Environmental and Experimental Botany 134 (2017) 21–32
Contents lists available at ScienceDirect
Environmental and Experimental Botany
journal homepage: www.elsevier.com/locate/envexpbot
Nitrogen enrichment alters plant N: P stoichiometry and intensifies
phosphorus limitation in a steppe ecosystem
Shuxia Zhana , Yang Wanga,b , Zhicheng Zhua,b , Wenhuai Lia , Yongfei Baia,*
a
b
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences, Beijing, 100093, China
University of Chinese Academy of Sciences, Beijing 100049, China
A R T I C L E I N F O
Article history:
Received 11 May 2016
Received in revised form 18 October 2016
Accepted 22 October 2016
Available online 22 October 2016
Keywords:
N and P limitation
N: P stoichiometry
Rhizosphere effect
Plant-soil feedbacks
Water and light availability
Primary production
Soil N and P availability
A B S T R A C T
Although many studies have demonstrated that N deposition decreases biodiversity and alters ecosystem
functioning, fewer studies have tested how N enrichment affect plant N and P limitation, N: P
stoichiometry and ecosystem functioning. We examined the independent and interactive effects of N and
P enrichment on plant N: P stoichiometry, nutrient limitation, and thereby ecosystem functioning based
on two N, P, and N + P addition experiments in a typical steppe. At the species level, N enrichment
increased leaf N: P ratio and P limitation of dominant species. The responses of plant tissue N: P ratio tend
to saturate at soil available N: P supply ratio of approximately 20 for leaf N: P and 10 for root N: P ratio. At
the community level, patterns of N and P limitation shifted from N limitation in a normal year to N and P
co-limitation in a wet year, triggered mainly by inter-annual changes in soil N and P availability. The
homoeostasis of N: P stoichiometry increases from plant leaves to roots and to microbes although the
available N: P supply ratio varied by 56-fold in soil. Given that N deposition rates are projected to increase
in upcoming decades, N deposition may further alter the stoichiometric balance of N and P and intensify P
limitation of steppe ecosystems in future.
ã 2016 Elsevier B.V. All rights reserved.
1. Introduction
The cycles of nitrogen (N) and phosphorus (P), both of which are
key limiting nutrient elements controlling plant growth and
primary production in most terrestrial ecosystems, have been
substantially altered by human activities (Aerts and Chapin, 2000;
Elser et al., 2007; Gleeson and Tilman, 1992; Harpole et al., 2011).
Human activities have doubled N availability globally over the
recent several decades through industrial and agricultural
processes (Bobbink et al., 2010). The elevated N availability may
alter the stoichiometric balance of nutrient elements, increase the
ratio of N to P in plants or soils, and potentially intensify ecological
limitation of P relative to N (Crowley et al., 2012; Vitousek et al.,
2010). Meanwhile, long-term atmospheric N deposition has lead to
widespread soil acidification, which may increase soil P availability
and thereby indirectly alleviate P limitation (Galloway et al., 2008;
Stevens et al., 2010). A previous study on the relationship of plant
* Corresponding author at: State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences, 20 Nanxincun,
Xiangshan, 100093, Beijing, China.
E-mail address: [email protected] (Y. Bai).
http://dx.doi.org/10.1016/j.envexpbot.2016.10.014
0098-8472/ã 2016 Elsevier B.V. All rights reserved.
diversity to the type of nutrient limitation suggests that P
limitation can contribute to species coexistence and the maintenance of plant diversity, and more endangered plant species persist
under P-limited than N-limited conditions in herbaceous terrestrial ecosystems of the Eurasian region (Wassen et al., 2005).
Atmospheric N deposition and N-induced soil acidification are
simultaneously altering the patterns of nutrient limitation and
biogeochemical cycling in many terrestrial ecosystems, such as
grassland and cropland (Crowley et al., 2012; Guo et al., 2010; Yang
et al., 2012). Thus, it is critical to understand how N and P
enrichment independently and interactively affect the patterns of
plant N and P limitation and consequently ecosystem functioning
(e.g. primary production).
The primary production of individual plants and communities
is limited by N and/or P in most terrestrial ecosystems, as indicated
by nutrient addition experiments (Aerts and Chapin, 2000; Chapin
et al., 1986). Plant production responses to high nutrient additions
that can not only saturate chemical and microbial immobilizations
but also meet the requirements of plants are commonly used for
assessing nutrient limitation in communities (Chapin et al., 1986).
If addition of an available nutrient causes increases in plant
responses such as primary production or tissue nutrient
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S. Zhan et al. / Environmental and Experimental Botany 134 (2017) 21–32
concentrations, then the plant or plant community is considered to
be limited by that nutrient (Chapin et al., 1986; Elser et al., 2007;
Vitousek et al., 2010). Plant growth responses following nutrient
addition indicate nutrient availability sufficient to saturate the
capacity for both soil and microbial nutrient immobilization, and
to meet the requirements of plants (Chapin et al., 1986).
Simultaneous co-limitation occurs if plant community biomass
increases only with addition of N and P together, while
independent co-limitation indicates a response to N or P added
individually (Harpole et al., 2011). Serial limitation (synergistic
limitation) suggests a combined response, where biomass
increases with N or P added alone, and increases further when
N and P are added together (Allgeier et al., 2011; Harpole et al.,
2011). It is important to differentiate between the different types of
co-limitation for predicting plant community responses to N and P
enrichment and for elucidating the potential mechanisms underlying the patterns of nutrient limitation.
