Science of the Total Environment 610611 (2018) 750758

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Science of the Total Environment

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Differential responses of soil microbial biomass, diversity, and compositions

to altitudinal gradients depend on plant and soil characteristics
Chengjie Ren, Wei Zhang, ZeKun Zhong, Xinhui Han , Gaihe Yang , Yongzhong Feng, Guangxin Ren
College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi, China
The Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling 712100, Shaanxi, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

Microbial biomass reects the C/N in

plant-soil systems along altitudinal gra-
dients.
Bacterial diversity varied more than
fungal diversity along altitudinal gradi-
ents.
Altitudinal gradient largely affected bac-
terial composition not fungal composi-
tion.
Microbial alpha diversity was mainly
coupled with plant diversity, SOC and
TN.
Microbial beta diversity and composi-
tion mainly differed with ST and SM.

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

Article history: Alt'itudinal gradients strongly affect plant biodiversity, but the effects on microbial patterns remain unclear, es-
Received 5 July 2017 pecially in the large scale. We therefore designed an altitudinal gradient experiment that covered three climate
Received in revised form 10 August 2017 zones to monitor soil microbial community dynamics and to compare those with plant and soil characteristics.
Accepted 11 August 2017
Illumina sequencing of the 16S rRNA gene and ITS gene was used to analyze soil microbial (bacterial and fungal)
Available online xxxx
diversity and composition, and fumigationextraction was used to determine microbial biomass; the plant com-
Editor: Elena Paoletti munity metrics (i.e., percent cover, Shannon-Wiener, grass biomass, and carbon/nitrogen in leaf and biomass)
and soil properties (i.e., soil moisture, soil temperature, bulk density, organic carbon, total nitrogen, and available
Keywords: nitrogen) were determined. The results showed that carbon/nitrogen in microbial biomass was higher at medi-
Altitudinal gradients um altitude and was positively related to carbon and nitrogen in both soil and grass biomass along the altitudinal
Illumina sequencing gradients. Soil bacterial alpha diversity was signicantly higher at medium altitude but fungal alpha diversity did
Soil microbial community not affected by altitudinal gradients; the effect of altitudinal gradients on bacterial beta diversity was larger than
Soil moisture that on fungal beta diversity, although both groups were signicantly affected by altitudinal gradients. Moreover,
Soil temperature
Alpha-proteobacteria, Beta-proteobacteria, and Gemmatimonadetes were signicantly more abundant in higher al-
titude than in lower altitude, both Acidobacteria and Actinobacteria signicantly declined with increasing altitude;
other bacterial taxa such as Chloroexi, Nitrospirae, Gamma-proteobacteria, and Delta-proteobacteria were signif-
icantly higher at medium altitudes. For fungal taxa, Basidiomycota and Ascomycota were the dominant phyla and
responded insignicantly to the altitudinal gradients. The responses of microbial alpha diversity were mostly as-
sociated with plant Shannon index, organic carbon, and total nitrogen, whereas microbial beta diversity and com-
position mainly depended on soil moisture and temperature. Overall, these results suggest that soil bacteria
rather than fungi can reect changes in plant and soil characteristics along altitudinal gradients.
2017 Elsevier B.V. All rights reserved.

Corresponding authors at: College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi, China.
E-mail addresses: hanxinhui@nwsuaf.edu.cn (X. Han), ygh@nwsuaf.edu.cn (G. Yang).

http://dx.doi.org/10.1016/j.scitotenv.2017.08.110
0048-9697/ 2017 Elsevier B.V. All rights reserved.
 C. Ren et al. / Science of the Total Environment 610611 (2018) 750758 751

available substrates and the above-ground plant community.

