota are considered.
© 2020 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding author.
E-mail address: shaoxinqing@163.com (X. Shao).
1
Present address: College of Grassland Science and Technology, China Agricultural University, 2 Yuan Ming Yuan West Road, Haidian District, Beijing
100193, China.

https://doi.org/10.1016/j.gecco.2020.e01003
2351-9894/© 2020 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
2 J. Li et al. / Global Ecology and Conservation 22 (2020) e01003

1. Introduction

Nitrogen (N) enrichment has been increasing globally, mainly due to the human activities (Canfield et al., 2010; Galloway
et al., 2008). N deposition increased drastically in most terrestrial ecosystems over the past three decades in China (Zhang
et al., 2017). From 1980 to 2010, the annual average nitrogen deposition increased year by year, and increased by approxi-
mately 8 kg ha
1 in China (Liu et al., 2013). Deposition of N not only influenced species diversity, biomass production and
nutrient availability of plants (Balota et al., 2004; Driscoll et al., 2003; Lal, 2004), but also influenced soil conditions (Tian et al.,
2016), including increases in soil N content and in nitrate levels, a reduction in soil pH and variations in soil microbial
communities (Bell et al., 2010; Turner and Henry, 2009). In addition, changes in annual precipitation are expected with global
climate change (Marvel and Bonfils, 2013). Water availability is one of the most important driving factors for the activities of
plants and microorganism in grasslands (Knapp et al., 2008), and is also the key driver for soil carbon cycling, N cycling, and
the composition of soil microbial community (Zhang et al., 2015).
The responses of soil microbes to nitrogen deposition and precipitation changes are important for predicting functional
feedback of ecosystems under global change, due to the important role they play in the biogeochemical cycles (Nie et al.,
2013). The increase in N deposition and precipitation, could directly impact microbial physiology, and indirectly impact
microbial community composition and diversity by altering some of the characteristics of plants and soil (Chen et al., 2015; Li
et al., 2017). Plantemicrobeesoil interactions are very important for the formation of plant community structure (Miki et al.,
2010). Vegetation type is a critical driving factor for the structure of soil microbial communities(Stone et al., 2015). Plant
residues are the main resource inputs to the soil, and the quantity and quality could affected by nitrogen and water availability
(Liu et al., 2016), which in turn can influence soil microbial communities’ structure and function (Bardgett and van der Putten,
2014). In addition, plant diversity and microbial diversity are mutually reinforcing relationships. Briefly, plant diversity
provides a variety of food to soil microorganisms, which promotes the diversity of soil microorganism (Klironomos et al.,
2011), and in turn, soil microorganism increased the diversity of the effective nutrient pools, which promotes the plant di-
versity (van der Heijden et al., 2008). It was found that the growth of plant community was promoted by the addition of
nitrogen, and the change of soil bacterial composition might be caused by the increase of labile carbon(Belay-Tedla et al.,
2009). Plant diversity is an important factor affecting soil food chain and soil function. Plants enrich soil organic carbon
through litter and root exudates, and high plant diversity helps accumulate soil organic matter and maintain soil biodiversity
and soil biological function (Eisenhauer et al., 2013; Lange et al., 2015). Studies have showed that fluctuations in substances
and secretions between soil microbes and the roots of plants provide abundant substrate inputs to modify the composition
and facilitate the reproduction of microorganisms (Bais et al., 2006; Broeckling et al., 2008). However, in alpine meadows of
China, the relationship between aboveground and underground communities still has not been fully researched in the
context of N enrichment and increased precipitation. Understanding the plantemicrobeesoil interactions is important to
assess the impacts of nitrogen enrichment and precipitation on the functions of alpine meadow ecosystem.
The Qinghai-Tibetan Plateau is “the third pole” on the earth, which is experiencing a sharp increase in N deposition and
changes in precipitation. The alpine meadow, occupying approximately 35% of the plateau, is one of the most important
ecological types on the Qinghai-Tibetan Plateau (Cao et al., 2004). The rate of N deposition has increased significantly, ranging
from 4 to 13.8 kg ha
1 yr
1 in the eastern Qinghai-Tibetan Plateau between the 1980s and 2000s (Liu et al., 2013). It is
predicted that the average annual precipitation will increase more than 10% at the end of 21st century (Ding et al., 2007). In
our study, a field simulation experiment was conducted in an alpine meadow of the Qinghai-Tibetan Plateau with three N and
two water addition levels, as well as their interactions. We evaluated the responses of the soil microbial community, diversity
of soil bacteria and fungi, and plant diversity. Our study addressed the questions below: (1) How do plant and soil microbial
community structure respond to nitrogen enrichment and water addition? (2) What is the relationship between soil mi-
crobial diversity and plant indexes (i.e., diversity and productivity) under nitrogen enrichment and water addition? Two
hypotheses are listed below (1) Water and nitrogen addition would change the structure of plant and soil microbial com-
munities, but the impact on plant diversity may be different. (2) Soil bacterial and fungal diversity would show an different
relationship with plant diversity and plant productivity.