Nitrogen-to-phosphorus (N: P) mass ratios of plant leaves and
biomass have been widely used to infer or assess the type of N or P
limitation at the community level when they are calibrated by
fertilization experiments (Güsewell, 2004; Koerselman and
Meuleman, 1996). For, example, plant growth and biomass
production are considered N-limited when leaf N: P ratio <14
(or community biomass N: P ratio <10), P limited when leaf N: P
ratio >16 (or community biomass N: P ratio >20), and co-limited by
N and P when leaf N: P ratios are between 14 and 16 (Güsewell,
2004; Koerselman and Meuleman, 1996). However, root N: P ratio
has rarely been used as an indicator of nutrient limitation. This is
because plants could allocate nutrients to meet the requirements
of leaf photosynthesis in nutrient limiting environments, resulting
in apparent inconsistencies in leaf and root N: P ratios (Aerts and
Chapin, 2000; Li et al., 2010). Furthermore, the indicators for
assessing N and P limitation are frequently complicated by other
factors, such as water and light availability, and interactions of N
and P (Güsewell, 2004; Vitousek et al., 2010). The multipleresource limitation hypothesis proposes that the increase in
biomass production in response to addition of one of several
resources can be arisen from plant physiological processes,
positive interactions among resources, and limitation of different
species by different resources (Vitousek et al., 2010). Plant resource
use strategies, limitation of different species by different resources
within an ecosystem, and synergistic or antagonistic interactions
between multiple limiting resources may all affect communitylevel biomass responses (Bloom et al., 1985; Harpole et al., 2011;
Vitousek et al., 2010).
Plants adapted to long-term low P environments can elevate
inorganic P concentrations in the rhizosphere through root
exudates (e.g. oxalate, citrate and malate), stimulation of root
growth, and increase in activity of soil microorganisms (Hinsinger,
2001; Jones, 1998; Marklein and Houlton, 2012; Richardson and
Simpson, 2011; Ström et al., 2005). Among these mechanisms,
rhizosphere acidification is an important process that can directly
affect the bioavailability of soil inorganic P availability to plants
(Hassan et al., 2012; Hinsinger, 2001). Plants exude carboxylates to
activate rhizosphere P availability and result in lower pH in the
rhizosphere than that in non-rhizosphere soils (Jones, 1998;
Tadano and Sakai, 1991); although the contribution of this process
to rhizosphere acidification is a matter for debate. Nitrogen
enrichment can stimulate phosphatase activity and thereby
increases soil inorganic P to plants (Marklein and Houlton,
2012). Nitrogen enrichment can also induce soil acidification,
which increases concentrations of diffusible inorganic phosphate
ions and extractable P (Malhi et al., 2003; Stroia et al., 2011). Few
studies, however, have comprehensively examined how rhizosphere effects and soil available P are influenced by N and P
enrichment.
Temporal scale is also an important dimension for understanding plant species, community and soil responses to N and P
enrichment because abiotic (e.g. water and light availability) and
biotic (shift in species composition) factors may vary among
different years (Bai et al., 2010, 2008; Sala et al., 2012). In arid and
semi-arid grasslands, precipitation is a key factor controlling the
interannual variation in primary production and plant community responses to N and P additions (Bai et al., 2008, 2004). It is also
important to note that multiple-year nutrient additions may
cause changes in species composition, which influence aboveground productivity responses to N and P (Avolio et al., 2014). A
recent study indicates that reduction in species richness and
changes in composition occurred after three years of continuous
additions of N in the Inner Mongolian grassland (Bai et al., 2010).
Therefore, the appropriate experiment would be annual additions
of N and P on new plots to test whether a species or community is
N- or P-limited, while controlling for potentially interacting
factors such as precipitation or species composition, which
change over time and may influence whether a system is limited
by N or P.
To test how N and P enrichment independently and interactively affect the patterns of plant N and P limitation and ecosystem
functioning, we carried out two independent N, P, and N + P
addition experiments in a typical steppe of the Inner Mongolia
grassland. Specifically, we address three questions: First, how do
patterns of N and P limitation vary with N and P enrichment and
environmental conditions (i.e. growing season precipitation,
photosynthetically active radiation, and soil N and P availability)?
Second, how do plant species respond to N and P enrichment in
terms of foliar and root N and P concentrations and N: P
stoichiometry? Third, how do N and P enrichment affect soil
acidity, species’ rhizosphere effects on P availability, and soil
microbial biomass N, P, and N: P stoichiometry?
2. Materials and methods
2.1. Study site
This study was conducted at the Inner Mongolia Grassland
Ecosystem Research Station (IMGERS, 43 380 N, 116 420 E) of the
Chinese Academy of Sciences, located in the Xilin River Basin, Inner
Mongolia Autonomous Region of China (Bai et al., 2004). Mean
annual precipitation (1970–2010) is 334.6 mm, with about 60–80%
of it occurring in the main growing season (May to August). Mean
annual temperature is 0.5 C and mean monthly temperatures
range from 21.5 C in January to 19.2 C in July. The soil is
classified as dark chestnut (Calcic Chernozem according to ISSS
Working Group RB, 1998). The study site has been fenced from
grazing by domestic animals since 1999. The plant community was
dominated by Leymus chinensis (perennial rhizomatous grass) and
Achnatherum sibiricum (perennial bunchgrass, PB), together
accounting for more than 70% of the total aboveground biomass.