Abbreviations
Serna-Chavez et al. (2013) reviewed that moisture availability and
soil nutrients rather than temperature primarily drove changes in
QVA Q aliena var. acutiserrata community
soil microbial biomass. However, other studies indicated that
QW Quercus wutaishanica community
climate-induced changes in soil temperature and moisture had no
AF Abies fargesii community
effects on Gram-positive bacteria because they have high tolerance
LC Larix chinensis community
and can spatially explore the soil for areas with increased water
and nutrients (Manzoni et al., 2012). While Shen et al. (2016) also
revealed that microbial functional genes dramatically increased
Plant characteristics
along an elevation gradient, but the bacterial taxonomic and phylo-
pshannon ShannonWiener diversity index
genetic diversity based on 16S rRNA gene sequencing did not exhibit
GB grass biomass
such a similar trend. Therefore, this leads to the further hypothesis
DBH diameter at breast height
that altitudinal gradients might have differential effects on soil
TCD tree crown density
microbial biomass and composition and the microbial process in
SCD shrub crown density
different ecosystems.
GC grass coverage
Recently, although several studies revealed the horizontal distribu-
tion of microbial communities (Bahram et al., 2012; Meng et al.,
2013), few studies have examined microbial diversity patterns along
Soil characteristics
the altitudinal gradients, especially in large altitudinal scales. A previous
SOC soil organic carbon
study found that soil bacterial richness signicantly decreased from
TN total nitrogen
lowest altitude (2460 m) to highest altitude (3380 m) in the Colorado
NH+ 4 ammonium
Rocky Mountains (Bryant et al., 2008), whereas another study found
NO 3 nitrate
that soil bacteria at medium altitude (820 m) showed the highest diver-
BD Soil bulk density
sity in subtropical mountainous forest of China (Meng et al., 2013).
ST soil temperature
Bahram et al. (2012) revealed that fungal diversity decreased monoton-
SM soil moisture
ically with increasing altitudes in the hyrcanian forests of northern Iran,
MBC microbial biomass carbon
however, Susan et al. (2015) observed an insignicant response of fun-
MBN microbial biomass nitrogen
gal community diversity along a short (300 m) altitudinal gradient in an
NMDS Nonmetric multidimensional scaling analysis
area Scots pine (Pinus sylvestris L.). These differences lead to the hypoth-
ANOSIM analysis of similarities
esis that spatial and elevation scales affect temperature uctuations and
climate types, which account for the different patterns of microbial rich-
ness and community (Classen et al., 2015). Particularly, large altitude
scales are associated with different climate zones and may contain
other complex abiotic and biotic factors, which greatly limit our under-
1. Introduction standing of microbial community dynamics along the altitudinal
gradients.
Altitudinal gradients are characterized by drastic changes in biotic To comprehensively understand changes in microbial biomass, di-
and abiotic characteristics over geographical distances, and thus have versity (bacteria and fungi), and community composition along altitudi-
been recognized as a natural experiments to assess ecological and evo- nal gradients, we examined large altitudinal gradients at Taibai
lutionary responses of the microbial community to changing environ- Mountain, which is the highest mountain in the Qinling Range of east-
ments (Krner, 2007; Siles and Margesin, 2017). It is presently fueled ern mainland China, and the elevation changes from 470 to 3767 m
by our emerging understanding of the complex interactions between above sea level (Tang and Fang, 2006). Several studies performed in
soil conditions and plant community in relation to changing altitude, this range have revealed the contrasting altitudinal above-plant diversi-
which can improve the predication for microbial process (Siles and ty patterns along large climate gradients (Qiao et al., 2015; Tang et al.,
Margesin, 2016; Sundqvist et al., 2013). Moreover, climatic factors, indi- 2012). However, soil microbial community responses to this type of al-
cated by air temperature, precipitation and UV radiation, can also vary titudinal/climatic gradient and associated climate zones were not well-
with altitudes, thus creating complex environmental changes along understood. In this study, we used fumigationextraction and Illumina
the altitudinal gradients (Krner, 2007). Under such circumstances, mi- sequencing to analyze the changes in soil microbial biomass, diversity
crobial species, especially for aerobic microbes, are mostly affected be- (bacterial and fungal), and community (bacterial and fungal) composi-
cause of the changing oxygen concentration (Bahram et al., 2012). tion along four altitudinal gradients (average of 1364, 2060, 2741, and
Thus, the altitudinal gradients can provide a framework for understand- 3320 m above sea level), which covered three climate zones (warm
ing the biodiversity patterns in earth's major environmental gradients temperate zone, cold temperate zone, and alpine cold zone). Based on
and further predict the diversity loss patterns resulting from climate previous studies related to soil microbes in terrestrial ecosystems
change (Mayor et al., 2017) (Bryant et al., 2008; Van der Heijden et al., 2008), we hypothesized
Being an integral part of forest ecosystems, soil microorganisms can that soil microbial biomass can be used to reect the plantsoil system
mediate biogeochemical cycles in terrestrial ecosystems (Van der and that, compared to fungal community, bacterial community may
Heijden et al., 2008). Thus, in dynamic environments, small shifts in vary more along the altitudinal gradients. Moreover, we also predicted
soil microbe may lead to signicant changes of nutrient transformation that soil bacterial and fungal community compositions characterizing
in plant- soil system (Bragazza et al., 2015; Deng et al., 2016). This leads this large altitudinal scale would respond differently to climate condi-
to a hypothesis that soil microbe might reect the changes of above- tions and plant and soil characteristics. Therefore, the objectives of
and below-ground properties along the altitudinal gradients. Moreover, this study were to: (1) understand how microbial biomass responded
several studies also documented the differential responses of soil micro- to altitudinal gradients, (2) compare the responses of microbial (bacte-
bial community to climatic factors and analyzed how these changed rial and fungal) diversity and community composition to altitudinal gra-
with environmental conditions (Bryant et al., 2008; Miki, 2012). For dients, and (3) evaluate the relationships between the soil microbial
instance, Cregger et al. (2012) predicated that differential responses of community and plant and soil characteristics along the altitudinal
the soil microbial community to climate change were dependent on gradients.
752 C. Ren et al. / Science of the Total Environment 610611 (2018) 750758
2. Methods and materials
2.1. Study area description
The study area is located in the Taibai Mountain (1071910758E
and 33453410N, 3767 m above sea level (a.s.l.), which is the highest
mountain in the Qinling Range of eastern mainland China. The Qinling
Mountains range shows an east-to-west arrangement and has high ele-
vation in central China; this range constitutes a large obstacle for the
north and south movement of air masses, which critically affects climate