2. Materials and methods

2.1. Site description

Our study was conducted at the Haibei Demonstration Zone of Plateau Modern Ecological Animal Husbandry Science and
Technology (36 550 N, 100 570 E), which located in Qinghai Province, China, with an altitude of 3040 m. The study site has a
plateau continental climate, which means the warm season is short and the cold season is long. The annual average tem-
perature is
0.45  C, and the annual average precipitation is about 400 mm. The grassland type belongs to alpine meadow,
and the dominant plant species belong to the Gramineae family and include Elymus dahuricus, Stipa capillata, Poa pratensis
and Agropyron cristatum. The soil at the study site is classified as Mat-Gryic Cambisol, with the texture of clay-loam (Ma et al.,
2017). The study area was fenced to exclude any grassland management and use practices.
 J. Li et al. / Global Ecology and Conservation 22 (2020) e01003 3

2.2. Experimental design

In 2017, we established a nitrogen and precipitation manipulation study. A 21  25 m area was divided into 3  3 m plots
arrayed into a five by six matrix (a total of 30 plots), with a 1-m-wide buffer zone between each plot. All the treatments were
randomly distributed. Our experiment consisted of two precipitation and three nitrogen addition gradients. Precipitation
treatment was water addition (precipitation þ increased 10% of annual precipitation), and the control was the natural pre-
cipitation. The water addition treatment was applied by artificially adding 10% of water to simulate the condition of an in-
crease in precipitation, which was described in detail by (Li et al., 2019). Nitrogen was added as ammonium nitrate to simulate
the condition of nitrogen enrichment, and at the rates of 0, 5 and 10 g N m
2 yr
1 for each plot. NH4NO3 was evenly added by
hand throughout each plot in late May during each growing season. There was a total of 6 treatments with 5 replicates per
treatment. Hereafter, the 6 treatments will be represented by the abbreviations: CK, control; W, added water; N5, added 5 g N
m
2 yr
1; N10, added 10 g N m
2 yr
1; N5W, added N5 and W; N10W, added N10 and W.

2.3. Plant and soil sampling

In each plot, there was a permanent quadrat (1  1 m) to survey the coverage of each plant species. All plants were
classified into four plant functional groups: grasses, forbs, legumes and cyperaceae and counted the species of each functional
group. Above-ground biomass was harvested in these permanent quadrants at the peak of plant biomass production. Shoots
were cut at the soil surface, and all samples were oven-dried at 65  C for 72h (Fig A1).
In mid-August of 2018, we collected three soil cores (0e15 cm, 5 cm in diameter) from each plot and mixed them evenly as
one composite soil sample for the plot. Hereafter, we transferred soil samples into the laboratory immediately, where we
picked out the roots and stones in each soil sample, and then sieved with the 2 mm sieve and divided into two parts. One was
used for the determination of soil physicochemical properties, and the other one was stored in a freezer at
80  C for the
extraction of soil DNA.