Other species include Stipa grandis (PB), Agropyron cristatum (PB),
Cleistogenes squarrosa (PB), Carex korshinskyi (sedge species),
Chenopodium glaucum (annual forbs).
During the two years of the experiment, annual precipitation
was much higher in 2012 (514.2 mm) than in 2011 (286.7 mm). The
growing season precipitation (May to August) was 234.1 mm in
2011, which is 6% lower than the long-term mean value
(249.1 mm). May-August precipitation was 343.6 mm in 2012,
which is 40% higher than the long-term mean value (Fig. S1 in
Supporting information). Patterns of mean monthly temperature
were similar between the two years. However, photosynthetically
active radiation (PAR) in June, July and August was significantly
higher in 2011 than in 2012 (F3,180 = 146.51, P < 0.0001; Fig. S1,
Supporting information).
S. Zhan et al. / Environmental and Experimental Botany 134 (2017) 21–32
2.2. Experimental design
In early June 2011, 44 plots (each 1.5 m 1.5 m) were laid out in
a 20 m 20 m area and separated by 1-m walkways. Each plot was
fenced with a 25-cm height iron sheet, of which 20 cm was driven
into soil. The experiment was a completely randomized design,
including N, P, N and P (i.e. N + P) addition treatments, and ambient
treatment (control). There were 10 replicates for each treatment
except for control, which had 14 replicates. Nitrogen was added as
commercial pelletized NH4NO3 fertilizer at the rate of 10.5 g N m2
yr1. Phosphorus was added at the rate of 13.9 g P m2 yr1 as
commercial calcium superphosphate (i.e. Ca(H2PO4)2H2O, in
which available P2O5 content is 16%). The N and P addition rates
correspond to levels assumed to be sufficient to overcome plant N
and P limitations (Avolio et al., 2014; Bai et al., 2010; Yu et al., 2010).
In the early growing season (early June) of 2011 and 2012, N and P
were uniformly applied to each treatment plot with manual
broadcasting (Lan and Bai, 2012).
To test the effect of precipitation by nutrient addition
interaction, we conducted the second experiment (new plots)
adjacent to the first experiment with the identical experiment
design in 2012. The first year data of the two experiments allow us
to assess how fluctuations in precipitation (e.g. wet versus dry
years) will affect plant community responses to N and P additions,
without considering any shift in species composition due to the
nutrient additions. Furthermore, the community biomass data of
the two experiments in the same year (i.e. 2012) allow us to
explore the first- versus second-year (cumulative) effects of N and
P additions on primary production under the identical precipitation regime.
2.3. Vegetation survey and plant tissue N and P concentrations
Aboveground vegetation was sampled in mid August each year
by clipping all plants at the soil surface using a 0.5 m 0.5 m
quadrat within each plot. All vascular plants were sorted to species,
oven-dried at 70 C for 48 h, and weighed. The total dry mass of all
living species per quadrat averaged over all replicates of each
treatment was used to estimate the aboveground community
biomass, which approximated to the aboveground net primary
production of this system (Bai et al., 2004). Leaf and root samples of
L. chinensis and A. sibiricum within each plot were also collected,
oven-dried at 70 C for 48 h and ground for N and P analysis. Total N
in plant leaf and root samples was determined following Kjeldahl
digestion by a Nitrogen Analyzer System (KJELTEC 2300 AUTO
SYSTEM II, Foss Tecator AB, Höganäs, Sweden). Total P was
determined by the H2SO4-HClO4 digestion method (Sparks et al.,
1996).
2.4. Soil sampling and analysis
To examine how N and P additions affect plant available soil N
concentrations, we measured net N mineralization rates by using
the in situ soil core incubation method (Hook and Burke, 1995).
Field sampling was conducted each month in the main growing
seasons (June to August) of 2011 and 2012. Sampling was carried
out on 15th of each month, with 30 days of incubation time in the
field. Within each plot, two sharp-edged PVC tubes (5.6 cm in
diameter and 12 cm in length) were driven into soil at the depth of
10 cm after plants and litter were clipped and removed (Shan et al.,
2011). Soil core inside each tube was 5 cm in diameter and 10 cm in
length. One of the two PVC cores was sealed by Parafilm
membrane to prevent water penetration and allow gas exchange,
incubated in the field for 30 days, and then taken back to the
laboratory. Another PVC core was directly taken back to the
laboratory and stored in a refrigerator at 4 C for measuring the
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initial inorganic N concentrations. All fresh soil samples were
processed within two days in the laboratory of IMGERS. Each fresh
soil core was sieved through 2 mm mesh and separated into two
parts. One was used for soil moisture (oven dried at 105 C for
48 h) and soil pH (air-dried) measurements. The other was
maintained fresh for measuring ammonium (NH4+–N) and nitrate
(NO3þ–N) concentrations. For each fresh soil sample, one 10 g subsample was extracted with 50 ml of 2-mol/L KCl, and the solution
was shaken for one hour with a reciprocal shaker. Then it was
settled for 10–15 min and soil suspension was firstly filtered using
a filter paper (12.5 cm in diameter) and further filtered using a
0.45 mm filter. Concentrations of NH4+–N and NO3þ–N were
determined using a flow injection autoanalyzer (FIAstar 5000
Analyzer, Foss Tecator, Denmark). Extractable NH4+–N and NO3þ–
N concentrations were converted to dry mass basis using soil
moisture data. The total soil available inorganic N for each plot and
each month was the sum of extractable soil ammonium (NH4+–N)
and nitrate (NO3þ–N) concentrations. The growing season soil
available inorganic N for each plot was the average of soil available
inorganic N in June, July, and August, which was used in the
statistical analyses.