zone distribution in eastern China. According to the Foping climate sta-
tion (10759E, 3332N, 1088 m a.s.l.), the annual mean temperature
and precipitation are 11.4 C and 910.6 mm, respectively. With increas-
ing altitude, climate zones of the northern slope of Taibai Mountain
changes from warm temperature, cold temperature, to alpine cold;
the vegetation types (named by the dominant plant species) changes
from Quercus aliena var. acutiserrata community (12531856 m a.s.l.),
Quercus wutaishanica community (19602265 m a.s.l.), Betula albo-
sinensis community (23562700 m a.s.l.), Abies fargesii community
(27003150 m a.s.l.), Larix chinensis community (31503362 m a.s.l.)
to a Rhododendron capitatum shrub (N 3400 m a.s.l.) (Ren, 2012)
2.2. Experimental design, plant investigation, and soil sampling
In August 2016, investigation and sampling were collected along
four altitudes at approximately 700 m intervals on the northern slope
of Taibai Mountain at average elevations of 1364, 2060, 2742, and
3320; the experiment was designed to cover four vegetation types (Q
aliena var. acutiserrata community: QVA; Quercus wutaishanica commu-
nity: QW; Abies fargesii community: AF; Larix chinensis community: LC)
and three climate zones (temperature zone, cold temperature zone and
alpine cold zone) (Table S1). To account for altitude effects, three inde-
pendent replicate sites (50 50 m each) were designed at each altitude
because the distance between any two plots exceeded the spatial de-
pendence (b 13.5 m) of most soil variables. For plant investigation in
situ, ten 1 1 m quadrants were randomly selected at each site to deter-
mine the ShannonWiener diversity index, grass biomass (GB), diame-
ter at breast height (DBH), tree crown density (TCD), shrub crown
density (SCD), and grass coverage (GC). Leaves were collected from
major tress species to determine carbon and nitrogen. All plant charac-
teristics were measured as previously described (Zhang et al., 2016;
Zhao et al., 2015).
For soil sampling, after removing the litter layer, 10 replicate sam-
ples (top 010 cm) were collected in an S shape by using a standard
soil auger (5 cm inner diameter) and then homogenized to provide
one composite sample per replicated site. The samples were sieved
through a 2-mm screen and roots and other debris were removed. Dur-
ing this sampling process, in order to avoid contamination and keep
samples fresh, we also used sterile paper to wipe the remains that
were attached to the auger and screen before colleting the next soil
samples. A portion of each soil sample was immediately transported
to the laboratory to determine soil moisture. Subsamples for molecular
analysis were transported from the eld to the laboratory in a portable
refrigerator containing ice and then stored at 80 C. A portion of each
soil sample was stored at 4 C for dissolved organic matter and microbial
biomass analyses, and soil subsamples were air-dried and stored at
room temperature prior to physical and chemical analysis.
2.3. Analysis of plant characteristics and soil physicochemical properties
All grass biomass and leaf were dried at 60 C until a constant weight
and the relative biomass of grass biomass was determined by weight
(g/cm3). The C and N content in both grass biomass and leaf were deter-
mined after grinding nely. The C content in both grass biomass and leaf
were determined using the K2Cr2O7 oxidation method, and the N con-
tent was determined using the Kjeldahl method (Bremner and
Mulvaney, 1982). Soil moisture (SM) was assessed by oven drying to
constant mass at 105 C, soil temperature (ST) at 10 cm depth was mea-
sured with a portable temperature probe. Soil pH, soil organic carbon
(SOC), and total nitrogen (TN) were performed as described by
(Zhang et al., 2011). The ammonium (NH+ 4 ) and nitrate (NO3 ) levels
were determined following extractions of fresh soil with 2 M KCl for
18 h and were analyzed colorimetrically using a continuous ow ana-
lyzer (Jones and Willett, 2006). Soil microbial biomass carbon and nitro-
gen were estimated from fresh soil samples using a chloroform
fumigation-extraction method as previously described (Vance et al.,
1987; Brookes et al., 1985). The fumigated and non-fumigated soil sam-
ples were extracted with 0.5 M potassium sulfate for 30 min, and the
measurements of microbial biomass carbon and nitrogen were deter-
mined using a TOC-TN analyzer (Shimadzu Corp.). Soil bulk density
(BD) was calculated from the gravimetric weight of the core before
and after oven drying at 105 C for 24 h and considering the individual
core volume (De Vos et al., 2005).
2.4. Soil DNA extraction, PCR amplication, and illumina sequencing
Microbial DNA was extracted from 0.5 g of fresh soil three times (for
a total of 1.5 g of soil) with E.Z.N.A soil DNA (OMEGA, USA), following
the manufacturer's instructions. The concentration and quality of the
DNA were assessed using a NanoDrop2000 spectrophotometer (Ther-
mo Scientic, Wilmington, DE, USA). PCR amplication of bacterial
16S rRNA targeting the V4 region was conducted by using primers
515F (5-GTGCCAGCMGCCGCGG-3) and 907R (5-CCGTC AATT
CMTTTRAGTTT-3) (Biddle et al., 2008). The fungal ITS-1 region was am-
plied by using primers ITS1F (50-ACTTGGTCATTTAGAG- GAAGTAA-
30) and ITS2 (50-BGCTGCGTTCTTCATCGATGC-30) (Mukherjee et al.,
2014). This primer set provides comprehensive coverage with the
highest taxonomical accuracy for bacterial and fungal sequences. The
PCR protocols that were used to amplify the 16S rRNA gene and ITS
rRNA genes were described previously (Mukherjee et al., 2014; Ren
et al., 2017). Finally, an equal amount of PCR product from each sample
was put in a single tube and sent to Illumina's MiSeq platform at Person-
al Biotechnology Co., Ltd. Shanghai, China.
Reads were demultiplexed, quality-ltered, and processed using
QIIME according to the following three criterions (Caporaso et al.,
2012). Sequence analysis was performed using the USEARCH v5.2.32
to lter and eliminate noise from the data by clustering similar se-
quences with b3% dissimilarity. Microbial Ecology pipeline software
was used to select 16S rRNA and ITS rRNA operational taxonomic
units from combining reads of clustered operational taxonomic units
with 97% similarity. Finally, the complete dataset was sent to the Se-
quence Read Archive (SRA) database of the National Center for Biotech-
nology Information (NCBI) under the accession numbers of SRP114805
for bacteria and SRP114821 for fungi.
2.5. Statistical analyses
Taxonomic alpha diversity was calculated as estimated community
diversity by the Shannon index using Mothur software (v.1.30.1).
Non-metric multi-dimensional scaling (NMDS) was used to illustrate
the clustering of different samples and further reecting the microbial
community structure, while the changes of microbial structure along
the altitudinal gradients was referred as microbial beta diversity. In ad-
dition, we used the analysis of similarities (ANOSIM) to determine the
signicance of separation along the climate gradients (Clarke et al.,
2014). Correlations between the soil microbial compositions and soil
characteristics were determined using Redundancy analysis (RDA)
(Clarke et al., 2014). The relationships between the microbial character-
istics (diversity and pupations) and the plant and soil properties were
performed by spearman correlation analysis.
Furthermore, the changes of soil properties, soil microbial biomass,
alpha diversity and microbial dominant phyla induced by altitudinal
 C. Ren et al. / Science of the Total Environment 610611 (2018) 750758 753