2.4. Determination of soil physicochemical properties

Soil was analyzed for soil water content(SWC), soil pH; soil organic carbon concentration (SOC), soil total nitrogen con-
centration (STN), and soil inorganic nitrogen concentration (NHþ

4 -N and NO3 -N) (SIN). Briefly, SWC was measured by oven

drying 10 g fresh soil at 105 C for 12 h. Soil pH was measured using a pH meter after shaking a 1:2.5 air-dried soil/water
suspension for 30min (Yang et al., 2019). SOC was measured with an automatic carbon analyzer (TOC, Elementar, Germany).
STN was measured with FOSS Kjeltec 2300 Analyzer Unit (FOSS, Hillerød, Sweden). SIN was extracted with 2 M KCl, and then
measured with Flow-Solution analyzer (Flowsys, Ecotech, Germany) (Yang et al., 2018a).

2.5. Soil microbial DNA extraction and sequencing

Soil microbial DNA was extracted using PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA). First, we accu-
rately weighed 0.30 g soil samples of each treatment (5 replicates). Then soil DNA integrity was detected by 0.8% agarose gel
electrophoresis. DNA purity and concentration were determined using Mini Dorp, constructing the library after passing the
test.
After the total DNA extraction of each soil sample, the specific primers of the Illumina MiSeq sequencing platform were
used to PCR-amplify, including the bacterial 16S rRNA V3þV4 region and the fungal ITS1 and ITS2 regions, respectively.
Reaction procedure: pre-denaturation at 95  C for 5 min; denaturation at 95  C for 45 s, annealing at 55  C for 50 s, extension
at 72  C for 45 s, 32 cycles; extension at 72  C for 10 min. The amplified products were purified, quantified and homogenized
to form a sequencing library, which was sequenced by an Illumina MiSeq platform of Beijing Allwegene Biotechnology Co.,
Ltd. The PCR amplification primers included: V3þV4 region 338F (50 -ACTCCTACGGGAGGCAGCAG-30 ) and 806R (50 -GGAC-
TACHVGGGTWTCTAAT-30 ) (Li et al., 2015b), and fungal ITS1 (50 -CTTGGTCATTTAGAGGAAGTAA-30 ) and ITS2 region (50 -
GCTGCGTTCTTCATCGATGC-30 ) (Sun et al., 2016).

2.6. Processing of sequencing data

Illumina Miseq sequencing produced double-ended sequence data. So, the obtained sequences were first filtered using the
Quantitative Insights into Microbial Ecology. In brief, 1) the bases of the reads tail mass value of less than 20 were filtered by
setting a 50 bp window. If the average quality value in the window is lower than 20, the back base was truncated from the
window to improve the quality of the sequence; 2) the pairs of reads were spliced into a sequence according to the overlap
relationship, and the minimum overlap length was 10 bp; 3) The maximum mismatch ratio allowed by the overlap area of the
splicing sequence was 0.1, and the non-conforming sequence was directly filtered in pairs; 4) the samples were distinguished
according to the barcode and primer at the beginning and the end of the sequence. UCLUST was used to classify the unique
sequence set as an OTU with a 97% identity threshold.
4 J. Li et al. / Global Ecology and Conservation 22 (2020) e01003

2.7. Statistical analysis

The Shannon index (H) calculation used the equation below (Wang et al., 2019) to measure the plant diversity (Fig A1) of
above-ground communities:
X
H¼
Pi ln Pi

Where Pi is the ratio of the coverage of species i to the total coverage.

Shannon and Chao 1 richness were calculated with the equations below to evaluate the diversity soil bacteria and fungi
(Dong et al., 2017).