To evaluate how plant available soil P varies with N and P
additions, we also collected non-rhizospheric bulk soils and
rhizospheric soils of L. chinensis and A. sibiricum for available P
analysis each year in mid August. Within each plot, bulk soil
samples were collected by randomly taking three soil cores from 0
to 20 cm depth using a 7-cm-diameter soil auger. After removal of
roots, the three soil samples were mixed in situ as a composite
sample. Rhizospheric soil, which consists of a narrow core of soil
that remained attached to roots, were sampled by collecting soils
adhering to root surfaces of each species using a brush (Bell et al.,
2014; Luster et al., 2009). For each species, five plant individuals
with roots and soils were collected within each plot, and
rhizospheric soils were separated from roots in the laboratory of
IMGERS. All fresh soil samples were passed through a 2-mm-mesh
sieve and air dried for available P and soil pH analysis. Soil available
P was extracted by 0.5 M NaHCO3 solutions and determined by a
continuous flow analyzer (Seal AutoAnalyser 3, Seal Analytical,
Norderstedt, Germany). Soil pH values were determined in water
suspension (water: soil = 2.5:1) at 25 C using a pH meter (FE20—
FiveEasy).
To further explore how N and P additions affect soil microbial
biomass carbon (C), N, and P and C: N: P stoichiometry, we also
measured soil biomass C, N and P in mid August of 2012. Soil
samples were collected using the same method and stored at 20
C before analysis. Soil microbial biomass C and N were measured
using the chloroform-extraction method according to Joergensen
(1996). Two 10 g samples were collected from each composite soil
sample. One sample was placed in a chloroform steam bath for 24 h
and another sample was kept non-fumigated. Microbial biomass C
and N were calculated from extractions of C and N from each
fumigated and non-fumigated samples with 0.5 M KCl solution for
30 min. Microbial C and N concentrations in extracts were
determined with a TOC analyzer (Elementar vario TOC, Hanau,
Germany). Microbial biomass C (MBC) and microbial biomass N
(MBN) were calculated as follows:
MBC (mg kg1 soil) = EC/kEC
(1)
MBN (mg kg1 soil) = EN/kEN
(2)
Where is EC the organic C extracted from fumigated soil minus
organic C extracted from non-fumigated soil, EN is the N extracted
from fumigated soil minus N extracted from non-fumigated soil,
kEC is the correction coefficient of microbial biomass C (kEC = 0.38)
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S. Zhan et al. / Environmental and Experimental Botany 134 (2017) 21–32
(Joergensen, 1996), and kEN is the correction coefficient of
microbial biomass N (kEN = 0.54)(Brookes et al., 1985).
Similarly, microbial biomass P was measured using the
chloroform-extraction method according to Hedley et al. (1982).
Microbial P was calculated from extractions of P from each
fumigated and non-fumigated samples with 0.5 M NaHCO3
solution for 30 min. P concentrations in extracts were determine
by a UV Spectrometer (6505 spectrometer, Jenway, Stone, UK)
using Molybdenum Blue Method. Microbial biomass P (MBP) was
calculated as:
MBP (mg kg1 soil) = EP/kEP
(3)
Where is EP the P extracted from fumigated soil minus P extracted
from non-fumigated soil, and kEP is the correction coefficient of
microbial biomass P (kEP = 0.40) (Hedley et al., 1982).
2.5. Statistical analysis
Statistical analyses were performed using SAS 9.2 (SAS Institute,
Cary, North Carolina, USA). Repeated measures analysis of variance
with mixed linear models (PROC MIXED) were used for response
variables, using N and P addition treatments, year and all
interactions as fixed effects. These response variables include soil
available N, P and N: P ratio, rhizosphere available P concentrations, soil pH, soil microbial biomass C, N, P and C: N: P
stoichiometry, leaf and root N and P concentrations and N: P ratios
of dominant species, and aboveground biomass of species and
community. The mixed linear models were also performed to
compare the effects of N and P additions on each response variables
for each year. One-way ANOVA was used to test whether the soil
available N and P concentrations in control plots differed
significantly between the two years. To discern the criteria for
the saturation levels of plant tissue N: P ratio, logarithm regression,
y = a + b* ln (x), was conducted using plant leaf or root N: P ratio as
the response variable and soil available N: P supply ratio as the
independent variable.
We used a mixed linear model to determine how community
biomass responses to N and P additions were affected by interannual variation in precipitation, using the first year data of the
two experiments. We also used the mixed linear model to assess
the first- versus second-year (cumulative) effects of N and P
additions on primary production under the identical precipitation
regime, using the community biomass data of the two experiments
in the same year (i.e. 2012).