gradients were tested through one-way ANOVA using the R v.3.1.3 pro-
gram. The regression analysis were used to reect the relationship be-
tween the microbial biomass and plant characteristics (Shannon
Wiener diversity index, GB, DBH, TCD, SCD, GC, leaf C, leaf N, biomass
C, biomass N) and SOC and TN. P b 0.05 was considered statistically
signicant.
3. Results
3.1. Changes in plant and soil characteristics
The altitudinal gradients greatly affected plant and soil characteris-
tics, but showed differential effects (Table 1). For plant characteristics,
diameter at breast height (DBH) and shrub crown density (SCD) were
high at lower altitude with warm temperature; the plant Shannon
index (understory herb layers) was higher in medium altitude with
cold warm temperature, whereas grass coverage and biomass signi-
cantly increased with altitude (p b 0.01). For soil characteristics, soil
temperature signicantly decreased with increasing altitude (F(3,11) =
82.3, p b 0.01), ranging from 6.90 to 16.50 at high (warm temperature)
and low altitude sites (alpine cold zones), respectively. Soil moisture
increased with increasing altitude (F(3,11) = 52.83, p b 0.01), ranging
from 21.60% to 52.70% at the high and low altitudinal sites, respectively.
Soil bulk density was higher at low than at high altitude sites and
responded signicantly to the altitudinal gradients (F(3,11) = 54.58,
p b 0.01). However, the altitudinal gradients had no effect on C: N
ratio, NH+ 4 , and NO3 , and a slight effect on soil pH. Moreover, soil
microbial biomass carbon and nitrogen were higher in medium altitude
at cold temperatures and were positively related to carbon and nitrogen
in the grass biomass and soil, respectively, but was unrelated to leaf
carbon and nitrogen (Fig. 1)
3.2. Effect of altitudinal gradients on microbial diversity and their responses
to plant and soil characteristics
After quality sequencing, both bacterial communities (a total of
457,279 sequences) and fungal communities (a total of 685,249
paired-end sequences) were obtained using the 338F/806R (bacterial
16S rRNA) and ITS1F/ITS2 (fungal ITS) primer sets across all soil sam-
ples. The number of bacterial sequences varied from 29,455 to 48,650
per sample (mean = 38,106), whereas the number of fungal sequences
varied from 47,351 to 78,595 per sample (mean = 57,104). For down-
stream analysis of bacterial sequences, datasets were rareed to
29,400 sequences, whereas for the downstream analysis of fungal se-
quences, datasets were rareed to 57,000 sequences.
Fig. 2 showed that the altitudinal gradients signicantly affected soil
bacterial alpha diversity but had no effects on fungal alpha diversity.
Bacterial alpha diversity was higher at medium altitude and was
lower at low and high altitudes, ranging from 4.80 to 6.40; fungal
alpha diversity changed from 1.98 to 3.52. Nonmetric multidimensional
scaling analysis (NMDS) was conducted to reect microbial beta diver-
sity (Fig. 3). For bacteria, the sampling sites were distant from each
other and were greatly affected by the altitudinal gradients; for fungi,
the aggregation degrees of the sample points from A. fargesii (AF)
were distant from those of the L. chinensis (LC) sample points, and
both land use types were very distant from Q. wutaishanica (QW) and
Q. aliena (QVA). Analysis of similarities (ANOSIM) with 999 permuta-
tions revealed that the inuence of the altitudinal gradients on bacterial
beta diversity (ANOSIM, R = 0.998, p b 0.01) was higher than the inu-
ence on fungal beta diversity (ANOSIM, R = 0.843, p b 0.01).
In the analysis of plant and soil characteristics effects (Table 2), we
found that bacterial alpha diversity (Shannon index) was signicantly
related to the plant Shannon-Wiener, biomass nitrogen, SOC, and TN;
soil bacterial beta diversity (NMDS1) was signicantly related to GB,
DBH, TCD, GC, pH, ST, and SM. However, soil fungal alpha diversity
(Shannon index) was not affected by plant and soil characteristics,
and soil fungal beta diversity (NMDS1) was affected by leaf carbon,
NO 3 , BD, ST, and SM.
3.3. Effect of altitudinal gradients on microbial compositions and responses
to plant and soil characteristics
Among all sequences, the dominant bacterial phyla (relative abun-
dance N 1%) were Acidobacteria, Proteobacteria, Actinobacteria,
Chloroexi, Gemmatimonadetes, Nitrospirae, Planctomycetes,
Verrucomicrobia, and Bacteroidetes, with contributions of 27.67%,
29.78%, 13.88%, 6.67%, 6.21%, 5.83%, 1.59%, 2.24%, and 1.99%, respective-
ly (Fig. 4, Table S2). Notably, except for the phylum Verrucomicrobia, all
bacterial dominant phyla were signicantly affected by the altitudinal
gradients. Among them, Acidobacteria and Actinobacteria signicantly
decreased from low altitude to high altitude; both Proteobacteria and
Gemmatimonadetes signicantly increased with altitude; Chloroexi
and Nitrospirae were higher in medium altitude. Furthermore, at the
class level (Table S2), both Alphaproteobacteria and Betaproteobacteria
were the most dominant classes and signicantly increased with in-
creasing altitude (p b 0.05), but two other Proteobacteria branches,
Gammaproteobacteria and Deltaproteobacteria, were higher at medium
altitude and lower at both low- and high-altitudinal sites. At the order
level (Table S2), the Rhizobiales and Rhodospirillales, the branches of
Alphaproteobacteria, were the most abundant but showed different
trends along altitudinal gradients. In details, with increasing altitude,
the Rhizobiales was signicantly increased but the Rhodospirillales
was signicantly declined (p b 0.05); similarities, other two orders
(Caulobacterales and Rhodobacterales) in Alphaproteobacteria also