XNi Ni
Shannon index ¼
ln
N N

Coverage ¼ 1
c=N

cðc
1Þ
Chao 1 ¼ Sobs þ
2ðd þ 1Þ

where N is the total OTUs of the sample, Ni is the number of individuals in group i; c is the number of OTUs with only one
sequence; N is the total number of OTUs; Sobs is the observed number of OTUs; d is the number of OTUs with only two
sequences.
Two-way Analysis of Variance (ANOVA) was conducted to test the impacts of the N, W, and their combination on soil
physicochemical properties and soil bacterial and fungal diversity. One-way ANOVA was used to test the effects of treatments
on all of the plant and soil variables, conducting with the SPSS 19.0 (IBM, Armonk, NY, USA). Pearson correlation analysis was
used to test the relationship between soil properties and dominant microbial phyla, conducting with the SPSS 19.0 (IBM,
Armonk, NY, USA). Principal component analysis (PCA) was used to illustrate the relationship among plant and microbial
community composition under nitrogen (N) and water (W) addition treatments, using CANOCO 5.0. We performed a
Redundancy analysis to analyze the relationships between dominant soil bacterial and fungal communities and soil prop-
erties (Dong et al., 2017), using CANOCO 5.0. The rarefaction curve and Coverage were used to indicate the sequencing depth,
which was calculated using Mothur 1.21.1.
We performed structural equation model (SEM) to test the relationships between soil microbial diversity and plant di-
versity. The SEM procedure compared the observed varianceecovariance matrix against the model-implied
varianceecovariance matrix. A maximum likelihood estimation method was used to fit data to the model. The c2 test was
conducted to test the goodness of fit for structural equation model. The model had a high goodness of fit index (0  c2  2;
0.05 < P  1.00) and with low root mean square error of approximation (0  RMSEA  0.05; 0.10 < P  1.00) (Yang et al.,
2018b). The initial model was simplified and re-evaluated by eliminating non-significant pathways gradually until all links
were significant contributors to the final model. The final model included two exogenous variable (N addition and W addition)
and four endogenous variables (soil bacterial diversity, soil fungal diversity, plant productivity and plant diversity). SEM
analyses were conducted using AMOS 21.0 (SPSS Inc., IBM Co., Armonk, NY, USA). All figures were produced using SigmaPlot
12.5 (Systat Soft-ware, Inc., San Jose, CA, USA).

3. Results

3.1. Soil physicochemical properties and plant diversity and community composition

Two-way analysis of variance showed that SOC, Soil C:N ratio and SIN were significant impacted by N addition, W addition
and their interactions (Table 1, P < 0.05). The addition of W significantly increased SWC (Table 1). Soil pH significantly
decreased under N5 and N10 addition, (Table 1). N enrichment significantly increased SOC concentration, but less so at N10
when W was added. STN concentration significantly increased compared with the control under the addition of W and N,
especially under the N10W treatment. (Table 1). SIN concentration with the W and N addition treatment was significantly
higher than the control, and reached the maximum under N5W treatment. Soil C:N ratio significantly decreased with W, N5,
N5W and N10W treatments (Table 1).
For different plant functional groups, the relative abundance of grasses significantly increased, while the relative abun-
dance of forbs significantly decreased under N10, N5W and N10W treatments. Under N10W treatment, the relative abun-
dance of legumes was significantly reduced, while the relative abundance of cyperaceae was significantly increased (Fig. 1
a).The result of PCA analysis showed plant community composition tended to be clustered separately from each other
under different N and W treatments (Fig. 1b).
 J. Li et al. / Global Ecology and Conservation 22 (2020) e01003 5

Table 1
Observed variables for soil under each treatment. Values are Mean ± SE (n ¼ 5) for each treatment. Lowercase letters indicate difference between treatments
(LSD, P < 0.05). Two-way analysis of variance was used to determining the influences of nitrogen (N), water (W) and their interactive impacts on all observed
variables (P values are listed in the last three lines).