The critical leaf or root N: P ratio, above which plant growth is
limited by P and below which plant growth is limited by N
(Güsewell, 2004), was calculated as the ratio between leaf or root N
concentration at the P addition treatment and P concentration at
the N addition treatment. The critical N: P ratio represents the ratio
between N concentration in N-limited plants grown at the optimal
supply of P and P concentration in P-limited plants grown at the
optimal supply of N (Güsewell, 2004). The rhizosphere effect on
plant P availability was calculated as the ratio of available P
concentration in rhizospheric soil to available P concentration in
non-rhizospheric bulk soil (Prhizo/Pnon-rhizo).
3. Results
3.1. Soil available N, P and rhizosphere P concentrations and soil pH
N and P additions significantly increased soil available N and P
(Table S1). For treatments that received N enrichment, soil
available N concentration increased by 143–187% in the first year
and by 8–21% in the second year (Fig. 1). For treatments that
received P addition, soil available P concentration increased by 89–
98% in the first year and by 42.7–49.3 times in the second year
(Fig. 1). No significant effect of N addition on soil available P
concentration was observed over the two years of experiment
(Fig. 1). Without N and P treatments, soil available N and P
concentrations in control plots also differed substantially between
the two years. Soil available N concentration, on average, was 33%
lower in 2011 than that in 2012 (one-way ANOVA: F1,26 = 6.53,
P = 0.0168). Soil available P concentration, in contrast, was 7.6 times
higher in 2011 than that in 2012 (F1,26 = 146.51, P < 0.0001). In
general, P addition significantly reduced soil available N: P ratio,
while N addition elevated soil available N: P ratio. For a given
treatment, soil available N: P ratio differed substantially over the
two years due to changes in both available N and P concentrations
(Fig. 1). The largest variation in soil available N: P ratio was
observed in control plots, in which the average soil available N: P
ratio varied from 4.8 in 2011 to 60.5 in 2012.
For both L. chinensis and A. sibiricum, P addition significantly
increased rhizosphere available P concentrations, while N addition
had no significant effect on rhizosphere available P concentrations
(Table S1; Fig. 1). Nitrogen addition significantly reduced bulk soil
pH in the second year but not in the first year (Table S1). Soil pH
reduction in N addition treatment (pH control pH treatment), on
average, was 0.4 in the second year (Fig. 1).
3.2. Plant species and community aboveground biomass
At the species level, aboveground biomass of L. chinensis
increased by 29% in the first year in the N addition treatment, but
was not significantly increased by N addition in the second year
(Table S2; Fig. 2). Phosphorus addition alone did not show a
significantly positive effect on aboveground biomass of L. chinensis
over two years. Aboveground biomass of A. sibiricum increased by
21–66% in the first and by 62–216% in the second year in
treatments with either N or N and P additions (Fig. 2). In contrast, a
significantly positive response in aboveground biomass of A.
sibiricum to P addition alone was only observed in the second year
(Fig. 2). During two years of study, the effects of N, P, and N + P
additions had no significant positive effects on aboveground
biomass of other species (Table S2, Fig. 2).
At the community level, N addition alone increased community
aboveground biomass by 20% in the first year (2011) and by 44% in
the second year (2012). Phosphorus addition alone increased
community aboveground biomass by 38% in the second year, with
no positive response in community biomass in the first year.
Without N and P treatments, community aboveground biomass, on
average, was 61% greater in 2011 than that in 2012 (one-way
ANOVA: F1,26 = 29.44, P < 0.0001). In both 2011 and 2012, the
greatest biomass response was found in treatment that received
N + P additions. On average, community aboveground biomass
increased by 70% in treatment of N + P additions (Fig. 2). Stepwise
multiple linear regressions, using soil available N and P as
independent variables, showed that across all treatments the
two variables explained 38% of the variance in community
aboveground biomass in 2011 (F1,42 = 25.52, P < 0.0001) and 35%
in 2012 (F2,41 = 10.9, P = 0.0002). In 2011, soil available N
concentration was the only variable left in model. In 2012,
however, soil available P concentration showed greater partial R2
(partial R2 = 0.20, P = 0.0021) than that of the soil available N
concentration (partial R2 = 0.14, P = 0.0047). For all N, P, and N + P
addition treatments, community aboveground biomass responses
were significantly greater in 2012 than those in 2011 (P < 0.05 for
all treatments).
Furthermore, the first year results of the two independent
experiments showed that community aboveground biomass was
significantly increased in 2011 by N and N + P additions (the first
experiment; Fig. 2), while community biomass was significantly
S. Zhan et al. / Environmental and Experimental Botany 134 (2017) 21–32
increased by N and P additions in 2012 when they were added
individually (the second experiment; Fig. S2). Within the same
year (2012), the increases in community biomass with N and P
additions were almost identical between the two experiments
(Table S3); although the duration of the two experiments differed.