Table 1
Characteristics of the plant and soil along the altitudinal gradients.

Vegetation typesa QVA QW AF LC F(3,11) p

Diameter at breast height (cm) 23.50 2.60A 19.85 4.94A 17.13 4.35A 8.16 3.20B 8.52 0.01b
Tree crown density (%) 89.00 6.08AB 92.67 1.53A 84.33 3.51B 75.00 2.88C 14.08 b0.01c
Shrub crown density (%) 26.67 6.81A 13.33 3.06B 10.30 4.56B 13.33 5.77B 5.85 0.02b
Grass coverage (%) 6.67 2.89C 11.67 7.64BC 25.00 13.23B 81.67 5.77A 51.91 b0.01c
Grass biomass (g/cm3) 9.64 1.31C 40.66 14.52B 45.86 4.29B 72.01 0.34A 33.99 b0.01c
Shannon-Wiener 0.78 0.32B 2.47 0.35A 2.52 0.28A 1.22 0.17B 27.25 b0.01c
C: N 3.88 2.24A 1.31 0.76A 4.93 2.85A 4.41 2.54A 1.28 0.35
NH+4 (mg/kg) 68.31 7.95A 73.11 32.57A 84.57 4.20A 67.41 5.58A 0.21 0.89
NO3 (mg/kg) 4.54 0.46B 6.33 2.75B 21.75 6.62A 11.98 3.71AB 3.67 0.06
pH 6.06 0.04A 5.99 0.05AB 5.93 0.02B 5.92 0.02B 3.23 0.08
Bulk density (g/cm3) 1.45 0.04A 1.06 0.02B 1.03 0.02B 1.05 0.02B 54.58 b0.01c
Soil temperature (C) 16.07 0.34A 14.6 0.52B 10.43 0.35C 7.6 0.47D 82.3 b0.01c
Soil moisture (%) 25.97 2.22C 25.5 1.54C 41.7 1.03B 49.6 1.57A 52.83 b0.01c
a
Means the different vegetation types along four altitudinal gradients, including Q aliena var. acutiserrata community (QVA), Quercus wutaishanica community (QW), Abies fargesii
community (AF), and Larix chinensis community (LC). Capital letters indicate the signicant difference among different vegetation types (P b 0.05).
b
Correlation is signicant at the 0.05 level.
c
Correlation is signicant at the 0.01 level.
754 C. Ren et al. / Science of the Total Environment 610611 (2018) 750758

Fig. 1. The relationships between the microbial biomass (carbon and nitrogen) and the carbon and nitrogen in soil, biomass and leaf. The microbial biomass carbon was positively
and signicantly correlated with soil organic carbon (y = 0.0759x 35.0505, R2 = 0.9531, p = 0.0158) and biomass carbon (y = 0.1353x + 357.2431, R2 = 0.9601, p = 0.0134);
microbial biomass nitrogen was positively and signicantly correlated with total nitrogen (y = 0.0490x 4.3173, R2 = 0.9194, p = 0.0272) and biomass nitogen (y = 0.1156x
+ 2.0287, R2 = 0.8990, p = 0.0342). QVA, QW, AF, and LC represent the abbreviations of Q aliena var. acutiserrata community, Quercus wutaishanica community, Abies fargesii
community, and Larix chinensis community, respectively.