1
Treatment SWC (%) Soil pH SOC (g kg ) STN (g kg
1) Soil C: N ratio SIN (mg kg
1)
CK 23.25 ± 0.006b 8.09 ± 0.01a 21.28 ± 0.48c 3.33 ± 0.05f 6.39 ± 0.14a 13.98 ± 0.74c
W 26.26 ± 0.005a 8.04 ± 0.01a 21.36 ± 0.04c 3.64 ± 0.01e 5.86 ± 0.02b 20.09 ± 0.91b
N5 23.05 ± 0.009b 7.92 ± 0.02bc 22.81 ± 0.52b 3.81 ± 0.04d 5.98 ± 0.13b 20.56 ± 0.80b
N10 21.69 ± 0.01b 7.86 ± 0.04c 24.98 ± 0.57a 4.05 ± 0.03c 6.17 ± 0.16 ab 23.07 ± 0.94a
N5W 26.68 ± 0.005a 7.95 ± 0.02b 21.64 ± 0.46bc 4.23 ± 0.02b 5.11 ± 0.11c 25.20 ± 0.78a
N10W 27.97 ± 0.006a 7.89 ± 0.02bc 22.01 ± 0.45bc 4.54 ± 0.04a 4.85 ± 0.08c 18.65 ± 0.50b
W < 0.001 0.923 0.001 < 0.001 < 0.001 0.003
N 0.988 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
WN 0.075 0.159 0.010 0.058 0.010 < 0.001

Note: CK, control; W, added water; N5, added 5 g N m
2 yr
1; N10, added 10 g N m
2 yr
1; N5W, added N5 and W; N10W, added N10 and W; SWC, soil
water content; SOC, soil organic carbon; STN, soil total nitrogen; SIN, soil inorganic nitrogen.

Fig. 1. Relative abundance of plant functional group composition (a) and (b) Principal component analysis (PCA) plot illustrating distances between plant
community composition under nitrogen (N) and water (W) addition treatments. Note: CK, control; W, added water; N5, added 5 g N m
2 yr
1; N10, added 10 g N
m
2 yr
1; N5W, added N5 and W; N10W, added N10 and W. Asterisks indicate significant differences under different treatments (P < 0.05).

3.2. Overview of soil microbial sequencing

A total of 4958 bacterial OTUs and 2365 fungal OTUs (at 97% sequence identity) were observed from the 30 soil samples,
with bacterial OTUs ranging from 2197 to 2277 and fungal OTUs ranging from 483 to 779 from all soil samples (Table A1).
Observed species is the number of OTUs actually observed as the increasing of sequencing depth. The number of bacterial
observed species ranged from 2117 to 2182, and fungal observed species ranged from 468 to 753 from all soil samples (Table
A1). The sequencing workload was sufficient to detect >96% in bacterial communities and >98% in fungal communities to
predict the richness of each sample (Table A1, Fig A2).

3.3. Soil microbial community composition and diversity

The relative abundances of the main dominant bacterial and fungal phyla under each treatment were depicted in Fig. 2.
The Acidobacteria phylum was most abundant, accounting for 22%~32% of total bacterial sequences among different treat-
ments, while Tectomicrobia was the minor phyla, accounting for 0.5%~0.8% of all bacterial sequences. Specifically, the
abundance of the Acidobacteria phylum decreased under two gradients of N addition and their interactions with W addition
(Fig. 2a). The Ascomycota phylum of fungal community had the highest abundances, accounting for 65%~75% of the total OTUs
among different treatments, and Kickxellomycota was the minor phylum, accounting for 0.01%~0.04% of all fungal sequences.
The Ascomycota and Mortierellomycota phylum both increased in abundance under N5W and N10W treatments (Fig. 2b). PCA
analysis revealed separation between samples from treatments for soil bacterial and fungal communities, respectively (Fig. 2c
and d).
Two-way ANOVA showed that W addition had significant effects on bacterial Chao 1 index (P ¼ 0.001), and bacterial Chao
1 and Shannon index was significantly impacted by N addition (P < 0.0001). W addition, N addition and W addition plus two
rates of N addition had significant effects on fungal Chao 1 and Shannon index (Fig. 3). One-way ANOVA showed that bacterial
Chao 1 index significantly decreased under N10W treatment, and Shannon index significantly decreased under N10, N5W and
N10W treatments comparing with CK (Fig. 3a and b). In addition, the fungal Chao 1 and Shannon index significantly decreased
under the addition of N5W and N10W treatments (Fig. 3c and d).
6 J. Li et al. / Global Ecology and Conservation 22 (2020) e01003

Fig. 2. Relative abundance of dominant bacterial phyla (a), fungal phyla (b) and Principal component analysis (PCA) plot illustrating distances between microbial
community composition for bacteria (c) and fungus (d) under nitrogen (N) and water (W) addition treatments. Note: CK, control; W, added water; N5, added
5 g N m
2 yr
1; N10, added 10 g N m
2 yr
1; N5W, added N5 and W; N10W, added N10 and W.