25
3.3. Leaf and root N and P concentrations and N: P stoichiometry
Patterns of leaf and root N and P concentrations and N: P
stoichiometry between the two dominant species, i.e. L.
chinensis and A. sibiricum, were mostly similar in their responses
Fig. 1. Effects of nitrogen (N) and phosphorus (P) additions on soil available N, P and rhizosphere available P concentrations (mg kg1 dry soil), and soil pH. Soil available N, P
and rhizoshphere available P concentrations for each treatment were the average of all replicates (error bars denote SEM). Significant differences are reported as NS, P > 0.1; +,
0.05 < P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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S. Zhan et al. / Environmental and Experimental Botany 134 (2017) 21–32
to N and P treatments over two years of study (Table S4). In
general, for both species, strong positive responses in leaf and
root N concentrations were found in treatments that received N
enrichment, while leaf and root P concentrations responded
positively to treatments that received P enrichment (Figs. S3 and
S4). Across all treatments, leaf N and P concentrations were
substantially greater than root N and P concentrations over two
years (Figs. S3 and S4).
For both L. chinensis and A. sibiricum, N addition elevated leaf
and root N: P ratios, while P addition reduced both leaf and root N:
Fig. 2. Effects of N and P additions aboveground biomass of L. chinensis, A. sibiricum, other species, and community. Aboveground biomass for each treatment was the average
of all replicates (error bars denote SEM). All symbols are derived as from Fig. 1.
S. Zhan et al. / Environmental and Experimental Botany 134 (2017) 21–32
27
Fig. 3. Effects of N and P additions on leaf and root N:P ratios of L. chinensis and A. sibiricum. Leaf and root N:P ratios for each treatment were the average of all replicates (error
bars denote SEM). All symbols are derived as from Fig. 1.
P ratios (Fig. 3). The critical leaf N: P ratio of the two species was
consistently 19 across the two- and single-year treatments. In
contrast, the critical root N: P ratio of the two species varied from
around 8 in 2011 to 16 in 2012. The N: P ratios were consistently
higher for leaves than for roots across all treatments (Fig. S5).
Furthermore, leaf and root N: P ratios of both species were
positively correlated with soil available N: P supply ratio across all
treatments in 2011 and 2012 (Fig. 4). However, little additional
response of leaf N: P ratio was found for soil available N: P supply
ratio >20 over two years of treatments. Root N: P ratio tended to
reach a plateau at soil available N: P supply ratio>10 in both 2011
and 2012 (Fig. 4).
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S. Zhan et al. / Environmental and Experimental Botany 134 (2017) 21–32
Fig. 4. Relationships of leaf and root N:P ratios of L. chinensis (black points) and A. sibiricum (red points) with soil available N: P supply ratio across all plots in 2011 and 2012.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. Rhizosphere effect on P availability
Rhizosphere effect on P availability was mostly modified by P
addition treatment. In control plots, the rhizospheric soil available
P concentration was five (A. sibiricum) to ten (L. chinensis) times
higher than that of the non-rhizospheric bulk soil (Fig. 5).
Treatments that received P or N + P additions significantly
decreased the rhizosphere effect on P availability in the second
year of experiment (Fig. 5). The rhizosphere P activation capability
of L. chinensis and A. sibiricum, on average, was decreased by 6-10
times in the second year in treatments that received P or N + P
additions. Without N and P enrichment, the rhizosphere P
activation capability of L. chinensis was substantially greater than
that of A. sibiricum in both 2011 (one-way ANOVA: F1,26 = 9.71,
P = 0.0044) and 2012(one-way ANOVA: F1,26 = 9.43, P = 0.0050).
Also, for both species, the rhizosphere P activation capability in
control plots increased by three times in 2012 when soil available P
was low, compared with rhizosphere P activation capability in 2011
(Fig. 5).
3.5. Soil microbial biomass C, N and P and C: N: P stoichiometry
The results showed that P addition slightly decreased soil
microbial biomass C (P = 0.0979) and N (P = 0.0183) but increased
soil microbial biomass P (P = 0.0690; Fig. 6). The effects of N and
N + P on soil microbial biomass C, N and P were non-significant
(Fig. 6). For treatments that received P or N + P additions, soil
microbial biomass P on average increased by 20% while soil
microbial biomass C and N decreased by 15% and 25%, respectively.
Without nutrient additions, soil microbial biomass C: N, N: P and C:
P ratios were 5.0, 4.5 and 21.4, respectively. Soil microbial biomass
C: N ratio was significantly increased (P = 0.0335) but N: P ratio was
reduced (P = 0.0007) by P addition. However, no significant effects
of N, P or N + P additions on microbial biomass C: P ratio was
observed (Fig. 6). In addition, relationships of soil available N, P and
N: P ratio with soil microbial biomass C, N, P, C: N, C: P and N: P
were all non-significant (P > 0.05).
4. Discussion
4.1. Patterns and controlling factors of community-level N and P
limitation
Our study, based on two independent N, P, and N + P addition
experiments in the Inner Mongolia grassland, demonstrated that
aboveground net primary production (ANPP) was co-limited by N
and P. The patterns of N and P limitation, however, shifted from
serial limitation by N in 2011 (a normal year) to independent N and
P co-limitation in 2012 (a wet year). In 2011, the increase in ANPP
occurred only to N enrichment when N and P were added
individually, while ANPP increased synergistically in treatment in
which N and P were added simultaneously. In 2012, the increase in
S. Zhan et al. / Environmental and Experimental Botany 134 (2017) 21–32
29
Fig. 5. Responses of rhizosphere effect on soil P availability to N and P additions. Rhizosphere effect on soil P availability was calculated as rhizosphere available P divided by
non-rhizosphere soil available P (Prhizo/Pnon-rhizo). All symbols are derived as from Fig. 1.