responded signicantly to altitudinal gradient (p b 0.05). The
Xanthomonadales and Myxococcales, the branches of Gammaproteobacteria
and Deltaproteobacteria, were signicantly higher in medium altitude.
Within Actinobacteria, The order Solibacterales was signicantly decreased
from low to high altitudes.
According to fungal community compositions, the dominant phyla
across all samples were Basidiomycota, Ascomycota, and Zygomycota,
with average contributions of 85.92%, 9.51%, and 3.63%, respectively
(Fig. 4 and Table S3). Further taxonomical classication revealed that
the Agaricomycetes was the dominant class, accounting for N90% of
the variation of the fungal classes (Table S3); the abundance of
Archaeorhizomycetes was signicantly higher at lower altitude (p =
0.01). Moreover, at the order level (Table S3), only Sordariales was sig-
nicantly higher at medium altitude (p b 0.001). Agaricales, Sebacinales,
and Russulales accounted for N 75% variations and other reads including
unknown taxa were not signicantly different among different plots
(mean = 13.58%). Overall,the altitudinal gradients had minor effect
on the fungal taxa across taxonomical levels (Phyla, Class, and Order)
(p b 0.05).
Redundancy analysis (RDA) showed that bacterial and fungal taxa
responded differently to changes in the plant and soil characteristics
at different taxonomic levels (Phylum, Class, and Order) (Fig. 5,
Fig. S3, S4, Table S4). These results demonstrated that plant characteris-
tics, particularly grass biomass, greatly affected soil bacterial communi-
ty composition, however, it had no effect on fungal community
composition. Soil characteristics, particularly soil temperature and
moisture, were responsible for much of the variations in bacterial com-
munity compositions rather than those observed in the fungal commu-
nity compositions (Table S4, Table S5). Fig. 5AB showed that grass
biomass, SCD and DBH in plant properties, and ST, SM, and BD in soil
properties were signicantly related to changes in Proteobacteria,
Nitrospirae, Bacteroidetes, Chloroexi, Acidobacteria, Actinobacteria, and
Planctomycetes. For Proteobacteria, grass biomass, ST and SM were asso-
ciated with changes in the abundance of Rhizobiales, Rhodospirillales,
Rhodobacterales, and Burkholderiales (Fig. S4), which were the branches
of Alphaproteobacteria and Betaproteobacteria, respectively (Table S2).
However, in contrast, among the plant and soil properties, only SM
was positively and signicantly correlated with Zygomycota but had
no effect on the abundance of Basidiomycota and Ascomycota
(Fig. 5CD). Among Ascomycota, only Sordariales was positively correlat-
ed with SOC, while Archaeorhizomycetes and Pezizomycetes classes and
their respective orders were also sensitive to changes of SM (Fig. S3, 4).
 C. Ren et al. / Science of the Total Environment 610611 (2018) 750758
Fig. 2. Changes of microbial (bacterial and fungal) alpha diversity (Shannon index)
along the altitudinal gradients. Different letters represent differences among different
altitudes. QVA, QW, AF, and LC represent the abbreviations of Q aliena var. acutiserrata
community, Quercus wutaishanica community, Abies fargesii community, and Larix
chinensis community, respectively.
4. Discussion
4.1. Synergistic responses of carbon and nitrogen in plantsoilmicroor-
ganism continuum to altitudinal gradients
Altitudinal gradients contributed to the distribution of vegetation
types and led to differential responses of soil microorganism in climatic
conditions (Bragazza et al., 2015; Bryant et al., 2008). The direction and
magnitude of such differential responses were associated with plant di-
versity (Thakur et al., 2015), which were also reected in current study.
Soil microbial biomass was higher at medium altitude and
corresponded the similar trends with higher plant Shannon index
along the altitudinal gradients (Fig. 1 and Fig. S1 and S2). This may be
because the higher plant diversity might be expected to have higher
root trait diversity, and which in turn results in the exudation of a
more diverse range of organic compounds into the soil, thereby sustain-
ing higher microbial biomass (Reynolds et al., 2003; Thakur et al., 2015).
In addition, it was generally accepted that belowground microbial com-
munity can affect the above-ground plant community by carrying out a
Fig. 3. Changes of soil bacterial (a) and fungal (b) beta diversity along the altitudinal
gradients. QVA, QW, AF, and LC represent the abbreviations of Q aliena var. acutiserrata
community, Quercus wutaishanica community, Abies fargesii community, and Larix
chinensis community, respectively.
755
wide spectrum of decomposition processes (Van der Heijden et al.,
2008; Miki, 2012). Thus, it was not surprising that a signicant relation-
ship between microbial biomass (MBC and MBN) and the C/N in grass
biomass and soil along was observed along the altitudinal gradients
(Fig. 1). However, no relationship was observed between microbial bio-
mass and leaf. These different effects can be ascribed to the physiologi-
cal characteristics of leaf, since changes in the tree leaves might compete
with the limited amounts of mineral nutrients with belowground biodi-
versity, and thus may cause unexpected changes in the microbial de-
composition (Miatto et al., 2016; Miki, 2012; Miki and Doi, 2016).
Altogether, these suggest supported the rst hypothesis that soil micro-
bial biomass can reect the carbon and nitrogen in plant-soil systems
along altitudinal gradients, but respond differently to plant tissues.
4.2. Differential responses of microbial diversity to altitudinal gradients de-
pend on plant and soil characteristics
Along the altitudinal gradients, differences in plant and soil charac-
teristics can explain the soil bacterial and fungal community character-
izing each altitudinal site (Bragazza et al., 2015; Siles and Margesin,
2017). Our results conrmed that bacterial alpha diversity was higher
at medium altitude but it was difcult to predict fungal alpha diversity
along the altitudinal gradients (Fig. 2); this was because altitudinal
gradient-induced changes of plant Shannon index, soil organic carbon,
and total nitrogen largely affected bacterial but not fungal alpha diversi-
ty (Table 2). The bacterial responses were suggested by the previous
ndings (Meng et al., 2013; Shen et al., 2013; Siles and Margesin,
2016). However, the fungal alpha diversity responses to climate gradi-
ents contradicted some of the previous studies (Bahram et al., 2012;
Siles and Margesin, 2016; Tang et al., 2012) and it was likely that
fungi responded to a combination of different variables (Susan et al.,
2015). These results proved the second hypothesis that soil bacterial
alpha diversity, compared with fungal alpha diversity, varied more
along the altitudinal gradients, and such responses were associated
with plant Shannon index, soil organic carbon, and total nitrogen.
Moreover, analysis of the community structure response to altitudi-
nal gradients revealed that soil microbial beta diversity changed signif-
icantly with the altitudinal gradients (Fig. 3) and was highly related to
soil temperature and moisture among environmental variables
(Table 2). Possible explanation can be ascribed to either changing eco-
logical strategy or suppressed oxygen supply for aerobic microbes, ulti-
mately driving differences in the microbial community (Meng et al.,
2013; Allen et al., 2002; Fierer et al., 2007). In addition, we also detected
that changes in soil pH and soil bulk density along the altitudinal gradi-
ents were responsible for differences in bacterial beta diversity
(Table 2), which was supported by the results of previous studies
(Ludwig et al., 2015; Shen et al., 2013). In contrast, we found that soil
bacterial beta diversity responded signicantly to plant characteristics
(i.e., grass biomass and coverage, diameter at breast height, and tree
crown density), whereas fungi did not (Table 2), suggesting that soil
bacterial beta diversity was more variable than fungal beta diversity
and can reect changes in plant diversity, community, and biomass
along the altitudinal gradients. Overall, these results demonstrated
that altitudinal gradients had major effects on soil bacterial beta diver-
sity but minor effects on fungal beta diversity, and such differential re-
sponses depended on climatic factors (temperature and moisture) and
plant characteristics.
4.3. Differential responses of microbial compositions to altitudinal gradients
depend on soil temperature and moisture
The positive response of soil microbial diversity to the altitudinal
gradients and their response to plant and soil characteristics are de-
scribed above; whether such responses represent a community-wide
response or are accompanied by changes in certain microbial taxa was
previously unclear. In this study, we found that the altitudinal gradients
756 C. Ren et al. / Science of the Total Environment 610611 (2018) 750758