3.4. Relationships between soil physicochemical properties and dominant microbial communities

Redundancy analysis showed obvious separation of soil bacterial and fungal communities under different treatments. The
first axis explained 25% and 15% changes in structure of bacterial and fungal community influenced by soil variables,
respectively (Fig. 4a and b). In the soil bacteria RDA biplot, soil pH was positively correlated with soil bacterial communities
under CK and W addition treatments, and influenced bacterial phyla including Acidobacteria, Nitrospirae, Gemmatimonadetes,
and Bacteroidetes. Soil C/N ratio was positively correlated with bacterial communities with the addition of W and N5,
including Acidobacteria, Verrucomicrobia, Planctomycetes and Chloroflexi. SIN and SOC showed positive correlations with
bacterial communities under N10 treatment, including Actinobacteria and Tectomicrobia. SWC and STN showed positive
correlations with Proteobacteria communities under N5W and N10W treatments (Fig. 4a). Pearson correlation analysis
revealed that Acidobacteria was positively correlated with soil pH (Table A2, P < 0.05), and negatively correlated with TN
(Table A2, P < 0.05). Actinobacteria and Tectomicrobia were negatively correlated with soil pH (Table A2, P < 0.05), but Acti-
nobacteria was positively correlated with TN and SIN (Table A2, P < 0.05).
As for soil fungus RDA biplot, soil pH and C/N ratio were positively correlated with fungal communities under CK, W and
N5 treatments, including Basidiomycota, Glomeromycota, and Rozellomycota. SOC showed positive correlation with fungal
communities under addition of N10, including Olpidiomycota, Chytridiomycota and Mortierellomycota. SIN positively related
with Ascomycota communities under N5W treatment. SWC and STN showed positive correlations with Kickxellomycota,
Cercozoa and Mucoromycota communities under N5W and N10W treatments (Fig. 4b). Pearson correlation analysis showed
that soil TN was negatively correlated with Basidiomycota and Glomeromycota, but positively correlated with Mucoromycota
(Table A3, P < 0.05). Soil C/N ratio was positively correlated with Basidiomycota and Glomeromycota, but negatively correlated
with Mucoromycota (Table A3, P < 0.05)

3.5. Relationship between soil bacterial and fungal diversities and plant diversity

The SEM indicated the direct or indirect relationships between microbial diversity, plant productivity and plant diversity
under nitrogen (N) and water (W) addition (Fig. 5). The model explained 19%, 64%, 53% and 25% of the variance in bacterial
diversity, fungal diversity, plant diversity and plant productivity respectively (Fig. 5a). N addition had a significant positive
effect on plant productivity (P < 0.05), but had significant negative effect on plant diversity (P < 0.05). W addition positively
directly impacted plant diversity (P < 0.05), but negatively directly impacted soil fungal diversity (P < 0.05). There was a
significant positive correlation between plant diversity and bacterial diversity (P < 0.05), but no significant relationship
between plant diversity and fungal diversity. There was a significant negative correlation between plant productivity and soil
fungal diversity (P < 0.01). Our SEM showed N addition has an indirect effect on the fungal diversity by directly affecting plant
 J. Li et al. / Global Ecology and Conservation 22 (2020) e01003 7

Fig. 3. Chao 1 richness (a), Shannon index (b) for soil bacterial community and Chao 1 richness (c), Shannon index (d) for soil fungal community under different
treatments. Values are Means ± SE (n ¼ 5) for each treatment. Lowercase letters indicate difference between treatments (LSD, P < 0.05). The influences of nitrogen
(N), water (W) and their interactive impacts on the diversity of soil bacteria and fungi are shown in each plot, ns: no significant difference. Note: CK, control; W,
added water; N5, added 5 g N m
2 yr
1; N10, added 10 g N m
2 yr
1; N5W, added N5 and W; N10W, added N10 and W.