ANPP occurred to both N and P treatments when they were added
individually. The synergistic interaction between N and P, however,
was statistically insignificant in 2012, implying that the effects of N
and P on ANPP in N + P addition treatment are likely to be additive.
The N and P co-limitation is further confirmed by the results from
the second experiment, which were almost identical to those from
the first experiment in year two. These results suggest two general
conclusions. First, the inter-annual shift in patterns of N and P
limitation was mainly triggered by soil N and P availability, both of
which varied considerably among years. ANPP was more limited by
N than by P in 2011, when soil P availability was high and soil
available N: P ratio was low; whereas it was more limited by P than
by N in 2012, when soil P availability was low, while soil N
availability and available N: P ratio were high. These findings
highlight the importance of soil N and P availability in controlling
the inter-annual shift in patterns of N and P limitation in semiarid
steppe ecosystems (Elser et al., 2007; Harpole et al., 2011).
Second, the inter-annual difference in ANPP responses to N and
P treatments was mainly caused by the interaction of water and
light availability but not by the accumulative effects of N and P
additions, at least in the short term. Our results indicate that the
inter-annual variation in precipitation alters soil N and P
availability and stoichiometric ratio of available N: P and thereby
indirectly determines the relative strength of N and P limitation to
ANPP. Previous studies have shown that N limitation is tightly
coupled with water availability in semiarid grassland ecosystems
(Bai et al., 2010; Hooper and Johnson, 1999; Lan and Bai, 2012; Sala
et al., 2012). ANPP responds more to added N in wet years than in
dry years (Hooper and Johnson, 1999; Lan and Bai, 2012). In this
study, however, ANPP responses to N and P treatments did not
30
S. Zhan et al. / Environmental and Experimental Botany 134 (2017) 21–32
Fig. 6. Responses of soil microbial biomass carbon (C), nitrogen and phosphorus concentrations (mg kg1 dry soil) and C: N, N: P and C: P ratios to N and P additions. For each
treatment, soil microbial biomass C, N and P concentrations and C: N, N: P and C: P ratios were the average of all replicates (error bars denote SEM). All symbols are derived as
from Fig. 1.
increase with precipitation, although the growing season precipitation was 41% higher in 2012 than in 2011. ANPP in control plots
was higher in 2011 than in 2012. How do we reconcile these
seemingly contradictory results? One possibility is that the lower
ANPP responses to N and P and lower ANPP in control plots in 2012
might be attributed to low photosynthetically active radiation
(PAR) in the growing season, which may limit the productivity
response to the increased precipitation and N and P treatments.
Indeed, the total PAR from June to August was 17% lower in 2012
than in 2011. This suggests that light availability is likely to have
limited primary productivity both in control plots and in N and P
treatment plots in the wet year when water limitation is lessened,
as indicated by previous studies (Paruelo et al., 1997; Wu et al.,
2008). These results are corroborated by a recent study that the
reduction in light availability substantially decreased community
biomass production in experimental grasslands (Roscher et al.,
2016). Therefore, findings from this study support the multipleresource limitation hypothesis that the interactions between
multiple limiting resources may all affect the responses of primary
production to N and P additions (Harpole et al., 2011; Vitousek
et al., 2010).
4.2. Species-level N and P limitation and leaf N: P stoichiometry
The aboveground biomass responses of the two dominant
species indicate that L. chinensis was primarily limited by N,
whereas A. sibiricum was co-limited by N and P, i.e. serial limitation
in 2011 and independent co-limitation in 2012. However, without
N and P additions leaf N: P ratio ranged from 17 to 19 for L. chinensis
and from 18 to 21 for A. sibiricum over the two years, suggesting
biomass production of both species might be limited by P alone
(Koerselman and Meuleman, 1996). Obviously, this is contrary to
the observed biomass responses. The discrepancy between the
results from the leaf N:P ratios and biomass responses of the two
species implies that the critical N: P ratios of perennial grasses in
semiarid grasslands might be higher than those in Koerselman and
S. Zhan et al. / Environmental and Experimental Botany 134 (2017) 21–32
Meuleman (1996), due to their inherently high P resorption
efficiency (Lü et al., 2013; Lü and Han, 2010). Indeed, our results
showed that, for both species, the critical leaf N: P ratio was
consistently 19 across the two experiments, which is consistent
with their biomass responses to N and P treatments. Previous
studies within the same plant community suggest that nutrient
resorption is an important pathway of plant-soil feedbacks in
semiarid ecosystems (Lü et al., 2013; Lü and Han, 2010). Nitrogen
addition increased leaf N concentration and N: P ratio, reduced leaf
N resorption efficiency, and elevated P resorption efficiency (Lü
and Han, 2010). Recent studies also showed that N addition
increased soil phosphatase activity and thereby delayed the onset
of P limitation to primary productivity in European grassland and
other terrestrial ecosystems (Fujita et al., 2010b; Marklein and
Houlton, 2012). In our system, whether N addition can increase soil
phosphatase activity and delay the onset of P limitation to primary
productivity is need to be further tested. In this study, we found
that P and N + P additions substantially reduced rhizosphere P
activation capacity of L. chinensis and A. sibiricum. Hence, plant N
and P cycles are tightly coupled through feedbacks between soil N
and P inputs, leaf N and P responses, and plant N and P resorption
efficiencies (Güsewell, 2004; Lü et al., 2013).