Table 2
Spearman's rank correlation coefcients (R) between microbial diversity (i.e., alpha diversity: Shannon index; beta diversity: NMDS1) and the plant and soil characteristics.

Characteristics Bacterial diversity Fungal diversity

Shannon index NMDS1 NMDS2 Shannon index NMDS1 NMDS2

Plant Shannon-Wiener 0.720b 0.224 0.825b 0.182 0.406 0.357

Grass biomass (GB) 0.354 0.925c 0.217 0.112 0.480 0.053
Diameter at breast height (DBH) 0.245 0.860c 0.182 0.245 0.469 0.063
Tree crown density (TCD) 0.179 0.743b 0.294 0.182 0.417 0.060
Shrub crown density (SCD) 0.559 0.409 0.491 0.018 0.527 0.434
Grass coverage (GC) 0.244 0.881c 0.096 0.209 0.531 0.011
Leaf carbon 0.559 0.413 0.357 0.161 0.664a 0.455
Leaf nitrogen 0.196 0.035 0.252 0.042 0.280 0.091
Biomass carbon 0.476 0.469 0.671a 0.077 0.503 0.007
Biomass nitrogen 0.685a 0.224 0.755b 0.119 0.063 0.322
Soil organic carbon 0.664a 0.343 0.734b 0.112 0.168 0.063
Total nitrogen 0.762b 0.217 0.853c 0.503 0.112 0.014
C: N 0.378 0.273 0.350 0.483 0.196 0.056
NH4 + 0.476 0.028 0.294 0.182 0.091 0.580a
NO3- 0.441 0.531 0.259 0.049 0.650a 0.657a
pH 0.441 0.669a 0.165 0.011 0.333 0.291
Bulk density (BD) 0.547 0.575 0.621a 0.226 0.660a 0.173
Soil temperature (ST) 0.203 0.909c 0.119 0.084 0.622a 0.203
Soil moisture (SM) 0.028 0.839b 0.028 0.091 0.741b 0.217
a
Correlation is signicant at the 0.05 level. Nonmetric multidimensional scaling analysis (NMDS).
b
Correlation is signicant at the 0.01 level.
c
Correlation is signicant at the 0.001 level.

had differential effects on bacterial and fungal community compositions
(Fig. 4, Table S2, Table S3), such differential responses can be largely ex-
plained by ST and SM rather than plant and soil properties (Fig. 5,
Table S4)
With respect to bacterial compositions, Acidobacteria was less abun-
dant when the soil was moister and cooler (Lauber et al., 2013; Placella
et al., 2012), thus the higher ST and lower SM at lower altitude might
promote Acidobacteria abundance (Fig. 5). The Proteobacteria signi-
cantly increased with altitude and was also largely affected by ST and
SM but not by measured SOC, despite that Proteobacteria was the carbon
availability preferences of this phylum (Fierer et al., 2007). This may be
related to the composition of Proteobacteria along the altitudinal gradi-
ents (Fig. S3 and Fig. S4). At the class level, Beta-Proteobacteria was
typically present at low abundance in dry soil (Lipson, 2006) and thus
was more sensitive to SM (Fig. S3). At the order level, both Rhizobiales
and Rhodospirillales, the branch of Alpha- Proteobacteria, were also sig-
nicantly correlated with ST and SM (Fig. S4), further supporting the
variations of Proteobacteria and their response to ST and SM along the
altitudinal gradients. The decrease in Actinobacteria with altitude was
related to changes in ST, SM, pH, and lower soil properties (Fig. 4,
Fig. 5, Table S2), and this phylum was adapted to resource-limited con-
ditions (Siles and Margesin, 2016; Stroobants et al., 2014). In addition,
other specic taxa, especially Gemmatimonadetes and Planctomycetes,
showed mostly closed to changes of SM and ST (Fig. 5), and these pat-
terns were likely due to their ecological strategies (DeBruyn et al.,
2011; Ivanova et al., 2016). Therefore, these results suggested that