Fig. 4. The influences of soil physicochemical properties on dominant bacterial phyla (a) and fungal phyla (b) is depicted in Redundancy analysis (RDA) plots. The
communities are represented by color symbols: white circles, CK; red squares, W; green diamonds, N5; purple down-triangles, N10; yellow left-triangles, N5W;
and pink up-triangles, N10W. Note: soil water content, SWC; soil organic carbon SOC; soil total nitrogen, STN; soil inorganic nitrogen, SIN; CK, control; W, added
water; N5, added 5 g N m
2 yr
1; N10, added 10 g N m
2 yr
1; N5W, added N5 and W; N10W, added N10 and W. (For interpretation of the references to color in
this figure legend, the reader is referred to the Web version of this article.)
8 J. Li et al. / Global Ecology and Conservation 22 (2020) e01003

Fig. 5. Structural equation model (SEM) indicates the direct and indirect impacts of nitrogen (N) addition and water (W) addition on plant productivity, plant
diversity, bacterial diversity and fungal diversity and (a), and standardized total effects derived from the SEM of bacterial diversity and fungal diversity (b). The
positive and negative relationships among variables are represented by continuous and dotted arrows, respectively. The width of the arrow is proportional to the
intensity of the path coefficient. Levels of significance: *P < 0.05, **P < 0.01.

productivity. Water addition has an indirect effect on bacterial diversity by directly affecting plant diversity. In addition, the
relationship between bacterial and fungal diversity and plant diversity was different under W and N additions. Standardized
total effects derived from the SEM revealed that soil bacterial diversity was primarily driven by plant diversity, and then by
plant productivity. In contrast, soil fungal diversity was driven primarily by plant productivity, and then by W addition
(Fig. 5b).

4. Discussion

4.1. Response of plant and soil microbial diversity to water and nitrogen additions

Previous researches have reported that N addition lowers plant diversity (Roth et al., 2013), which was supported by our
study. W addition had non-significant effect on plant diversity, but there was an increasing trend compared with the CK. The
addition of N and W together also significantly decreased plant diversity, but not to the same degree at the highest level.
Adding N and water together removed the positive effect of water addition on plant diversity in our study, which is consistent
with the finding of a meta-analysis (DeMalach et al., 2017). One possible explanation is that N enrichment increased
aboveground biomass and thus promoted the photosynthetic rate of plants, which caused a higher transpiration rate and
faster depletion of water from the root zone (Harpole et al., 2007), offsetting the effect of water addition alone.
Previous studies showed that N deposition and changes in precipitation could alter the microbial diversity (Evans and
Wallenstein, 2014; Li et al., 2016). Here, we found that soil bacterial richness decreased with high gradient of N and W
addition together. However, W addition alone has no effects on soil bacterial diversity. Some research has shown that N
application reduces soil bacterial diversity (Sun et al., 2015; Zhou et al., 2015). While another study indicated that increasing N
deposition may not affect soil bacterial diversity (Fierer et al., 2012). We thought that these results are explained by differ-
ences in the spatial scale. It has been shown that water usually limits soil microbial communities with a variety of factors
(Zhang et al., 2015), and soil gram-negative bacteria significantly decreased when N enrichment and precipitation changes
occurred together (Shi et al., 2018). N addition alone significantly increased fungal Shannon index at 100e200 kg N$ ha
1 yr

1
, and the Chao1 index at 100e300 kg N$ ha
1 yr
1, however, when water was added, these indices did not significantly
change following N enrichment (Zhang et al., 2018). In this study, soil fungal Chao 1 richness and Shannon index significantly
responded to the increased N deposition and precipitation. Soil fungal abundance was significantly altered by N enrichment
and changes in precipitation (Jia et al., 2017). According to the researches, soil fungal communities have an elastic response to
increased environmental changes. (i.e. nitrogen deposition and precipitation changes) (Jumpponen and Jones, 2014; Li et al.,
2015a). How soil fungi respond to N and water addition is probably influenced by local environmental conditions, plant
communities, intensity and frequency of N and water addition, experimental duration and other unknown factors (Hawkes
et al., 2011; Zhao et al., 2018).
 J. Li et al. / Global Ecology and Conservation 22 (2020) e01003 9