Furthermore, our results showed that N addition reduced soil pH
by 0.4 units in the second year of experiment, which increased
rhizosphere available P concentration of L. chinensis. However, even
for L. chinensis, the increased rhizosphere available P did not cause a
significant increase in leaf P concentration and/or a decrease in leaf
N: P ratio. We also noted that the short-term experimental duration
in this study limits our ability to assess the effects of N and Ninduced soil acidification on species N and P limitation. Therefore,
more studies are needed to evaluate how N enrichment and soil
acidification interactively affect plant species and community N and
P limitation, nutrient use efficiencies, and N and P cycles (Chen et al.,
2013; Elser et al., 2007; Fujita et al., 2010a).
4.3. Linkages among plant, soil, and microbial biomass N: P
stoichiometry
Are N:P ratios of plants, soils, and microbes closely related in
semiarid steppe ecosystems? Our results showed that, for both L.
chinensis and A. sibiricum, leaf N: P is more sensitive than root N: P
to changes in soil N and P availability. For both species, leaf N: P
ratio tends to saturate at soil available N: P supply ratio of
approximately 20, while root N: P ratio become saturated at soil
available N: P supply ratio of around 10. The low sensitivity of root
N: P ratio to changes in soil available N: P supply ratio is likely due
to their rhizosphere effects to elevate available P concentrations.
For example, the available P concentration in rhizosphere soil
increased 4.4-fold for L. chinensis and 2.5-fold for A. sibiricum in
2011 when available P in bulk soils was relatively high. However,
the available P concentration in rhizosphere soil increased 13.9fold for L. chinensis and 7.9-fold for A. sibiricum in 2012 when
available P in bulk soils was substantially low. The high rhizosphere
effect on P availability may explain why L. chinensis was more
limited by N, compared with A. sibiricum which was co-limited by
N and P. These results corroborate several previous findings that
plants can adapt to P deficient conditions through rhizosphere
processes to increase the availability and uptake of P (Neumann
and Romheld, 1999; Richardson et al., 2009; Ström et al., 2005).
Soil microbial biomass N: P ratio, in contrast, was not
significantly correlated with leaf and root N:P ratios and soil
available N:P supply ratio. This implies that soil microbial biomass
N:P stoichiometry is less affected by plant tissue and soil N:P
stoichiometry. A previous study also proposes that soil microbial
N:P ratios do not correlate with soil N: P supply ratio (Cleveland
and Liptzin, 2007). In this study, the reduction in microbial
31
biomass N: P ratio with P and N + P additions might be caused by a
shift in microbial community structure, such as fungal: bacteria
ratio. Two recent studies suggest that P and N + P additions altered
soil microbial community structure and increased fungal: bacteria
ratio in forest ecosystems (Li et al., 2015; Liu et al., 2012). Across all
treatments, we only observed 5–35% reductions in microbial
biomass N:P ratio although soil available N:P supply ratio varied by
56-fold. Together, these findings indicate that microbial biomass N:
P ratio is strongly homoeostatic, compared with 3-fold variations
in leaf N:P ratio and 2-fold variations in root N: P ratio of the two
dominant species. Our results are consistent with findings from
previous studies that microbial biomass N:P ratio is less variable
than plant tissue and soil N:P ratios (Cleveland and Liptzin, 2007;
Makino et al., 2003).
4.4. Implication for global change
Our findings have important implications for understanding the
influence of N deposition and global climatic change on ecosystem
structure and functioning of semiarid grasslands. First, chronic N
deposition alters plant functional group composition and substantially decreases biodiversity and ecosystem stability in semiarid
grasslands (Bai et al., 2010; Lan and Bai, 2012). The results from this
study further demonstrated that N enrichment lessened N
limitation of plant species and the plant community but intensified
P limitation, and effects of N and P on primary production were
tightly coupled. This suggests that chronic N deposition, which has
occurred over several decades, may further alter the stoichiometric
balance of N and P and intensify P limitation of steppe ecosystems
in this century (Elser et al., 2007). Second, our results suggest that
shifts in N and P limitation and plant N: P stoichiometry are likely
mediated by water and light availability through direct effect on
plant growth and indirect effect on soil N and P availability. Future
changes in precipitation amount and seasonal distribution may
have profound impacts on N and P limitation and ecosystem
functioning of arid and semiarid grasslands (Bai et al., 2008;
Easterling et al., 2000).
Author contribution
Y. F. Bai and S. X. Zhan designed the experiment. S. X. Zhan and
W. H. Li conducted the experiment. Y. F. Bai, S. X. Zhan, and Y. Wang
analyzed the data, drafted the manuscript, and revised the paper.
Acknowledgement
We are grateful to Xue Bai and staff at the Inner Mongolia
Grassland Ecosystem Research Station (IMGERS), Chinese Academy
of Sciences for their help with field work. This study was supported
by the National Key Research and Development Program of China
(2016YFC0500804) and the National Natural Science Foundation of
China (31320103916). S. X. Zhan and Y. Wang contributed equally
to this work.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
envexpbot.2016.10.014.
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