Fig. 4. Changes of microbial (bacterial and fungal) taxonomic composition along altitudinal gradients. Mean relative abundances of taxa within site. The abundance of each taxon was
calculated as the percentage of sequences per gradient for a given bacterial group. Acidobacteria (Acid), Proteobacteria (Prot), Actinobacteria (Acti), Chloroexi (Chlo), Gemmatimonadetes
(Gemm), Nitrospirae (Nitr), Planctomycetes (Plan), Verrucomicrobia (Verr), Bacteroidetes (Bact); Basidiomycota (Basi), Ascomycota (Asco), Zygomycota (Zygo). QVA, QW, AF, and LC
represent the abbreviations of Q aliena var. acutiserrata community, Quercus wutaishanica community, Abies fargesii community, and Larix chinensis community, respectively.
 C. Ren et al. / Science of the Total Environment 610611 (2018) 750758 757

Fig. 5. Ordination plots of the results from the redundancy analysis (RDA) to identify the relationships among the microbial (bacterial and fungal) taxa (Black arrows) and the
plant and soil characteristics (Red arrows). A and B: the relationships between the soil bacterial taxa and the plant and soil properties; these are the abbreviations of bacterial taxa:
Acidobacteria (Acid), Proteobacteria (Prot), Actinobacteria (Acti), Chloroexi (Chlo), Gemmatimonadetes (Gemm), Nitrospirae (Nitr), Planctomycetes (Plan), Verrucomicrobia (Verr),
Bacteroidetes (Bact); C and D: the relationships between the soil fungal taxa and the plant soil properties; these are the abbreviations of bacterial taxa: Basidiomycota (Basi),
Ascomycota (Asco), Zygomycota (Zygo). The abbreviations of plant and soil characteristics: plant Shannon-Wiener (Plant Shannon), Diameter at breast height (DBH), Tree crown
density (TCD), Shrub crown density (SCD), Grass coverage (GC), Grass biomass (GB), soil organic carbon (SOC), total nitrogen (TN), bulk density (BD), soil temperature (ST), and soil
moisture (SM). QVA, QW, AF, and LC represent the abbreviations of Q aliena var. acutiserrata community, Quercus wutaishanica community, Abies fargesii community, and Larix
chinensis community, respectively. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

differential responses of soil bacterial community composition to the al-
titudinal gradients were largely dependent on climate factors (soil tem-
perature and moisture) rather than on the dynamics of soil carbon and
nitrogen.
For fungal community composition, the dominant phyla along the
altitudinal gradients (basically comprised of Basidiomycota, Ascomycota,
and Zygomycota) was in line with that described for temperate forests at
global scale (Tedersoo et al., 2014) (Fig. 4). However, these dominant
phyla responded insignicantly to climate-induced changes in vegeta-
tion types (Table S3) and were mainly affected by SM (Table S4).
These results conicted with the functions of dominant fungal phyla;
in detail, Basidiomycota and Ascomycota metabolized the organic sub-
strates of rhizodeposition (Clemmensen et al., 2013) and their abun-
dances were affected by soil organic matter dynamics as a result of
decomposition of plant residues (Hannula et al., 2012). Possible expla-
nation is that soil moisture and temperature may be the primary and
potential factors affecting soil fungal community composition, and the
effect of climate factors (moisture and temperature) on the soil fungal
community composition was larger than the effect of soil nutrients
(Bahram et al., 2012; Cregger et al., 2012) (Fig. 5, Table S4). In addition,
among the top most abundant classes, only one that showed a signi-
cant correlation with altitude was Archaeorhizomycetes, and here the
shift with altitude was negatively governed by ST and SOC (Fig. S3), sug-
gesting that Archaeorhizomycetes might be stimulated at relatively high
temperature and was shown to be able to grow in carbon-rich condi-
tions (Rosling et al., 2011). At the order level, we detected that the
Sordariales was also signicantly higher in medium altitude and posi-
tively correlated with SOC (Fig. S4, Table S3), this was because the
Sordariales had the ability to decompose the plant debris and as a result
increasing the soil carbon (Wang et al., 2017). The large variations of
Agaricales and Cantharellales in class Agaricomycetes can be explained
by their close relationships with SM, despite they were not affected by
altitudinal gradients, further supporting the above discussions that SM
could cause the changes of soil fungal community compositions along
the altitudinal gradients. Together with bacterial community composi-
tion responses, these ndings claried the third hypothesis that soil mi-
crobial (bacterial and fungal) community composition were majorly
affected by ST and SM and minorly affected by soil nutrients levels
along the altitudinal gradients.
5. Conclusions
These results have demonstrated that altitudinal gradients in a large
scale has caused different alterations in microbial biomass, diversity
(bacterial diversity), and community compositions. Soil microbial bio-
mass dynamics can reect the carbon and nitrogen content in the
plant-soil systems along the altitudinal gradients; however, it
responded differently to plant traits. From the analysis of the patterns
of microbial diversity, our results indicated that bacterial alpha diversity
was higher in the sites at medium altitude, and varied more than the
fungal alpha diversity along the altitudinal gradients; these differential
responses were mainly dependent on the dynamics of plant diversity,
SOC, and TN. The changes in soil temperature and moisture induced
by altitudinal gradients were considered as the potential factors for
the prediction of microbial beta diversity but with different effects,
which can be explained by the fact that changes in microbial communi-
ty composition, especially for bacterial composition, were mainly de-
pendent on SM and ST along the altitudinal gradients. Our ndings
758 C. Ren et al. / Science of the Total Environment 610611 (2018) 750758
highlighted the different patterns of microbial communities, such as
bacteria and fungi to altitudinal gradients, and further shed light on
the microbial biodiversity and their ecological roles in climate change.
Acknowledgements
The authors especially thank Yadong Xu (Northwest A&F University)
for their help with the eldwork and this work was supported by the
National Natural Science Foundation of China (No. 41571501) and the
State Key Laboratory of Soil Erosion and Dryland Farming on Loess Pla-
teau (No. A314021402-1709).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2017.08.110.
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