4.2. Relationship between soil microbial diversity and plant diversity

In this study, we found that N addition indirectly impact fungal diversity by directly affecting plant productivity, however
W addition indirectly impact bacterial diversity by directly affecting plant diversity. A study has reported N addition resulted
in a significant decline in fungal alpha diversity (Chen et al., 2018). The difference of our results was that the addition of N
alone had no significant effect on fungal diversity, but the interaction of water and N addition reduced the fungal diversity
significantly. In addition, the SEM also showed that there was no significant direct relationship between N addition and fungal
diversity. Water addition promote plant diversity and may eliminate the water stress or substrate limitation for microor-
ganisms and therefore benefit bacteria growth (Zhao et al., 2017). Thereby indirectly promoted bacterial diversity by directly
affecting plant diversity. However, there was a significant negative correlation between water addition and fungal diversity.
The possible reasons may be bacteria and fungi are the two main constitutes of soil microbes, and the differences in cell
structure, physiological traits and community diversity bacterial versus fungal communities likely experience different
susceptibility to water addition (Kooijman et al., 2016).
Aboveground-belowground interactions play a key role in controlling the composition and function of terrestrial eco-
systems. However, the basic relationship between plant diversity and soil microbial diversity remains elusive. Plant diversity
is the key driving factor for the construction of microbial community (Chen et al., 2017). The high plant diversity is helpful to
soil organic matter accumulation and soil biodiversity maintenance (Lange et al., 2015). In our case, soil bacterial diversity was
positively correlated with plant diversity, but soil fungal diversity showed no significant correlation with plant diversity. The
mechanism of plant diversity affecting bacterial diversity may be: (i) a more diverse plant community returning a more
heterogeneous mixture of resources to the soil; and (ii) this more diverse resource mixture in turn influencing bacterial
diversity (Wardle, 2006). It was reported that plant-fungi biotic linkages did not exist under N enrichment at surface soil
(Chen et al., 2018). However, other study found the positive relationship between soil fungal diversity and plant indexes (i.e.
plant richness & plant composition) (Zhang et al., 2018). In our study, we found there was no significant relationship between
plant diversity and fungal diversity. This indicated that the relationship between plant diversity and soil biodiversity is
different in different spaces and this relationship is complex and should not be generalized. Our study found that plant
productivity was an important factor affecting fungal diversity, and it was negatively correlated with fungal diversity. As
reported previously, under N addition alone, the plant community was the main factor that directly influenced fungal OTU
richness (Zhang et al., 2018). Collectively, the nature of the interaction effects of nitrogen and water on soil microbial com-
munities, is very complex, and occurs through changes in plant communities and soil properties.

5. Conclusions

Our simulated nitrogen deposition and increased precipitation experiment has studied the relationship between plant
indexes (i.e., plant diversity and plant productivity) and soil microbial diversity in an alpine meadow. N and W additions
changed the community structure of soil microbes and the relative abundance of plant functional group composition. Soil
bacterial and fungal diversity lowered under two gradients of N and W additions together. Our study indicated that N addition
has an indirect effect on the fungal diversity by directly affecting plant productivity. Water addition has an indirect effect on
bacterial diversity by directly affecting plant diversity. Soil bacterial diversity showed a positive correlation with plant di-
versity, while soil fungal diversity had no significant correlation with plant diversity, but had a negative correlation with plant
productivity. It also indicates that the strength of feedbacks between aboveground and belowground biodiversity will vary
depending on which groups of soil biota are considered.

Funding

This work was supported by the National Key Research and Development Program of China [2016YFC0501902].

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

We are very grateful to Haibei Demonstration Zone of Plateau Modern Ecological Animal Husbandry Science and Tech-
nology for providing us with a research platform, and sincerely thank the staff there for their enthusiastic help in our field
experiment treatment and sample collection.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.gecco.2020.e01003.

10 J. Li et al. / Global Ecology and Conservation 22 (2020) e01003

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